Liwen Lai

Interests

Research

Mutation Analysis and Genotype/Phenotype Correlation of Genetic Diseases; Uniparental Disomy and Imprinted Genes as a Mechanism for Human Diseases; Animal Models of Mendelian Diseases; Molecular Basis of Hematological Neoplasms

Teaching

Molecular Diagnosis in the Era of Precision Medicine; Clinical Molecular Genetics and Genomics; Clinical Cytogenetics and Genomics; Cytogenetic and Molecular Diagnosis of Hematological Neoplasms; Cancer Genetics

Courses

Scholarly Contributions

Journals/Publications

• Alswied, A., Rehman, A., Lai, L., Duran, J., Sardar, M., & Proytcheva, M. A. (2021). Rare monosomy 7 and deletion 7p at diagnosis of chronic myeloid leukemia in accelerated phase. Cancer genetics, 252-253, 111-114.
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  Clonal cytogenic evolution with the development of additional chromosomal abnormalities (ACAs) in chronic myelogenous leukemia (CML) is a marker for disease progression and is known to impact therapy and survival. The presence of ACAs has been shown to affect the responses to tyrosine kinase inhibitors (TKI) in patients with newly diagnosed CML in accelerated phase (CML-AP). We report a rare case of a CML patient who presented in CML-AP and was found to have multiple ACAs including monosomy 7, deletion 7p, trisomy 8, and an extra Philadelphia chromosome (Ph) in separate Ph-positive cell line, respectively. Six months after combined chemotherapy with TKI, the patient achieved a major cytogenetic response with disappearance of monosomy 7/deletion 7p with no major molecular response.
• Mustacich, D. J., Lai, L., Bernas, M. J., Jones, J. A., Myles, R. J., Kuo, P. H., Williams, W. H., Witte, C. L., Erickson, R. P., & Witte, M. H. (2021). Digenic Inheritance of a FOXC2 Mutation and Two PIEZO1 Mutations Underlies Congenital Lymphedema in a Multigeneration Family. The American journal of medicine. doi:doi: 10.1016/j.amjmed.2021.09.007
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  The lymphatic system is essential for maintaining the balance of interstitial fluid in tissues and for returning protein-rich fluids (lymph) to the bloodstream. Congenital lymphatic defects lead to accumulation of lymph in peripheral tissues and body cavities, termed primary lymphedema. To date, only a limited number of individual genes have been identified in association with primary lymphedema. However, variability of age of onset and severity of lymphatic abnormalities within some families suggests that multiple mutations or genes may be responsible, thus hampering efforts to identify individual associated genes.
• Otsuji, J., Girard, N., Perry, C. S., Fuchs, D., & Lai, L. (2021). Sibling Donor-Derived Myeloid Sarcoma After Hematopoietic Stem Cell Transplant. Human Pathology, 24. doi:https://doi.org/10.1016/j.ehpc.2021.200512
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  Janelle Otsuji, MD (jjotsuji@pathology.arizona.edu); Nicole Girard, MD; Catherine Spier, MD; Deborah Fuchs, MD; Li-Wen Lai, PhD. Department of Pathology, The University of Arizona, Tucson, Arizona.Myeloid sarcoma (MS) occurs in 8% to 20% of post–allogeneic hematopoietic stem cell transplant (allo-HSCT) patients, mostly from recipient's relapsed leukemia. Donor-derived MS (DDMS) is extremely rare. We report 2 cases of DDMS after successful allo-HSCT from human leukocyte antigen (HLA)-identical, sex-mismatched siblings. Donor origin was confirmed by both cytogenetic and molecular chimerism tests. Case 1 was a 50-year-old man with history of KMT2A-rearranged acute myeloid leukemia (AML) presented with bilateral knee soft tissue masses 5 years post–allo-HSCT. Knee biopsy revealed infiltrative large cells with blastic morphology expressing CD4, CD10, CD38, CD33 (dim), subset MPO, while negative for CD117 and CD34 (Table). Interphase fluorescence in situ hybridization (FISH) analysis showed gain of chromosomes 5/6/8/15q/20q/21q and loss of 17q/20q with 95.5% cells showing donor origin. Concurrent bone marrow biopsies showed donor-derived karyotype//46,XX,del(20)(q11.2q13.3)[4]/46,XX[16] versus 46,XY,t(6;11)(q27;q23)[18]/46,XY[2] at diagnosis 8 years prior (Table). Case 2 was a 40-year-old man with history of AML with myelodysplasia-related changes (AML-MRC) presented with a testicular mass 5 years post-HSCT. Orchiectomy yielded a mass of 163 g and 8.7 cm in diameter. Microscopically, there was diffuse effacement of the testis architecture with large cells demonstrating an immature myeloid phenotype CD34+, CD117+, MPO dim subset, and aberrant CD7. FISH analysis showed 100% of cells showing XX donor cells. Concurrent bone marrow biopsies showed 100% normal XX donor cells. Both cases developed DDMS 5 years after allo-HSCT from siblings. Verifying donor origininAML relapseiscriticalas transformed donor cells may have different genetic alterations and behaviors from initial AML. Our case 2 is likely the first testicular sibling-DDMS in the literature.
• Islam, F., Htun, S., Lai, L., Krall, M., Poranki, M., Martin, P. M., Sobreira, N., Wohler, E. S., Yu, J., Moore, A. T., & Slavotinek, A. M. (2020). Exome sequencing in patients with microphthalmia, anophthalmia, and coloboma (MAC) from a consanguineous population. Clinical genetics, 98(5), 499-506.
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  Next-generation sequencing strategies have resulted in mutation detection rates of 21% to 61% in small cohorts of patients with microphthalmia, anophthalmia and coloboma (MAC), but despite progress in identifying novel causative genes, many patients remain without a genetic diagnosis. We studied a cohort of 19 patients with MAC who were ascertained from a population with high rates of consanguinity. Using single nucleotide polymorphism (SNP) arrays and whole exome sequencing (WES), we identified one pathogenic variant in TENM3 in a patient with cataracts in addition to MAC. We also detected novel variants of unknown significance in genes that have previously been associated with MAC, including KIF26B, MICU1 and CDON, and identified variants in candidate genes for MAC from the Wnt signaling pathway, comprising LRP6, WNT2B and IQGAP1, but our findings do not prove causality. Plausible variants were not found for many of the cases, indicating that our current understanding of the pathogenesis of MAC, a highly heterogeneous group of ocular defects, remains incomplete.
• Erickson, R. P., Lai, L., Mustacich, D., Bernas, M. J., Kuo, P. H., & Witte, M. H. (2019). Sex-limited penetrance of lymphedema to females with CELSR1 haploinsufficiency: A second family.. Clinical Genetics, 96(5), 478-482. doi:10.1111
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  A second multigeneration family with hereditary lymphedema (LE) secondary to a variant in the planar polarity gene, CELSR1, is described. Dominant inheritance of the variant was discovered using whole-exome sequencing and confirmed by Sanger sequencing. In contrast to heterozygous males, all heterozygous females showed LE during physical examination albeit variable in severity and age of onset. Lymphscintigraphy in affected females showed previously undescribed lymphatic abnormalities consistent with lymphangiectasia, valve dysfunction, and thoracic duct reflux.
• Lee, C. T., Ng, H. Y., Lee, Y. T., Lai, L. W., & Lien, Y. H. (2016). The role of calbindin-D28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics. American journal of physiology. Renal physiology, 310(3), F230-6.
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  Calbindin-D28k (CBD-28k) is a calcium binding protein located in the distal convoluted tubule (DCT) and plays an important role in active calcium transport in the kidney. Loop and thiazide diuretics affect renal Ca and Mg handling: both cause Mg wasting, but have opposite effects on Ca excretion as loop diuretics increase, but thiazides decrease, Ca excretion. To understand the role of CBD-28k in renal Ca and Mg handling in response to diuretics treatment, we investigated renal Ca and Mg excretion and gene expression of DCT Ca and Mg transport molecules in wild-type (WT) and CBD-28k knockout (KO) mice. Mice were treated with chlorothiazide (CTZ; 50 mg · kg(-1) · day(-1)) or furosemide (FSM; 30 mg · kg(-1) · day(-1)) for 3 days. To avoid volume depletion, salt was supplemented in the drinking water. Urine Ca excretion was reduced in WT, but not in KO mice, by CTZ. FSM induced similar hypercalciuria in both groups. DCT Ca transport molecules, including transient receptor potential vanilloid 5 (TRPV5), TRPV6, and CBD-9k, were upregulated by CTZ and FSM in WT, but not in KO mice. Urine Mg excretion was increased and transient receptor potential subfamily M, member 6 (TRPM6) was upregulated by both CTZ and FSM in WT and KO mice. In conclusion, CBD-28k plays an important role in gene expression of DCT Ca, but not Mg, transport molecules, which may be related to its being a Ca, but not a Mg, intracellular sensor. The lack of upregulation of DCT Ca transport molecules by thiazides in the KO mice indicates that the DCT Ca transport system is critical for Ca conservation by thiazides.
• Lee, C., Lien, Y. H., Lai, L., Ng, H., Chiou, T. T., & Chen, H. (2013). Variations of dietary salt and fluid modulate calcium and magnesium transport in the renal distal tubule. Nephron - Physiology, 122(3-4), 19-27.
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  PMID: 23774784;Abstract: Background: The renal distal tubule fine-tunes renal epithelial calcium transport. Dietary intake of salt and fluid varies day-to-day and the kidney adapts accordingly to maintain homeostasis. The alternations in salt and fluid balance affect calcium and magnesium transport in the distal tubule, but the mechanisms are not fully understood. Methods: Sprague-Dawley rats were grouped into high-salt, low-salt and dehydration treatment. Daily intake, water consumption and urine output were recorded. At the end of the experiment, blood and urine samples were collected for hormonal and biochemical tests. Genetic analysis, immunoblotting and immunofluorescence studies were then performed to assess the alterations of calcium and magnesium transport-related molecules. Results: High-salt treatment increased urinary sodium, calcium and magnesium excretion. Low-salt treatment and dehydration were associated with decreased urinary excretion of all electrolytes. High-salt treatment was associated with increased intact parathyroid hormone levels. A significant increase in gene expression of TRPV5, TRPV6, calbindin-D28k and TRPM6 was found during high-salt treatment, while low salt and dehydration diminished expression. These findings were confirmed with immunofluorescence studies. High-salt and low-salt intake or dehydration did not cause any significant changes in WNK1, WNK3 and WNK4. Conclusions: Alternations in salt and water intake affect renal calcium and magnesium handling. High-salt intake increases the distal delivery of the divalent cations which upregulates distal tubule calcium and magnesium transport molecules, while the opposite effects are associated with low-salt intake or dehydration. Copyright © 2013 S. Karger AG, Basel.
• Lai, L., Yong, K., & Lien, Y. H. (2012). Pharmacologic recruitment of regulatory T cells as a therapy for ischemic acute kidney injury. Kidney International, 81(10), 983-992.
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  PMID: 22189844;PMCID: PMC3340526;Abstract: Regulatory T cells (Tregs) are key components of the peripheral tolerance system and have become an immunotherapeutic agent for treating inflammatory processes. This therapeutic option, however, is hampered by problems arising from isolating and expanding desirable Tregs. Here we used an alternative approach with a pharmacologic agent to stimulate Tregs to achieve immunosuppressive effects. Pretreatment of mice with the naturally occurring sphingosine N,N-dimethylsphingosine (DMS) was found to increase both tissue-infiltrating T effectors (Teffs, CD4 + Foxp3 - ) and Tregs (CD4 + Foxp3 - ) in the early phase of bilateral renal ischemia/reperfusion injury. DMS itself had no effects on renal function or histopathology, but rapidly and transiently increased both Teffs and Tregs and increased the expression of chemokines CXCL9, CCL5, and CXCL10 in non-ischemic kidneys (sham operation). This renoprotection was abolished by administration of the Treg suppressing agents, anti-CTLA-4 or anti-CD25 monoclonal antibodies, suggesting that Tregs play a key role in DMS-induced renoprotection. Thus, Tregs recruited to the kidney by DMS ameliorate acute kidney injury and provide a new approach to control inflammatory diseases. © 2012 International Society of Nephrology.
• Lee, C., Chen, H. C., Ng, H., Lai, L., & Lien, Y. H. (2012). Renal adaptation to gentamicin-induced mineral loss. American Journal of Nephrology, 35(3), 279-286.
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  PMID: 22378246;PMCID: PMC3357145;Abstract: Background: Gentamicin, a well-known nephrotoxic drug, affects calcium and magnesium homeostasis. Although gentamicin induces urinary calcium and magnesium wasting immediately, it rarely causes significant hypocalcemia or hypomagnesemia clinically. Methods: We conducted an animal study to investigate the renal adaptation in calcium and magnesium handling after gentamicin treatment and effects on the expression of calcium and magnesium transport molecules in distal tubule. Gentamicin (40 mg/kg) was injected daily in male Sprague-Dawley rats (220-250 g) for up to 7 days. Results: This treatment did not affect serum creatinine, calcium, or magnesium levels. Gentamicin induced significant hypercalciuria (14-fold) and hypermagnesiuria (10-fold) in 6 h, which was associated with upregulation of TRPV5 (175 ± 3%), TRPV6 (170 ± 4%), TRPM6 (156 ± 4%) and calbindin-D28k (174 ± 3%; all p < 0.05 vs. control). This gene upregulation was maintained with daily injection of gentamicin for 7 days. The gentamicin-induced urinary calcium loss was reduced by 80% at days 3 and 7, while magnesium loss was reduced by 52 and 57% at days 3 and 7, respectively. On the other hand, urinary loss of potassium became worse on day 7 (2-fold), and phosphorus loss worse from day 3 to day 7 (3-fold). Conclusion: There is a rapid adaptation to gentamicin-induced hypercalciuria and hypermagnesiuria. The upregulation of distal tubule transport molecules, TRPV5, TRPV6, TRPM6 and calbindin-D28k occurs within 6 h of gentamicin treatment. This renal adaptation prevents further mineral loss due to gentamicin treatment. Copyright © 2012 S. Karger AG.
• Mustafa, E., Lai, L., & Lien, Y. H. (2012). Rapid recovery from acute kidney injury in a patient with metformin-associated lactic acidosis and hypothermia. American Journal of Medicine, 125(2), e1-e2.
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  PMID: 22269627;PMCID: PMC3381612;
• Lee, C., Ng, H., Lien, Y., Lai, L., Wu, M., Lin, C., & Chen, H. (2011). Effects of cyclosporine, tacrolimus and rapamycin on renal calcium transport and vitamin D metabolism. American Journal of Nephrology, 34(1), 87-94.
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  PMID: 21691056;Abstract: Background: Abnormalities in mineral metabolism are common complications of organ transplantation. The role of immunosuppressive agents in alteration of mineral metabolism is not clear. Methods: We conducted an animal study to investigate the effects of cyclosporine A (CsA), tacrolimus, and sirolimus on renal calcium, magnesium and vitamin D metabolism. Results: CsA and tacrolimus induced a 2- to 3-fold and 1.6- to 1.8-fold increase in urinary calcium and magnesium excretion, respectively, while rapamycin had no effects on calcium, but doubled the urinary magnesium excretion. CsA and tacrolimus, but not rapamycin, elevated serum 1,25(OH) 2 vitamin D without affecting the parathyroid hormone level. CsA and tacrolimus reduced mRNA abundance in TRPV5 (CsA: 64 ± 3% of control; tacrolimus: 50 ± 3%) calbindin-D28k (CsA: 62 ± 4%; tacrolimus: 43 ± 3%), and vitamin D receptor (CsA: 52 ± 3%; tacrolimus: 58 ± 2%, all p < 0.05). Rapamycin did not affect gene expression in any of studied proteins. The immunofluorescence staining study demonstrated a 50% reduction of TRPV5 and calbindin-D28k by CsA and tacrolimus. Conclusion: The suppression of VDR by calcineurin inhibitors is probably the underlying mechanism of renal calcium wasting. In spite of an increased 1,25(OH) 2 vitamin D level, the kidney is not able to reserve calcium, suggesting a role of vitamin D resistance that may be related to bone loss. Copyright © 2011 S. Karger AG, Basel.
• Lien, Y. H., & Lai, L. (2011). Pathogenesis, diagnosis and management of paraneoplastic glomerulonephritis. Nature Reviews Nephrology, 7(2), 85-95.
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  PMID: 21151207;PMCID: PMC3058941;Abstract: Paraneoplastic glomerulonephritis is a rare complication of malignancy that is frequently mistaken for idiopathic glomerulonephritis. Failure to recognize paraneoplastic glomerulonephritis can subject patients to ineffective and potentially harmful therapy. The pathology of paraneoplastic glomerulonephritis varies between different types of malignancies. This Review discusses the association of glomerulonephritis with both solid tumors and hematological malignancies. The pathogenetic mechanisms of many glomerular lesions seem to relate to altered immune responses in the presence of a malignancy. Studies in the Buffalo/Mna rat model of spontaneous thymoma and nephrotic syndrome indicate that polarization of the immune response toward a T-helper-2 (TH 2) profile has an important role in the development of thymoma-associated glomerular lesions. Furthermore, overexpression of the TH 2 cytokine interleukin 13 in rats induces minimal change disease. Such findings from experimental studies might facilitate the identification of biomarkers that can distinguish paraneoplastic glomerulonephritis from idiopathic and other secondary glomerulonephritides. This Review describes potential pathogenetic mechanisms for paraneoplastic glomerulonephritides associated with different malignancies and highlights the need for a multidisciplinary approach to the management of patients with paraneoplastic glomerulonephritis. © 2011 Macmillan Publishers Limited. All rights reserved.
• Lai, L., Yong, K., & Lien, Y. H. (2008). Site-specific expression of IQGAP1, a key mediator of cytoskeleton, in mouse renal tubules. Journal of Histochemistry and Cytochemistry, 56(7), 659-666.
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  PMID: 18413651;PMCID: PMC2430167;Abstract: IQGAP1 is a multifunctional junction molecule that is involved in cell migration, proliferation, differentiation, cell polarity, and cell-cell adhesion. It is highly expressed in the kidney and has recently been identified in the glomerular basement membrane as a nephrin-associated protein. However, the distribution of IQGAP1 in renal tubular epithelial cells is unknown. We performed confocal microscopic studies to localize IQGAP1 in each nephron segment using dual immunofluorescence staining with various antibodies against segment-specific markers. We found that IQGAP1 was strongly expressed in the distal convoluted tubule (DCT), collecting duct, and macula densa and moderately in the thick ascending limb and proximal tubule. In the DCT, the IQGAP1-F-actin complex forms a comb-like structure with multiple parallel spikes sitting on the basal membrane. In the macula densa cells, IQGAP1 is strongly expressed in the apical membrane, whereas in type A intercalated cells, IQGAP1 is expressed in the basolateral membrane, where it colocalizes with anion exchanger 1, and in principal cells, it is diffusely expressed. In conclusion, we showed the expression and subcellular localization of IQGAP1 in various nephron segments. The site-specific expression pattern of this potent modulator of multiple biological pathways in the renal tubules suggests that IQGAP1 may have multiple important roles in various renal functions. © The Histochemical Society, Inc.
• Margolis, D. S., Geffre, C. P., Szivek, J. A., Grana, W. A., Lai, L. W., & Lien, Y. H. (2008). In vivo bone strain measurements collected from mouse femora using CPC coated strain Gauges. 8th World Biomaterials Congress 2008, 3, 1326-.
• Margolis, D. S., Szivek, J. A., Lai, L., & Lien, Y. H. (2008). Phenotypic characteristics of bone in carbonic anhydrase II-deficient mice. Calcified Tissue International, 82(1), 66-76.
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  PMID: 18175028;Abstract: Carbonic anhydrase II (CAII)-deficient mice were created to study the syndrome of CAII deficiency in humans including osteopetrosis, renal tubular acidosis, and cerebral calcification. Although CAII mice have renal tubular acidosis, studies that analyzed only cortical bones found no changes characteristic of osteopetrosis. Consistent with previous studies, the tibiae of CAII-deficient mice were significantly smaller than those of wild-type (WT) mice (28.7 ± 0.9 vs. 43.6 ± 3.7 mg; p < 0.005), and the normalized cortical bone volume of CAII-deficient mice (79.3 ± 2.2%) was within 5% of that of WT mice (82.7 ± 2.3%; p < 0.05), however, metaphyseal widening of the tibial plateau was noted in CAII-deficient mice, consistent with osteopetrosis. In contrast to cortical bone, trabecular bone volume demonstrated a nearly 50% increase in CAII-deficient mice (22.9 ± 3.5% in CAII, compared to 15.3 ± 1.6% in WT; p < 0.001). In addition, histomorphometry demonstrated that bone formation rate was decreased by 68% in cortical bone (4.77 ± 1.65 μm3/μm2/day in WT vs. 2.07 ± 1.71 μm3/μm2/day in CAII mice; p < 0.05) and 55% in trabecular bone (0.617 ± 0.230 μm 3/μm2/day in WT vs. 0.272 ± 0.114 μm 3/μm2/day in CAII mice; p < 0.05) in CAII-deficient mice. The number of osteoclasts was significantly increased (67%) in CAII-deficient mice, while osteoblast number was not different from that in WT mice. The metaphyseal widening and changes in the trabecular bone are consistent with osteopetrosis, making the CAII-deficient mouse a valuable model of human disease. © 2007 Springer Science+Business Media, LLC.
• Phull, H., Lien, Y. H., Salkini, M. W., Escobar, C., Lai, L., & Ramakumar, S. (2008). Delivery of Intercellular Adhesion Molecule-1 Antisense Oligonucleotides Using a Topical Hydrogel Tissue Sealant in a Murine Partial Nephrectomy/Ischemia Model. Urology, 72(3), 690-695.
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  PMID: 18336877;Abstract: Objectives: Ischemia/reperfusion injury is a leading cause of renal damage which can be improved with antisense oligonucleotide gene therapy. We have shown that polyethylene glycol (PEG) hydrogel, which also functions as a tissue sealant, is an effective topical delivery vehicle for oligonucleotides in a murine partial nephrectomy model. The objective of this study was to use and evaluate this method against intercellular adhesion molecule-1 (ICAM-1) to prevent tissue damage. Methods: Sixty mice underwent left upper pole partial nephrectomy with 45 minutes of warm ischemia, randomized to treatment with 50 μg ICAM-1 antisense oligonucleotides embedded in PEG hydrogel, no therapy, or sham surgery. Kidneys were harvested at 24 hours and 3, 4, and 5 days. The specimens were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) for ICAM-1 messenger ribonucleic acid (mRNA), immunohistochemical staining for ICAM-1 protein, and standard histology. Results: At 24 hours, qRT-PCR and immunohistochemistry data showed a significant reduction in ICAM-1 mRNA and protein expression in the antisense group versus the ischemia group, but no difference at 3 to 5 days. Histologically there was reduced inflammation and necrosis in the cortex at 24 hours. The outer and inner medulla also showed improvement at 3 to 5 days in the antisense group as opposed to the ischemia group. Conclusions: Topical PEG hydrogel delivery of antisense ICAM-1 oligonucleotides demonstrated decreased ICAM-1 mRNA expression, reduced ICAM-1 protein staining, and decreased cellular damage. The application of gene therapy through this novel topical delivery system holds potential for a highly specific, localized method of preventing tissue damage after ischemia/reperfusion injury. © 2008 Elsevier Inc. All rights reserved.
• Sorrell, V. L., Ross, W. D., Gregoire, J., Lai, L., & Lien, Y. H. (2008). Unexpected severe LVH in the elderly: Consider Fabry's disease. Echocardiography, 25(3), 345-348.
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  PMID: 18177384;
• Lai, L. -., Yong, K. -., Igarashi, S., & Lien, Y. -. (2007). A sphingosine-1-phosphate type 1 receptor agonist inhibits the early T-cell transient following renal ischemia-reperfusion injury. Kidney International, 71(12), 1223-1231.
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  PMID: 17377506;Abstract: T cells are thought to be involved in the pathogenesis of renal ischemia-reperfusion injury (IRI); however, earlier studies have not found significant T-cell numbers in the kidney following injury. In this study we test the hypothesis that T cells transiently infiltrate the kidney following reperfusion and leave behind T-cell-derived cytokines such as interferons and interleukins, thus triggering an inflammatory reaction. An early rise of infiltrating T cells was coupled with a decrease in both circulating lymphocytes and CD4+ cells of periarterial lymphocyte aggregates. The renal expression of several chemokines was rapidly and markedly increased by ischemia-reperfusion (IR). Sphingosine-1-phosphate type 1 receptor agonists have been shown to protect kidneys from injury. One of these agonists given before IR significantly reduced histologically assessed renal injury, circulating lymphocyte numbers, and renal T-cell infiltration. This pretreatment did not, however, affect the increase in T-cell chemokines but caused an increase in CD4+ cells in the renal lymphatic system. We conclude that T-cell infiltration is an early event after IRI and is mediated by several chemokines. Sphingosine-1-phosphate receptor agonists reduce renal injury and T-cell infiltration in spite of chemokine generation by inhibiting T-cell mobilization from both renal and extra-renal lymphoid tissue. © 2007 International Society of Nephrology.
• Lee, C., Chen, H., Lai, L., Yong, K., & Lien, Y. H. (2007). Effects of furosemide on renal calcium handling. American Journal of Physiology - Renal Physiology, 293(4), F1231-F1237.
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  PMID: 17652376;Abstract: Furosemide is a loop diuretic agent that has been used to treat hypercalcemia because it increases renal calcium excretion. The effect of furosemide on calcium transport molecules in distal tubules has yet to be investigated. We conducted studies to examine the effects of furosemide on renal calcium excretion and expression of calcium transport molecules in mice. Mice were administered with a single dose of furosemide (15 mg/kg) and examined 4 h later or were given twice-daily furosemide injections for 3 days. To evaluate the effects of volume depletion, drinking water was supplemented with salt. Our results showed that, in acute experiments, furosemide enhanced urinary calcium excretion, which was associated with a significant increase in mRNA levels of TRPV5, TRPV6, and calbindin-D28k but not calbindin-D9k as measured by real-time PCR (TRPV5 and TRPV6 are transient receptor potential vanilloid 5 and 6). Chronic furosemide administration induced three-to fourfold increases in urinary calcium excretion and elevated mRNA levels of TRPV5, TRPV6, calbindin-D28k, and calbindin-D9k without or with salt supplement. Similar upregulation of calcium transport molecules was observed in mice with gentamicin-induced hypercalciuria. Coadministration of chlorothiazide decreased furosemide-induced calciuria, either acutely or chronically, although still accompanied by upregulation of these transport molecules. Immunofluorescent staining studies revealed comparably increased protein abundance in TRPV5 and calbindin-D28k. We conclude that furosemide treatment enhances urinary calcium excretion. Increased abundance of calcium transport molecules in the distal convoluted tubule represents a solute load-dependent effect in response to increased calcium delivery and serves as a compensatory adaptation in the downstream segment. Copyright © 2007 the American Physiological Society.
• Lee, C. -., Lien, Y. -., Lai, L. -., Chen, J. -., Lin, C. -., & Chen, H. -. (2006). Increased renal calcium and magnesium transporter abundance in streptozotocin-induced diabetes mellitus. Kidney International, 69(10), 1786-1791.
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  PMID: 16557223;Abstract: Diabetes is associated with renal calcium and magnesium wasting, but the molecular mechanisms of these defects are unknown. We measured renal calcium and magnesium handling and investigated the effects of diabetes on calcium and magnesium transporters in the thick ascending limb and distal convoluted tubule in streptozotocin (STZ)-induced diabetic rats. Rats were killed 2 weeks after inducing diabetes, gene expression of calcium and magnesium transporters in the kidney was determined by real-time polymerase chain reaction, and the abundance of protein was assessed by immunoblotting. Our results showed that diabetic rats had significant increase in the fractional excretion for calcium and magnesium (both P < 0.01), but not for sodium. Reverse transcriptase-polymerase chain reaction revealed significant increases in messenger RNA abundance of transient potential receptor (TRP) V5 (223 ± 10%), TRPV6 (177 ± 9%), calbindin-D28k (231 ± 8%), and TRPM6 (165 ± 8%) in diabetic rats. Sodium chloride cotransporter was also increased (207 ± 10%). No change was found in paracellin-1 (cortex: 108 ± 8%; medulla: 110 ± 10%). Immunofluorescent studies of renal sections showed significant increase in calbindin-D28k (238 ± 10%) and TRPV5 (211 ± 10%), but no changes in paracellin-1 in Western blotting (cortex: 110 ± 7%; medulla: 99 ± 7%). Insulin administration completely corrected the hyperglycemia-associated hypercalciuria and hypermagnesiuria, and reversed the increase of calcium and magnesium transporter abundance. In conclusion, our results demonstrated increased renal calcium and magnesium transporter abundance in STZ-induced diabetic rats, which may represent a compensatory adaptation for the increased load of calcium and magnesium to the distal tubule. © 2006 International Society of Nephrology.
• Lien, Y. -., Yong, K. -., Cho, C., Igarashi, S., & Lai, L. -. (2006). S1P₁-selective agonist, SEW2871, ameliorates ischemic acute renal failure. Kidney International, 69(9), 1601-1608.
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  PMID: 16572108;Abstract: The pathogenesis of renal ischemia/reperfusion (I/R) injury involves activating several signal transduction cascade systems in endothelial cells. Sphingosine 1-phospate (S1P) maintains endothelial cell integrity and inhibits lymphocyte egress via the specific S1P1 receptor, and may play a role in reducing ischemic renal injury. We examined the protective effects of a newly identified S1P1-selective agonist, SEW2871, on mouse renal I/R injury. Kidneys were harvested 1-4 days after I/R injury for histopathology, immunofluorescence studies, and quantitative real-time reverse transcriptase-polymerase chain reaction analyses to assess the change in gene expression profiles of inflammation-associated cytokines and adhesion molecules. SEW2871 improved renal function with a 40% reduction in plasma creatinine levels (P
• Margolis, D. S., Kim, D., Szivek, J. A., Lai, L., & Lien, Y. H. (2006). Functionally improved bone in Calbindin-D28k knockout mice. Bone, 39(3), 477-484.
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  PMID: 16631426;PMCID: PMC2367120;Abstract: In vitro studies indicate that Calbindin-D28k, a calcium binding protein, is important in regulating the life span of osteoblasts as well as the mineralization of bone extracellular matrix. The recent creation of a Calbindin-D28k knockout mouse has provided the opportunity to study the physiological effects of the Calbindin-D28k protein on bone remodeling in vivo. In this experiment, histomorphometry, μCT, and bend testing were used to characterize bones in Calbindin-D28k KO (knockout) mice. The femora of Calbindin-D28k KO mice had significantly increased cortical bone volume (60.4% ± 3.1) compared to wild-type (WT) mice (45.4% ± 4.6). The increased bone volume was due to a decrease in marrow cavity area, and significantly decreased endosteal perimeters (3.397 mm ± 0.278 in Calbindin-D28k KO mice, and 4.046 mm ± 0.450 in WT mice). Similar changes were noted in the analysis of the tibias in both mice. The bone formation rates were similar in the femoral and tibial cortical bones of both mice. μCT analysis of the trabecular bone in the tibial plateau indicated that Calbindin-D28k KO mice had an increased bone volume (35.2% ± 3.1) compared to WT mice (24.7% ± 4.9) which was primarily due to increased trabecular number (8.99 mm-1 ± 0.94 in Calbindin-D28k KO mice compared to 6.75 mm-1 ± 0.85 in WT mice). Bone mineral content analysis of the tibias indicated that there is no difference in the calcium or phosphorus content between the Calbindin-D28k KO and WT mice. Cantilever bend testing of the femora demonstrated significantly lower strains in the bones of Calbindin-D28k KO mice (4135 μstrain/kg ± 1266) compared to WT mice (6973 μstrain/kg ± 998) indicating that the KO mice had stiffer bones. Three-point bending demonstrated increased failure loads in bones of Calbindin-D28k KO mice (31.6 N ± 2.1) compared to WT mice (15.0 N ± 1.7). In conclusion, Calbindin-D28k KO mice had increased bone volume and stiffness indicating that Calbindin-D28k plays an important role in bone remodeling. © 2006 Elsevier Inc. All rights reserved.
• Lien, Y. H., & Lai, L. (2005). Bilateral femoral head and distal tibial osteonecrosis in a patient with Fabry disease.. American journal of orthopedics (Belle Mead, N.J.), 34(4), 192-194.
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  PMID: 15913175;Abstract: Fabry disease is a lysosomal storage disease caused by alpha-galactosidase A deficiency. The classic presentation of Fabry disease involves multiple organs, including kidneys, heart, skin, eyes, and nervous system. Osteonecrosis is rarely reported in patients with Fabry disease. In this article, we describe the case of a 37-year-old white man who had Fabry disease and no risk factors for osteonecrosis but who developed osteonecrosis in both femoral heads and in an unusual site, bilateral distal tibiae. Results of mutation analysis showed a nonsense mutation (R227X) in the alpha-galactosidase A gene. This case suggests that Fabry disease may be a risk factor for development of osteonecrosis. The enzyme replacement therapy currently available may be an effective method of preventing this complication.
• Ramakumar, S., Phull, H., Purves, T., Funk, J., Copeland, D., Ulreich, J. B., Lai, L., & Lien, Y. H. (2005). Novel delivery of oligonucleotides using a topical hydrogel tissue sealant in a murine partial nephrectomy model. Journal of Urology, 174(3), 1133-1136.
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  PMID: 16094080;Abstract: Purpose: Ischemia/reperfusion injury is a leading cause of renal damage and antisense gene therapy has been shown to ameliorate its effects. However, this approach has been limited by current delivery methods that require high concentrations of intravenous nucleic acids lacking specificity for targeting tissues. To overcome these limitations we developed a novel murine partial nephrectomy model to evaluate polyethylene-glycol (PEG) hydrogel tissue sealant as a topical oligonucleotide delivery system. Materials and Methods: A total of 18 male C57BL/6 mice underwent left partial nephrectomy with vascular occlusion. Hydrogel primer and then sealant were applied to the cut surface and photopolymerized. Using this method 16 additional mice received hydrogel primer mixed with Cy5 labeled fluorescent oligonucleotide (10 to 100 μg). Kidneys were harvested at various time points and assessed for oligonucleotide penetration using fluorescence microscopy. Results: A survival rate of 100% (34 subjects) was obtained using this mouse model of partial nephrectomy. PEG hydrogel provided adequate protection against renal hematoma and intraperitoneal blood. Fluorescent images revealed that 50 μg was the minimum dose resulting in complete progressive cellular penetration with time. In addition to direct diffusion from the application site, movement of oligonucleotide through the subcapsular space into the cortex was an observed mechanism of distribution. Conclusions: A murine partial nephrectomy model was successfully created using PEG hydrogel. In addition to achieving hemostasis, hydrogel served as a successful depot for delivering oligonucleotides throughout the kidney. Copyright © 2005 by American Urological Association.
• Lai, L., Lien, Y. H., & Lai, L. -. (2004). Gene therapy for renal disorders. Expert opinion on biological therapy, 4(6).
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  During the last 20 years there have been major improvements in renal replacement therapy, including dialysis and kidney transplantation; however, the treatment options for renal diseases are still limited. Gene therapy is a potential modality for many renal diseases for which we are as yet unable to offer specific treatment. This article reviews the recent data on gene therapy in animal models applicable to human renal diseases and evaluates its efficacy, safety and clinical relevance. Several approaches appear to be promising, including adeno-associated viral vectors for long-term gene expression, electroporation for muscular gene delivery, ultrasound/microbubble-mediated gene targeting, macrophage-based gene therapy and small interfering RNAs.
• Lee, C. T., Shang, S., Lai, L. W., Yong, K. C., & Lien, Y. H. (2004). Effect of thiazide on renal gene expression of apical calcium channels and calbindins. American journal of physiology. Renal physiology, 287(6), F1164-70.
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  Thiazide diuretics are specific inhibitors of the Na-Cl cotransporter in the distal convoluted tubule (DCT). In addition to producing diuresis and natriuresis, they have a hypocalciuric effect. Recently, two apical calcium channels have been identified, transient receptor potential vanilloid 5 (TRPV5) and TRPV6; both are expressed in the DCT. We studied the effects of thiazides on mouse renal calcium handling and renal gene expression of TRPV5 and TRPV6, as well as calbindin-D(28k) and calbindin-D(9k), both of which are calcium transport facilitators located in the DCT. Upregulation of renal TRPV5 was found 4 h after intraperitoneal injection of chlorothiazide (CTZ) at both 25 and 50 mg/kg, but not at 100 mg/kg. Chronic treatment with CTZ at 25 mg/kg twice daily for 3 days, with or without salt supplementation of 0.8% NaCl and 0.1% KCl in the drinking water, caused hypocalciuria, but the gene expression patterns were different. Without salt supplementation, mice developed volume contraction and there were no changes in gene expression. When volume contraction was prevented by salt supplementation, there was a significant increase in gene expression of TRPV5, calbindin-D(28k), and calbindin-D(9k). Salt supplementation alone also induced significant upregulation of TRPV5, TRPV6, and both calbindins. The upregulation of TRPV5 by CTZ and salt supplementation and salt alone was further confirmed with immunofluorescent staining studies. Our studies suggest that thiazides induce hypocalciuria through different mechanisms depending on volume status. With volume contraction, increased calcium reabsorption in the proximal tubule plays the major role. Without volume contraction, hypocalciuria is probably achieved through increased calcium reabsorption in the DCT by the activation of a transcellular calcium transport system and upregulation of apical calcium channel TRPV5, calbindin-D(28k), and calbindin-D(9k).
• Lee, C., Shang, S., Lai, L., Yong, K., & Lien, Y. H. (2004). Effect of thiazide on renal gene expression of apical calcium channels and calbindins. American Journal of Physiology - Renal Physiology, 287(6 56-6), F1164-F1170.
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  PMID: 15265769;Abstract: Thiazide diuretics are specific inhibitors of the Na-Cl cotransporter in the distal convoluted tubule (DCT). In addition to producing diuresis and natriuresis, they have a hypocalciuric effect. Recently, two apical calcium channels have been identified, transient receptor potential vanilloid 5 (TRPV5) and TRPV6; both are expressed in the DCT. We studied the effects of thiazides on mouse renal calcium handling and renal gene expression of TRPV5 and TRPV6, as well as calbindin-D 28k and calbindin-D 9k, both of which are calcium transport facilitators located in the DCT. Upregulation of renal TRPV5 was found 4 h after intraperitoneal injection of chlorothiazide (CTZ) at both 25 and 50 mg/kg, but not at 100 mg/kg. Chronic treatment with CTZ at 25 mg/kg twice daily for 3 days, with or without salt supplementation of 0.8% NaCl and 0.1% KCl in the drinking water, caused hypocalciuria, but the gene expression patterns were different. Without salt supplementation, mice developed volume contraction and there were no changes in gene expression. When volume contraction was prevented by salt supplementation, there was a significant increase in gene expression of TRPV5, calbindin-D 28k, and calbindin-D 9k. Salt supplementation alone also induced significant upregulation of TRPV5, TRPV6, and both calbindins. The upregulation of TRPV5 by CTZ and salt supplementation and salt alone was further confirmed with immunofluorescent staining studies. Our studies suggest that thiazides induce hypocalciuria through different mechanisms depending on volume status. With volume contraction, increased calcium reabsorption in the proximal tubule plays the major role. Without volume contraction, hypocalciuria is probably achieved through increased calcium reabsorption in the DCT by the activation of a transcellular calcium transport system and upregulation of apical calcium channel TRPV5, calbindin-D 28k, and calbindin-D 9k.
• Lien, Y. H., & Lai, L. (2004). Gene therapy for renal disorders. Expert Opinion on Biological Therapy, 4(6), 919-926.
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  PMID: 15174973;Abstract: During the last 20 years there have been major improvements in renal replacement therapy, including dialysis and kidney transplantation; however, the treatment options for renal diseases are still limited. Gene therapy is a potential modality for many renal diseases for which we are as yet unable to offer specific treatment. This article reviews the recent data on gene therapy in animal models applicable to human renal diseases and evaluates its efficacy, safety and clinical relevance. Several approaches appear to be promising, including adeno-associated viral vectors for long-term gene expression, electroporation for muscular gene delivery, ultrasound/microbubble-mediated gene targeting, macrophage-based gene therapy and small interfering RNAs.
• Margolis, D. S., Lien, Y. H., Lai, L., & Szivek, J. A. (2004). Bilateral symmetry of biomechanical properties in mouse femora. Medical Engineering and Physics, 26(4), 349-353.
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  PMID: 15121061;Abstract: Bone healing and remodeling are commonly examined in animal models by comparing one femur (experimental) to the contralateral femur (control) with the assumption that they are identical with respect to their biomechanical properties. While past studies have characterized the symmetry in geometrical properties in many types of animal bones, few studies have compared the symmetry in the biomechanical properties. The purpose of this study was to determine whether there is symmetry in the mechanical properties of mouse femora. Strain gauges were attached to the posterior surface of the femora of C57BL/6 mice, parallel to the long axis of the bone. The femora were mechanically tested in cantilever bending while strain values were recorded. Moments of inertia, cortical areas, and moduli of elasticity were determined from strains and cross-sectional properties. Mouse femora demonstrated an average strain difference of 0.4% in tension and 1.4% in compression. Elastic moduli differed by 6.6% and 0.9% in tension and compression, respectively, and failure strength differed by an average of 2.0%. Statistical analysis showed there were no significant differences in strain, modulus, or failure load values for the mice, indicating mechanical and geometrical symmetry of mouse femora in cantilever bending. © 2003 IPEM. Published by Elsevier Ltd. All rights reserved.
• Margolis, D. S., Szivek, J. A., Lien, Y. H., Lai, L. W., Kastis, G. A., & Barrett, H. H. (2004). Bone remodeling in CAII deficient and calbindin-D28k KO mice. Transactions - 7th World Biomaterials Congress, 797-.
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  Abstract: Two genetically altered mice were studied to determine the effects of inhibited osteoclast and osteoblast function on bone phenotype. Carbonic anhydrase II (CAII) deficient mice were used as a model of inhibited osteoclast function as CAII is a key enzyme used to acidify resorption lacunae and activate the lysosomal enzymes that resorb bone. Calbindin-D28k knockout mice were also studied. Calbindin-D28k is a protein that is expressed in osteoblasts and has been shown to mitigate osteoblast apoptosis.
• Rosenthal, D., Lien, Y. H., Lager, D., Lai, L., Shang, S., Leung, N., & Fervenza, F. C. (2004). A novel α-galactosidase a mutant (M42L) identified in a renal variant of Fabry disease. American Journal of Kidney Diseases, 44(5), E85-E89.
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  PMID: 15492942;Abstract: A 65-year-old man presented to our institution for workup of proteinuria. His serum creatinine level was 1.7 mg/dL (130 μmol/L), and he had proteinuria with protein of almost 5 g/24 h. Fabry disease was diagnosed by means of kidney biopsy and low serum and leukocyte levels of α-galactosidase A. Review of his history, family history, physical examinations, and diagnostic studies did not show other findings typical of this disease. His renal function continued to decline, and he eventually underwent a living unrelated renal transplantation 5 years later. Three years after transplantation, his creatinine level is 1.7 mg/dL (130 μmol/L), and corrected iothalamate clearance is 53 mL/min/1.73 m 2. Genetic studies showed that he has a novel missense mutation (M42L) in exon 1. Methionine at codon 42 is highly conserved in eukaryotic α-galactosidase A orthologues. This genotype predicts a minor misfolding of α-galactosidase A because of a small difference in hydrophobicity between methionine and leucine. His mutation resulted in a very low, but detectable, serum level of α-galactosidase A (0.002 U/L; normal range, 0.016 to 0.2 U/L). Cases of Fabry disease that present with predominantly renal manifestations are rare and require a high index of suspicion for diagnosis. Because treatment for Fabry disease recently has become available, it is important for clinicians to be aware of this disease and pursue the diagnosis in cases of otherwise unexplained renal dysfunction. © 2004 by the National Kidney Foundation, Inc.
• Wu, M., Lai, L., & Lien, Y. H. (2004). Effect of Calbindni-D_28K on cyclosporine toxicity in cultured renal proximal tubular cells. Journal of Cellular Physiology, 200(3), 395-399.
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  PMID: 15254967;Abstract: Cyclosporine A (CsA) is known to have direct toxicity to renal tubular cells. Its toxicity may be mediated by intracellular calcium because CsA increases intracellular calcium concentration and enhances the activities of calcium-dependent calpains and caspases. Calbindin-D28k, a cytosolic calcium binding protein, has been used as an intracellular Ca2+ buffer to reduce calcium-mediated cytotoxicity in non-renal cells such as neuronal cells. We investigated the effects of gene transfer of calbindin-D 28k cDNA on CsA cytotoxicity and intracellular calcium concentration ([Ca2+]i) in cultured murine proximal tubular (MCT) cells. A plasmid containing calbindin-D28k cDNA under the control of CMV promoter was transfected to MCT cells with liposomes. Cytotoxicity was assessed by LDH release and cell viability assay, and [Ca2+]i was measured ratiometrically with fura-2. Compared with MCT cells, cells transfected with calbindin-D28k cDNA showed a reduction in LDH release by 27, 30, 32, 33, and 19% (all P < 0.05), respectively, after 24 h exposure to 1, 2.5, 5, 10, and 25 μM CsA. Cell viability after CsA treatment was also significantly higher in CB cells. A mock transfection using plasmid without calbindin-D28k cDNA insert did not affect the LDH release or cell viability after CsA treatment. CsA treatment did not affect the protein and mRNA abundance of transfected calbindin-D28k cDNA. The expression of calbindin-D28k did not affect the baseline [Ca2+] i, but significantly suppressed CsA-induced elevation in [Ca 2+]i. The expression of calbindin-D28k in renal tubular cells provides cytoprotective effects against CsA toxicity, probably through its buffering effects on [Ca2+]i. © 2004 Wiley-Liss, Inc.
• Lai, L. -., Whitehair, O., Wu, M. -., O'Meara, M., & Lien, Y. H. (2003). Analysis of splice-site mutations of the α-galactosidase A gene in Fabry disease. Clinical Genetics, 63(6), 476-482.
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  PMID: 12786754;Abstract: Fabry disease is an X-linked disease caused by a defective lysosomal enzyme, α-galactosidase A, and characterized by skin lesions and multiorgan involvement, including kidney, heart, and the central nervous system. Currently more than 200 genotypes have been identified, including several aberrant splicing. However, most of the mutation analyses were performed using genomic sequencing only, and therefore some of the splicing mutations were misclassified as missense mutations. In order to predict the splicing event caused by each mutation, we conducted a literature search for all published mutations located near the splice sites, including exonic point mutations, and performed a splice-site score (SSS) analysis. The literature search identified 13 donor-site mutations, including four exonic mutations (S65T, D183S, K213N, and M267I), located at the end of exons 1, 3, 4, and 5, respectively, six acceptor-site mutations, and one new exon creation. All mutated splice sites, except for the one associated with the new exon creation, had a lower SSS than their respective natural sites. Cryptic or newly created sites were identified with SSS from 0.09 to 1.0. The predictions, based on SSS analysis, are in agreement with all six mutations with known cDNA sequence from the literature, including five mutations with exon skipping and one mutation with creation of a new acceptor site. For the S65T genotype, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis using RNA isolated from the whole-blood sample. We verified that a weak cryptic site (SSS = 0.09) 14 nucleotides downstream was activated and resulted in an insertion of 14 bp and a frameshift stop at codon 106. This change is more consistent with the clinical presentation of the patient, the classical Fabry disease, than the amino acid substitution (S65T), which does not affect the enzyme function. In conclusion, the SSS analysis is very useful for predicting splicing events and genotype/phenotype correlation in Fabry disease. As different mechanisms may be involved in pre-mRNA splicing, it is important to obtain cDNA sequencing for molecular diagnosis.
• Lai, L., Lien, Y. H., & Lai, L. -. (2003). Renal gene transfer: nonviral approaches. Molecular biotechnology, 24(3).
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  Gene therapy has the potential to become an important modality for treating both hereditary and acquired renal diseases. Since renal diseases may involve different cell types in the kidney, it is critical to achieve efficient gene transfer specifically to each cell type. We reviewed the literature on nonviral gene transfer techniques, which are designed to target the kidney specifically. A variety of approaches have been developed to target glomeruli, tubules, renal vasculature, and interstitium with different degree of success. Besides using delivery systems based on liposomes, polycations, and viral fusion proteins, investigators have adopted newer approaches including electroporation and hydrodynamic-based gene transfer, and demonstrated that they are efficient and safe in animal models. Potential clinical applications and safety concerns of gene therapy for renal diseases are discussed.
• Lien, Y. H., & Lai, L. (2003). Renal gene transfer: Nonviral approaches. Applied Biochemistry and Biotechnology - Part B Molecular Biotechnology, 24(3), 283-294.
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  PMID: 12777694;Abstract: Gene therapy has the potential to become an important modality for treating both hereditary and acquired renal diseases. Since renal diseases may involve different cell types in the kidney, it is critical to achieve efficient gene transfer specifically to each cell type. We reviewed the literature on nonviral gene transfer techniques, which are designed to target the kidney specifically. A variety of approaches have been developed to target glomeruli, tubules, renal vasculature, and interstitium with different degree of success. Besides using delivery systems based on liposomes, polycations, and viral fusion proteins, investigators have adopted newer approaches including electroporation and hydrodynamic-based gene transfer, and demonstrated that they are efficient and safe in animal models. Potential clinical applications and safety concerns of gene therapy for renal diseases are discussed.
• Lien, Y. H., Lai, L., & Silva, A. L. (2003). Pathogenesis of renal ischemia/reperfusion injury: Lessons from knockout mice. Life Sciences, 74(5), 543-552.
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  PMID: 14623025;Abstract: Ischemia/reperfusion-induced acute renal failure is a common clinical problem associated with a high morbidity and mortality. Upon hypoxic injury, the depletion of ATP causes mitochondrial dysfunction, and accumulation of intracellular sodium, calcium and reactive oxygen species. Subsequently, multiple enzyme systems including proteases, nitric oxide synthases, phospholipases and endonuclease are activated and responsible for cytoskeleton disruption, membrane damage, and DNA degradation, and eventually cell death. Ischemia/reperfusion injury also activates complement, cytokines, and chemokines, which are cytotoxic themselves, but also attract leukocytes into the ischemic area to cause further damage. The vascular endothelial cell injury and dysfunction prolong ischemia and induce vascular congestion, edema, and further infiltration of inflammatory cells. Many players in renal ischemia/reperfusion injury and their mechanisms have been investigated using genetically manipulated mouse models. In this review, we focus on the information gathered from these studies. Deficiency of the Na/Ca exchanger, inducible nitric oxide synthase, Caspase-1, A3 adenosine receptor, C3, C5, C6, Factor B, or medkine protects the kidney against I/R injury. Conversely, deficiency of the interleukin-1 receptor, osteopontin, C4, or recombination activation gene-1 is not protective, while the absence of adrenomedullin or endothelin receptor B delays the recovery of ischemia/reperfusion injury. The knowledge obtained from these studies provides new direction for designing potential therapeutic agents for treating ischemia/reperfusion injury. © 2003 Elsevier Inc. All rights reserved.
• Yang, C. -., Lai, L. -., Whitehair, O., Hwu, W. -., Chiang, S. -., & Lien, Y. H. (2003). Two novel mutations in the α-galactosidase A gene in Chinese patients with Fabry disease. Clinical Genetics, 63(3), 205-209.
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  PMID: 12694230;Abstract: Fabry disease is an X-linked disorder caused by a deficiency of the lysosomal α-galactosidase A [EC 3.2.1.22]. The molecular diagnosis of Fabry disease is important for genotype/phenotype correlation, prenatal or early diagnosis, and detection of carrier status. Although more than 200 genotypes of the α-galactosidase A gene have been identified, mutation data on the Chinese population is sparse. We recently identified two unrelated Chinese families with Fabry disease. Mutation analysis was performed by polymerase chain reaction (PCR) sequencing of the seven exons and adjacent introns of the α-galactosidase A gene. Two novel mutations were identified: in family I, a C-to-A transversion resulted in an early termination at amino acid 222 (Y222X), while in family II, an A-to-G transition resulted in a substitution of alanine for threonine at amino acid 410 (T410A). Carrier status was identified in all four females in the two families. The genotype Y222X is associated with classic Fabry disease, with unexpectedly rapid deterioration of visual acuity, while T410A is associated with a milder Fabry disease, with ventricular hypertrophy and neuropathic pain.
• Lai, L., & Lien, Y. H. (2002). Chimeric RNA/DNA oligonucleotide-based gene therapy. Kidney International, 61(SUPPL. 1), S47-S51.
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  PMID: 11841612;Abstract: Background. Chimeric RNA/DNA oligonucleotides, emerging as a potential strategy for gene therapy, have been shown to induce site-specific correction of point mutations in several genetic disease models. Methods. Six recent studies of chimeric RNA/DNA oligonucleotide-based gene therapy in genetic disease models are reviewed. Chimeric RNA/DNA oligonucleotides, complementary to 25 to 30 residues of genomic DNA flanking the mutation site with the exception of a mismatch in the center, were delivered via different routes and delivery vehicles to target different tissues and organs. Corrections of the mutation at genotypic and phenotypic levels were assessed using various methods, including allele-specific polymerase chain reaction assay, restriction enzyme digestion, colony-lifting assays, sequencing, Northern and Western blot analyses, enzyme activity assay, immunohistochemical staining, and functional studies. Results. The gene correction frequency varied, ranging from less than 1% to more than 40%. This represented several magnitudes higher conversion rate compared with homologous recombination frequency, which is in the range of 10-5 to 10-6. The resulting phenotype changes lasted longer than one year in some studies. Conclusion. Chimeric RNA/DNA oligonucleotide-based gene therapy has the potential to develop into powerful therapeutic modality for genetic diseases. It can offer permanent expression and normal regulation of corrected genes in appropriate cells or tissues. Further efforts to elucidate the mechanisms of chimeric RNA/DNA oligonucleotide-based gene therapy are warranted in order to increase the efficacy and safety of this method.
• Lai, L., Lien, Y. H., & Lai, L. -. (2002). Gene therapy for renal disorders: what are the benefits for the elderly?. Drugs & aging, 19(8).
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  Chronic renal failure is one of the major health problems for the elderly. Currently, about 50% of all patients receiving chronic dialysis for end-stage renal disease (ESRD) are aged 65 years or older. Their first-year mortality rate is as high as 30%. The leading causes of ESRD in the elderly are diabetic nephropathy, hypertension and large vessel diseases, and glomerulonephritis. The elderly are also prone to developing acute renal failure induced by ischaemic injury or nephrotoxic drugs. Gene transfer in experimental animals have been tested in all of these conditions, as well as in animal kidney transplantation models, with various degrees of success. However, there are many obstacles to be overcome before gene therapy can be tested clinically for renal disorders. In particular, the major challenges include determining how to prolong and control transgene expression or antisense inhibition and how to minimise the adverse effects of viral or nonviral vectors. Once these problems are solved, gene therapy will have a role in treating age-related renal impairment.
• Lee, C., Huynh, V. M., Lai, L., & Lien, Y. H. (2002). Cyclosporine A-induced hypercalciuria in calbindin-D28k knockout and wild-type mice. Kidney International, 62(6), 2055-2061.
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  PMID: 12427129;Abstract: Background. It is known that cyclosporine A (CsA) treatment induces high bone-turnover osteopenia and hypercalciuria. It has been proposed that down-regulation of renal calbindin-D28k by CsA results in renal calcium wasting. We investigated the role of the kidney and bone in CsA-induced hypercalciuria in calbindin-D28k knockout (KO) and wild-type (WT) mice. Methods. Two sets of experiments were performed. In experiment 1, KO and WT mice were treated with CsA 20 mg/kg/day intraperitoneally (IP) for 7 days. In experiment 2, to eliminate the CsA effect on bone resorption, pamidronate (APD) 2.5 mg/kg IP was given every 4 days with the first dose given 4 days prior to the 7-day course of CsA. Serum levels of creatinine, calcium, and osteocalcin, as well as renal calcium excretion were measured to assess CsA's effects on calcium homeostasis. Effects of CsA on the expression of calbindin-D28k, and two calcium channels in the apical membrane of the distal tubule, epithelial calcium channel (ECaC) and α1G-subunit of a voltage-dependent Ca channel (α1G), in the kidney were examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Results. KO mice had a threefold increase in renal calcium excretion when compared with WT mice at the baseline. This difference disappeared when calcium load was reduced by overnight fasting. After the CsA treatment, both WT and KO mice had a significant increase of renal calcium excretion (urine Ca/Cr ratio in WT, 0.11 ± 0.01 to 1.29 ± 0.17; in KO, 0.39 ± 0.04 to 1.18 ± 0.13; both P < 0.01). CsA treatment decreased renal calbindin-D28k mRNA by 61%, but did not affect the expression of ECaC and α1G. Baseline serum osteocalcin level of KO mice was significantly lower than that of WT mice. After CsA treatment, both groups had a 50% increase in the serum osteocalcin level, indicating increased bone turnover. When mice were treated with both CsA and APD, the increase in serum osteocalcin level was prevented, and renal calcium excretion was significantly lower than that in mice treated with CsA alone. However, there was still a significant increase in the urine Ca/Cr ratio in WT and KO mice compared with pretreatment levels (urine Ca/Cr in WT, 0.11 ± 0.01 to 0.76 ± 0.05, P < 0.01; in KO, 0.39 ± 0.05 to 0.79 ± 0.06; P < 0.01). Conclusion. Calbindin-D28k KO mice have diet-dependent hypercalciuria and a lower bone turnover rate. CsA treatment suppresses the expression of calbindin-D28k in mice, but has no effects on ECaC and α1G gene expression at the mRNA level. The pathogenesis of CsA-induced hypercalciuria involves both down-regulation of calbindin-D28k with subsequent impaired renal calcium reabsorption and CsA-induced high turnover bone disease. Additionally, our results suggest that mechanism(s) independent of calbindin-D28k within the kidney also may contribute to the CsA-induced calcium leak.
• Lien, Y. H., & Lai, L. (2002). Gene therapy for renal disorders: What are the benefits for the elderly?. Drugs and Aging, 19(8), 553-560.
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  PMID: 12207549;Abstract: Chronic renal failure is one of the major health problems for the elderly. Currently, about 50% of all patients receiving chronic dialysis for end-stage renal disease (ESRD) are aged 65 years or older. Their first-year mortality rate is as high as 30%. The leading causes of ESRD in the elderly are diabetic nephropathy, hypertension and large vessel diseases, and glomerulonephritis. The elderly are also prone to developing acute renal failure induced by ischaemic injury or nephrotoxic drugs. Gene transfer in experimental animals have been tested in all of these conditions, as well as in animal kidney transplantation models, with various degrees of success. However, there are many obstacles to be overcome before gene therapy can be tested clinically for renal disorders. In particular, the major challenges include determining how to prolong and control transgene expression or antisense inhibition and how to minimise the adverse effects of viral or nonviral vectors. Once these problems are solved, gene therapy will have a role in treating age-related renal impairment.
• Wu, M., Lai, L., & Lien, Y. H. (2002). Cytoprotective effects of calbindin-d_28k against antimycin-A induced hypoxic injury in proximal tubular cells. Life Sciences, 71(5), 559-569.
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  PMID: 12052440;Abstract: Intracellular calcium plays an important role on the pathogenesis of hypoxia-induced cellular injury. Calbindin-D28k, a cytosolic vitamin D-dependent calcium binding protein, can serve as a buffer to limit a surge in intracellular Ca2+ concentration ([Ca2+]i) induced by various stimulations. To evaluate the possible cytoprotective effect of calbindin-D28k against hypoxic injury in proximal tubular cells, a plasmid containing calbindin-D28k cDNA under the control of CMV immediate-early gene promoter was transfected into the murine proximal tubular epithelial (MCT) cells. The expression of calbindin-D28k in the transfected cells was verified with Northern blot analysis, Western blot analysis, and immunofluorescent staining. The non-transfected and transfected MCT cells were subjected to chemical hypoxia induced by antimycin A (10 μM) and glucose deprivation for 30-120 min. The transfection of calbindin-D28k reduced lactate dehydrogenase (LDH) release by 41%, 41%, 24%, and 24%, respectively, at 30, 60, 90 and 120 min after hypoxia when compared to the non-transfected cells (all p < 0.05). Cell viability after hypoxic injury was also significantly higher in transfected cells than non-transfected cells. Transfection with the plasmid without calbindin-D28k cDNA did not affect LDH release or cell viability after chemical hypoxic injury. [Ca+2]i was measured ratiometrically with fura-2 after exposure to chemical hypoxia. The rate of initial rise in [Ca2+]i and final [Ca+2]i at 30-120 min were significantly lowered in transfected cells. In conclusion, this study demonstrated that transfection of calbindin-D28k gene into MCT cells provide protective effects against chemical hypoxic injury probably through its buffering effects on [Ca+2]i. © 2002 Elsevier Science Inc. All rights reserved.
• Lai, L., & Lien, Y. H. (2001). Therapeutic application of chimeric RNA/DNA oligonucleotide based gene therapy. Expert Opinion on Biological Therapy, 1(1), 41-47.
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  PMID: 11727546;Abstract: Chimeric RNA/DNA oligonucleotides, or chimera, have emerged as a breakthrough technology for treating genetic disorders. Chimera have been shown to induce correction of point mutations in several genetic disease models without utilising the viral vectors. Recent studies of chimera-based gene therapy in genetic disease models are reviewed. Chimera were delivered intravenously, intramuscularly, intradermally, or topically with or without vehicles. Correction of the mutation at genotypic and phenotypic levels was assessed using various methods. The gene correction frequency varied, ranging from 1 - 40%. The resulting phenotype changes lasted longer than one year in some studies. The most dramatic phenotypic change is the reduction of serum bilirubin level by 50% in the Gunn rat, a model for Crigler-Najjar syndrome. Chimera based gene therapy has the potential to develop into powerful therapeutic modality for genetic diseases. 2001 © Ashley Publications Ltd. Papers of special note have been highlighted as: • of interest • of considerable interest.
• Lai, L., O'Meara, M., & Lien, Y. H. (2001). Gene symbol: GLA Disease: Fabry disease. Human Genetics, 109(4), 469-.
• Lien, Y. H., Lai, L., & Lui, C. Y. (2001). Unexpected diagnosis of fabry disease in an 80-year-old man with syncope. Cardiology, 96(2), 115-116.
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  PMID: 11740141;
• Zhou, Q., Clarke, L., Nie, R., Carnes, K., Lai, L., Lien, Y. H., Verkman, A., Lubahn, D., Fisher, J. S., Katzenellenbogen, B. S., & Hess, R. A. (2001). Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function. Proceedings of the National Academy of Sciences of the United States of America, 98(24), 14132-14137.
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  PMID: 11698654;PMCID: PMC61180;Abstract: Estrogen receptor α (ERα) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogen's bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogen's role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na+/H+ exchanger-3 (NHE3) and the rate of 22Na+ transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased in ERα knockout (αERKO) efferent ductules. Targeted gene-deficient mice were compared with αERKO, and the NHE3 knockout and deficient mice showed αERKO-like fluid accumulation, but only the NHE3 knockout and αERKO mice were infertile. Northern blot analysis showed decreases in mRNA for NHE3 in αERKO and antiestrogen-treated mice. The changes in AQP1 and CAll in αERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in αERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na+ transport. Finally, these data raise the possibility of targeting ERα in developing a contraceptive for the male.
• Lai, L., & Lien, Y. H. (1999). Homologous recombination based gene therapy. Experimental Nephrology, 7(1), 11-14.
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  PMID: 9892808;Abstract: Background/Aims: Most of the current expression vector based gene therapy protocols fail to achieve clinically significant transgene expression required for treating genetic diseases. Homologous recombination, initially considered to be of limited use for gene therapy because of its low frequency in mammalian cells, has recently emerged as a potential strategy for developing gene therapy. Methods: Six recent studies of homologous recombination in mammalian cells are reviewed. Different approaches have been used in these studies including RNA/DNA chimeric oligonucleotides, small or large homologous DNA fragments, or adeno-associated viral vectors. Results: Most of these studies show a reasonable frequency of homologous recombination which warrants further in vivo testing. Conclusions: Homologous recombination based gene therapy has the potential to develop into a powerful therapeutic modality for genetic diseases. It can offer permanent expression and normal regulation of corrected genes in appropriate cells or organs and probably can be used for treating dominantly inherited diseases such as polycystic kidney disease.
• Lien, Y. -., & Lai, L. -. (1999). Entering the gene therapy era. Journal of the Formosan Medical Association, 98(11), 718-721.
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  PMID: 10705686;Abstract: Gene therapy is a new modality with the potential for treating a variety of diseases, inherited or acquired. Although the initial clinical trials, particularly those for cystic fibrosis, have not been successful, the preliminary results of more recent studies of gene therapy for myocardial infarction are encouraging. New plasmids, viral and nonviral vectors, and applications are being developed at a rapid pace. A switch is now inserted in the plasmids so that the therapeutic gene can be turned on and off as needed. A chimeric RNA/DNA oligonucleotide has also been designed so that mutated genes can be converted to normal sequences. These and other novel approaches soon will propel the gene therapy industry into reality. Healthcare providers and educators need to be prepared for the coming of the gene therapy era.
• Shayakul, C., Breton, S., Brown, D., Alper, S. L., Lien, Y. -., & Lai, L. -. (1999). Gene therapy of inherited renal tubular disease. American Journal of Kidney Diseases, 34(2), 374-379.
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  PMID: 10430991;
• Lai, L., Erickson, R. P., Venta, P. J., Tashian, R. E., & Lien, Y. H. (1998). Promoter activity of carbonic anhydrase II regulatory regions in cultured renal proximal tubular cells. Life Sciences, 63(2), 121-126.
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  PMID: 9674946;Abstract: Carbonic anhydrase II (CAII) plays an important role in the acid-base homeostasis of the body and its deficiency results in renal tubular acidosis. In order to identify the regulatory regions in the CAII gene for the future development of kidney-targeted gene therapy, we investigated the 5' region of the gene for its promoter activity. Deletion constructs with various lengths of the 5' flanking region of the human CAII promoter were ligated to the CAT reporter gene and lipofected in primary cultures of mouse proximal renal tubular cells and in cells of the established porcine proximal tubular cell line, LLC-PK1. The CAT activity was measured 48 hours after gene transfection. The -12000/CAT and -1300/CAT constructs expressed the highest CAT activity in both types of renal tubular cells (143- and 180-fold increase, respectively, in mouse proximal tubular cells; 50- and 70-fold increase, respectively, in LLC-PK1 cells) but not the -420/CAT, -270/CAT, or -180/CAT constructs (9, 12, and 9% of that of -1300/CAT construct, respectively, in mouse proximal tubular cells and, 23, 9, and 8%, respectively, in LLC-PK1 cells, all p
• Lien, Y. H., & Lai, L. (1998). Respiratory acidosis in carbonic anhydrase II-deficient mice. American Journal of Physiology - Lung Cellular and Molecular Physiology, 274(2 18-2), L301-L304.
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  PMID: 9486217;Abstract: To investigate the role of carbonic anhydrase (CA) II on pulmonary CO2 exchange, we analyzed arterial blood gases from CA II-deficient and normal control mice. CA II-deficient mice had a low arterial blood pH (7.18 ± 0.06) and HCO3/- concentration ([HCO3/-]; 17.5 ± 1.9 meq/l) and a high PCO2 (47.4 ± 5.3 mmHg), consistent with mixed respiratory and metabolic acidosis. To eliminate the influence of metabolic acidosis on arterial blood gases, NaHCO3 (4 mmol/kg body weight) was given intraperitoneally, and arterial blood gases were analyzed 4 h later. Normal mice had a small increase in pH and were able to maintain PCO2 and [HCO3/-]. The metabolic acidosis in CA II-deficient mice was corrected ([HCO3/-], 22.9 ± 2.4 meq/l), and respiratory acidosis became more profound (PCO2, 50.4 ± 2.4 mmHg). These results indicate that CA II-deficient mice have a partial respiratory compensation for metabolic acidosis. We conclude that CA II-deficient mice have a mixed respiratory and metabolic acidosis. It is most likely that CO2 retention in these animals is due to CA II deficiency in both red blood cells and type II pneumocytes.
• Lai, L. -., Moeckel, G. W., & Lien, Y. -. (1997). Kidney-targeted liposome-mediated gene transfer in mice. Gene Therapy, 4(5), 426-431.
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  PMID: 9274719;Abstract: To develop gene therapy targeted to the kidney, we compared three different routes of liposome-mediated gene delivery to the kidney in mice, ie intra-renal-pelvic, intra-renal-arterial, and intra-renal-parenchymal injections. A plasmid construct, pCMVβgal, containing a cytomegalovirus (CMV) immediate-early gene promoter and a β-galactosidase reporter gene was mixed with a 1:1 liposome mixture of N[1-(2,3-dioleoyloxy)propyl]-N, N,trimethylammonium chloride (DOTMA)/dioleoyl phosphatidyl ethanolamine (DOPE). The pCMVβgal-liposome complex was injected into the left kidney via three different routes. The efficacy of gene transfer was assessed using 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) staining on frozen kidney sections 3 to 42 days after injections. Cells with β-galactosidase activity were detected in the cortex and outer medulla in both intra-renal-pelvic and intra-renal-arterial groups, but not in the intra-renal-parenchymal group or in the contralateral noninjected kidney. Evidence of gene transfer was observed only in tubular epithelial cells, but not in glomerular, vascular, or interstitial compartments. The levels of β-galactosidase expression started to decrease 3 weeks after injection. The gene transfer in the kidney was not associated with nephrotoxicity as assessed by blood urea nitrogen levels and renal histology. We conclude that both intra-renal-pelvic and intra-renal-arterial injections provide a transient gene transfer to the renal tubular cells and are suitable routes for kidney-targeted gene therapy.
• Lien, Y. -., & Lai, L. -. (1997). Gene therapy for renal diseases. Kidney International, Supplement, 51(61), S-85-S-88.
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  PMID: 9328975;Abstract: Gene therapy is a promising therapeutic approach for a variety of renal diseases including both inherited and acquired diseases. In vivo gene transfer in the kidney using viral or non-viral vectors have been reported. These approaches have been tested in a few animal models of renal diseases, including experimental glomerulonephritis, ischemic renal failure, and carbonic anhydrase II deficiency. Selection of vectors, routes, and therapeutic genes is critical to the success of gene therapy targeted to the specific compartment of the kidney. Limitations of gene therapy for renal diseases exist and consist of: duration of transgene expression is short, transfection efficiency is not adequate, immune reactions are induced by adenoviral vector, and insertional mutagenesis may be caused by retroviral and adeno-associated vital vectors. Further studies are needed for improvement of gene delivery, minimization of side effects and development of cell-specific and long-term regulated gene expression.
• Lien, Y. H., & Lai, L. (1997). Liposome-mediated gene transfer into the tubules. Experimental Nephrology, 5(2), 132-136.
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  PMID: 9108995;
• Moeckel, G. W., Lai, L., Guder, W. G., Kwon, H. M., & Lien, Y. H. (1997). Kinetics and osmoregulation of Na ⁺- And Cl -dependent betaine transporter in rat renal medulla. American Journal of Physiology, 272(1 PART 2), F94-F99.
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  Abstract: -Betaine is one of the maior organic osmolytes that accumulate in the renal medulla in response to high extracellular tonicity. Recent studies in MDCK cells have shown that betaine is taken up by an Na +- and Cl~-dependent transporter located on the basolateral membrane. We demonstrate here the presence of Na +-Cl"dependent betaine transporter(s) in tubule suspensions prepared from the rat outer and inner medulla. The betaine transport activity was two to three times higher in the inner medulla compared with the outer medulla. The removal of Na + and Cl~ reduced betaine uptake in the outer medullary tubules by 86% and 82%, respectively. The betaine uptake was decreased by 39% in hypotonie buffer (189 mosmol/ kgH2O) and increased by 82% in hypertonic buffer (545 mosmol/kgHO), compared with isotonic buffer (308 mosmol/ kgH2O). Kinetic studies of Na +-dependent betaine uptake in the outer medullary tubules revealed both a low- and a high-affinity component as follows: low-affinity and highvolume component with Michaelis constant (Kmi) of 8.6 mM and maximal uptake rate (Vmaxi) of 112 pmol -ug protein"1 -h"1; and a low-volume and high-affinity component with Km% of 0.141 mM and Vmax2 of 10 pmol-pg protein"'-h"1. To investigate whether the Na +-Cl"-dependent betaine transporter is regulated by tonicity in vivo, we quantitated its mRNA in rat renal cortex and outer and inner medulla using both canine and rat Na +-Cl~-dependent betaine transporter cDNAprobes. A single band of 3.0 kb was seen in the Northern blots prepared from both outer and inner medulla, but not in the cortex. Water deprivation for 3 days increased the abundance of this mRNA in the outer and inner medulla by 140% and 170%, respectively, but did not affect its expression in the cortex. In conclusion, Na +-Cl~-dependent betaine transporter(s) is present in rat outer and inner medullary tubules, and betaine transporter mRNA abundance is regulated by the hydration state in vivo. Copyright © 1997 the American Physiological Society.
• Moeckel, G. W., Lai, L., Guder, W. G., Kwon, H. M., & Lien, Y. H. (1997). Kinetics and osmoregulation of Na⁺- and Cl^--dependent betaine transporter in rat renal medulla. American Journal of Physiology - Renal Physiology, 272(1 41-1), F100-F106.
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  PMID: 9039055;Abstract: Betaine is one of the major organic osmolytes that accumulate in the renal medulla in response to high extracellular tonicity. Recent studies in MDCK cells have shown that betaine is taken up by an Na+- and Cl--dependent transporter located on the basolateral membrane. We demonstrate here the presence of Na+-Cl--dependent betaine transporter(s) in tubule suspensions prepared from the rat outer and inner medulla. The betaine transport activity was two to three times higher in the inner medulla compared with the outer medulla. The removal of Na+ and Cl- reduced betaine uptake in the outer medullary tubules by 86% and 82%, respectively. The betaine uptake was decreased by 39% in hypotonic buffer (189 mosmol/kgH2O) and increased by 82% in hypertonic buffer (545 mosmol/kgH2O), compared with isotonic buffer (308 mosmol/kgH2O). Kinetic studies of Na+-dependent betaine uptake in the outer medullary tubules revealed both a low- and a high-affinity component as follows: low-affinity and high-volume component with Michaelis constant (K(m1)) of 8.6 mM and maximal uptake rate (V(max1)) of 112 pmol · μg protein-1 · h-1; and a low-volume and high-affinity component with K(m2) of 0.141 mM and V(max2) of 10 pmol · μg protein-1 · h-1. To investigate whether the Na+-Cl--dependent betaine transporter is regulated by tonicity in vivo, we quantitated its mRNA in rat renal cortex and outer and inner medulla using both canine and rat Na+-Cl--dependent betaine transporter cDNA probes. A single band of 3.0 kb was seen in the Northern blots prepared from both outer and inner medulla, but not in the cortex. Water deprivation for 3 days increased the abundance of this mRNA in the outer and inner medulla by 140% and 170%, respectively, but did not affect its expression in the cortex. In conclusion, Na+-Cl--dependent betaine transporter(s) is present in rat outer and inner medullary tubules, and betaine transporter mRNA abundance is regulated by the hydration state in vivo.
• Zwingman, T., Fujimoto, H., Lai, L. -., Boyer, T., Ao, A., Stalvey, J. R., Blecher, S. R., & Erickson, R. P. (1994). Transcription of circular and noncircular forms of Sry in mouse testes. Molecular Reproduction and Development, 37(4), 370-381.
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  PMID: 7516683;Abstract: Although its expression in adult testis was immediately apparent, the role for Sry (sex determining region, Y) in testicular function remains elusive. We have performed transcriptional studies in an effort to elucidate potential roles of Sry by studying the time and location of its transcription in mouse testes. Northern analyses and more sensitive nuclease protection assays detected transcripts in 28-day-old testes and beyond. The highly sensitive technique of reverse transcription polymerase chain reaction (RTPCR) could not detect Sry expression in 14-day testes when primers for the most conserved portion of the gene, the high mobility group (HMG) box, were used, but primers for the circular form detected Sry transcription at all postnatal stages studied. The same HMG box primers were able to detect expression of Sry in XX, Sxra or Sxrb testes. This suggested that Sry is expressed in cells other than germ cells, which was confirmed with studies on fractionated cells-RTPCR detected transcription of Sry in the highly pure interstitial cell fraction. However, Leydig cells and a Leydig cell tumor were negative for Sry expression. We performed in situ studies in an attempt to localize the expression of Sry in the testes. Abundant expression of an Sry cross- hybridizing transcript was found in spermatogonia, in early spermatocytes, and in some interstitial cells with antisense probes to the HMG box or a more specific, 3' region, whereas the sense probe gave little or no hybridization. It is probable that the circular transcripts, which are seen in reverse transcriptase positive (RT-) and RT- reactions by PCR because of the RT activity of Taq polymerase, are responsible for the hybridization seen in spermatogonia and spermatocytes, whereas linear and circular forms are detected later. Thus Sry is expressed in pre- and postmeiotic germ cells and in somatic cells of the testes.
• Erickson, R. P., Lai, L. -., & Grimes, J. (1993). Creating a conditional mutation of Wnt-1 by antisense transgenesis provides evidence that Wnt-1 is not essential for spermatogenesis. Developmental Genetics, 14(4), 274-281.
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  PMID: 8222343;Abstract: We have used mice transgenic for an antisense construct for Wnt-1 to study the role of this gene in post-meiotic sperm development. The human PGK-2 promoter provided levels of Wnt-1 antisense mRNA in testes in 5 transgenic lines greatly in excess of Wnt-1 mRNA concentrations, and Wnt-1 mRNA levels were greatly decreased in the lines, by 98% in three of them. There was a general correlation between copy number of the insert, levels of antisense RNA, and decreases in mRNA. There was little effect of the antisense transgene on fertility or testicular histology suggesting that normal levels of Wnt-1 transcript are not essential for spermatogenesis.
• Lai, L. -., Erickson, R. P., & Cassidy, S. B. (1993). Clinical correlates of chromosome 15 deletions and maternal disomy in Prader-Willi syndrome. American Journal of Diseases of Children, 147(11), 1217-1223.
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  PMID: 7901987;Abstract: We conducted restriction fragment length polymorphism and methylation pattern analyses on 26 typical and atypical patients with Prader-Willi syndrome who did not have a cytogenetically detectable 15q11-13 deletion and on four patients who did have this deletion and were clinically atypical. Maternal disomy for chromosome 15 was identified in 12 patients and paternal deletions in 15q11-13 were found in three cases. Patients with chromosome 15 abnormalities had typical or near typical presentations, based on published diagnostic criteria. Most of the absent criteria in this group were age- dependent features. The remaining 15 patients, including four previously thought to have a cytogenetically apparent 15q11-13 deletion, had neither chromosome 15 molecular abnormality, and these patients were atypical. Patients with maternal disomy had advanced maternal age, suggesting that nondisjunction is part of the etiology of uniparental disomy. This study suggests that molecular diagnosis is critical in patients with Prader-Willi syndrome who appear clinically atypical or who lack a cytogenetically detectable 15q deletion. Methylation pattern analysis is a useful adjunct diagnostic tool for Prader-Willi syndrome.
• Patterson, D., Hart, I., Lai, L. -., Brahe, C., Moscetti, A., Tassone, F., Raimondi, E., & Jones, C. (1993). Molecular and cytogenetic characterization of a Chinese hamster/human hybrid cell line containing a der (21)t(Ypter → cenY::cen21 → 21 qter) chromosome. Genomics, 15(1), 177-179.
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  PMID: 8432530;Abstract: Human/rodent somatic cell hybrids have been exceedingly useful in assigning human genes and DNA sequences to specific human chromosomes. As new technologies for analyzing the human chromosome complement of such human/rodent hybrid cells become available, it is of critical importance that these be applied to enhance characterization of existing hybrids. This is particularly important since human chromosomes in such hybrids have been observed to rearrange with time. We report here the use of fluorescence in situ hybridization of DNA probes to metaphase chromosomes to analyze one hybrid designated 72532X6. This analysis shows that the chromosome suspected to be a normal human chromosome 21 in this hybrid is actually a translocation chromosome containing Yp and 21q. In addition, the hybrid contains a fragment of human chromosome 9 translocated to a Chinese hamster chromosome. Analysis of the chromosomes from the human donor indicates that his chromosomes are normal. Thus, this translocation chromosome appears to have arisen after formation of the hybrid.
• Cassidy, S. B., Lai, L. -., & Erickson, R. P. (1992). Clinical correlates of molecular genetic defects in Prader-Willi syndrome. Dysmorphology and Clinical Genetics, 6(2), 77-.
• Cassidy, S. B., Lai, L., Erickson, R. P., Magnuson, L., Thomas, E., Gendron, R., & Herrmann, J. (1992). Trisomy 15 with loss of the paternal 15 as a cause of Prader-Willi syndrome due to maternal disomy. American Journal of Human Genetics, 51(4), 701-708.
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  PMID: 1357962;PMCID: PMC1682792;Abstract: Uniparental disomy has recently been recognized to cause human disorders, including Prader-Willi syndrome (PWS). We describe a particularly instructive case which raises important issues concerning the mechanisms producing uniparental disomy and whose evaluation provides evidence that trisomy may precede uniparental disomy in a fetus. Chorionic villus sampling performed for advanced maternal age revealed trisomy 15 in all direct and cultured cells, though the fetus appeared normal. Chromosome analysis of amniocytes obtained at 15 wk was normal in over 100 cells studied. The child was hypotonic at birth, and high-resolution banding failed to reveal the deletion of 15q11-13, a deletion which is found in 50%-70% of patients with PWS. Over time, typical features of PWS developed. Molecular genetic analysis using probes for chromosome 15 revealed maternal disomy. Maternal nondisjunction with fertilization of a disomic egg by a normal sperm, followed by loss of the paternal 15, is a likely cause of confined placental mosaicism and uniparental disomy in this case of PWS, and advanced maternal age may be a predisposing factor.
• Lai, L., Hart, I. M., & Patterson, D. (1991). A gene correcting the defect in the CHO mutant Ade^-H, deficient in a branch point enzyme (adenylosuccinate synthetase) of de novo purine biosynthesis, is located on the long arm of chromosome 1. Genomics, 9(2), 322-328.
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  PMID: 2004783;Abstract: Somatic hybrids between human cells and the Chinese hamster ovary (CHO) K1 mutant, Ade-H cells, were selected for purine prototrophy by growth in adenine-free medium. The Ade-H mutant is defective in the enzyme adenylosuccinate (AMPS) synthetase (ADSS; EC 6.3.4.4), which carries out the first of a two-step sequence in the biosynthesis of AMP from IMP, and therefore requires exogenous adenine for growth. The presence of the long arm of human chromosome 1 in the hybrids is 100% concordant for the ability to growth in adenine-free medium and restoration of the enzyme activity. Hybrid segregants that lose the ability to grow in adenine-free medium lose all or a portion of chromosome 1 and enzyme activity. Southern blot hybridization with a chromosome 1-specific probe, BCMI, confirms the existence of human chromosome 1 in these hybrids. Analysis of a human/CHO translocation chromosome that arose in one of the hybrids suggests that the gene correcting the defect lies in the region 1 cen-1q12. In summary, we have shown by cytogenetics, segregant analysis, biochemical assay, and Southern blot analysis that human chromosome 1, most likely in the region 1 cen-1q12, corrects the defect in ADSS-deficient mutant Ade-H cells. © 1991.
• Lien, Y. H., Lai, L. W., Cheung, C., Patterson, D., & Chan, L. (1991). Role of purine synthesis on renal function: effect of adenylosuccinate synthetase inhibition.. Contributions to nephrology, 95, 112-119.
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  PMID: 1807901;
• Lai, L., & Rosenstein, B. S. (1990). Induction of DNA strand breaks and DNA-protein cross-links in normal human skin fibroblasts following exposure to 254 nm UV radiation. Journal of Photochemistry and Photobiology, B: Biology, 6(4), 395-404.
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  PMID: 2120408;Abstract: Levels of DNA strand breaks and DNA-protein cross-links (DPCs) were measured using the alkaline elution assay in normal human skin fibroblasts irradiated with 0-200 J m-2 of 254 nm UV radiation and incubated for 0-24 h. On incubation, the yields of both single-strand breaks (SSBs) and DPCs increased with similar kinetics and remained elevated. In addition, when SSBs were measured under conditions in which DPCs were not eliminated by treatment with proteinase K, a measurable yield of SSBs could not be detected. Hence, the SSBs that form in the UV-irradiated cells following incubation appear to be associated with the DPCs. © 1990.

Others

• Lai, L., Lien, Y. H., & Lai, L. -. (2005, Apr). Bilateral femoral head and distal tibial osteonecrosis in a patient with Fabry disease. American journal of orthopedics (Belle Mead, N.J.).
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  Fabry disease is a lysosomal storage disease caused by alpha-galactosidase A deficiency. The classic presentation of Fabry disease involves multiple organs, including kidneys, heart, skin, eyes, and nervous system. Osteonecrosis is rarely reported in patients with Fabry disease. In this article, we describe the case of a 37-year-old white man who had Fabry disease and no risk factors for osteonecrosis but who developed osteonecrosis in both femoral heads and in an unusual site, bilateral distal tibiae. Results of mutation analysis showed a nonsense mutation (R227X) in the alpha-galactosidase A gene. This case suggests that Fabry disease may be a risk factor for development of osteonecrosis. The enzyme replacement therapy currently available may be an effective method of preventing this complication.