Ashwani Kumar Gupta

Interests

Research

Ashwani Kumar Gupta, PhD, is a Research Assistant Professor of Surgery at the University of Arizona. Dr. Gupta’s research work has been focused to study kidney development and disease. Diseases of the kidneys are common and debilitating, often with limited treatment options. There is no curative treatment available for the patients with chronic kidney diseases except renal transplantation. The lack of availability of transplant organs warrants research into technologies to understand how new kidney tissues can be generated. In the recent years, generation of kidney organoids from stem cells has become an important technology, with potential translational applications. Dr. Gupta’s research interest is to understand the pathways that regulate kidney stem/progenitor cell self-renewal and differentiation, in order to generate transplantable kidney tissues. His current research is focused to generate 3D organs using de-cellularized organ scaffolds, physiologically functional kidney organoids with glomerular filtration apparatus and interconnecting nephrons with collecting ducts/ureter. Dr. Gupta’s primary goal is to generate transplantable kidney tissues for the patients with end stage renal diseases.

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Scholarly Contributions

Journals/Publications

• Gupta, A. K., Ivancic, D. Z., Naved, B. A., Wertheim, J. A., & Oxburgh, L. (2021). An efficient method to generate kidney organoids at the air-liquid interface. Journal of biological methods, 8(2), e150.
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  The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.
• Ryan, A. R., England, A. R., Chaney, C. P., Cowdin, M. A., Hiltabidle, M., Daniel, E., Gupta, A. K., Oxburgh, L., Carroll, T. J., & Cleaver, O. (2021). Vascular deficiencies in renal organoids and ex vivo kidney organogenesis. Developmental biology, 477, 98-116.
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  Chronic kidney disease (CKD) and end stage renal disease (ESRD) are increasingly frequent and devastating conditions that have driven a surge in the need for kidney transplantation. A stark shortage of organs has fueled interest in generating viable replacement tissues ex vivo for transplantation. One promising approach has been self-organizing organoids, which mimic developmental processes and yield multicellular, organ-specific tissues. However, a recognized roadblock to this approach is that many organoid cell types fail to acquire full maturity and function. Here, we comprehensively assess the vasculature in two distinct kidney organoid models as well as in explanted embryonic kidneys. Using a variety of methods, we show that while organoids can develop a wide range of kidney cell types, as previously shown, endothelial cells (ECs) initially arise but then rapidly regress over time in culture. Vasculature of cultured embryonic kidneys exhibit similar regression. By contrast, engraftment of kidney organoids under the kidney capsule results in the formation of a stable, perfused vasculature that integrates into the organoid. This work demonstrates that kidney organoids offer a promising model system to define the complexities of vascular-nephron interactions, but the establishment and maintenance of a vascular network present unique challenges when grown ex vivo.
• Ye, M., Wysocki, J., Wertheim, J. A., Wang, Y., Randall, G., Nicoleascu, V., Hassler, L., Gupta, A. K., & Batlle, D. (2021). A Novel Soluble ACE2 Variant with Prolonged Duration of Action Neutralizes SARS-CoV-2 Infection in Human Kidney Organoids.. Journal of the American Society of Nephrology : JASN, 32(4), 795-803. doi:10.1681/asn.2020101537
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  There is an urgent need for approaches to prevent and treat SARS-CoV-2 infection. Administration of soluble ACE2 protein acting as a decoy to bind to SARS-CoV-2 should limit viral uptake mediated by binding to membrane-bound full-length ACE2, and further therapeutic benefit should result from ensuring enzymatic ACE2 activity to affected organs in patients with COVID-19..A short variant of human soluble ACE2 protein consisting of 618 amino acids (hACE2 1-618) was generated and fused with an albumin binding domain (ABD) using an artificial gene encoding ABDCon, with improved albumin binding affinity. Human kidney organoids were used for infectivity studies of SARS-CoV-2 in a BSL-3 facility to examine the neutralizing effect of these novel ACE2 variants..Whereas plasma ACE2 activity of the naked ACE2 1-618 and ACE2 1-740 lasted about 8 hours, the ACE2 1-618-ABD resulted in substantial activity at 96 hours, and it was still biologically active 3 days after injection. Human kidney organoids express ACE2 and TMPRSS2, and when infected with SARS-CoV-2, our modified long-acting ACE2 variant neutralized infection..This novel ACE2 1-618-ABD can neutralize SARS-CoV-2 infectivity in human kidney organoids, and its prolonged duration of action should ensure improved efficacy to prevent viral escape and dosing convenience.
• Kumar Gupta, A., Sarkar, P., Wertheim, J. A., Pan, X., Carroll, T. J., & Oxburgh, L. (2020). Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids. Communications biology, 3(1), 231.
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  A fundamental challenge in emulating kidney tissue formation through directed differentiation of human pluripotent stem cells is that kidney development is iterative, and to reproduce the asynchronous mix of differentiation states found in the fetal kidney we combined cells differentiated at different times in the same organoid. Asynchronous mixing promoted nephrogenesis, and heterochronic organoids were well vascularized when engrafted under the kidney capsule. Micro-CT and injection of a circulating vascular marker demonstrated that engrafted kidney tissue was connected to the systemic circulation by 2 weeks after engraftment. Proximal tubule glucose uptake was confirmed, but despite these promising measures of graft function, overgrowth of stromal cells prevented long-term study. We propose that this is a technical feature of the engraftment procedure rather than a specific shortcoming of the directed differentiation because kidney organoids derived from primary cells and whole embryonic kidneys develop similar stromal overgrowth when engrafted under the kidney capsule.
• Brown, A. C., Gupta, A. K., & Oxburgh, L. (2019). Long-Term Culture of Nephron Progenitor Cells Ex Vivo. Methods in molecular biology (Clifton, N.J.), 1926, 63-75.
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  Nephrons differentiate from the cap mesenchyme of the fetal kidney. Nephron progenitor cells that populate the cap mesenchyme efficiently balance self-renewal and epithelial differentiation to enable repeated rounds of nephron formation during development. Here we describe a method to isolate and propagate these cells from the embryonic mouse kidney. Using this method, nephron progenitor cells from a single litter of mice can be propagated to hundreds of millions of cells that express appropriate markers of the undifferentiated state and retain epithelial differentiation capacity in vitro.
• Gupta, A. K., Coburn, J. M., Davis-Knowlton, J., Kimmerling, E., Kaplan, D. L., & Oxburgh, L. (2019). Scaffolding kidney organoids on silk. Journal of tissue engineering and regenerative medicine, 13(5), 812-822.
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  End stage kidney disease affects hundreds of thousands of patients in the United States. The therapy of choice is kidney replacement, but availability of organs is limited, and alternative sources of tissue are needed. Generation of new kidney tissue in the laboratory has been made possible through pluripotent cell reprogramming and directed differentiation. In current procedures, aggregates of cells known as organoids are grown either submerged or at the air-liquid interface. These studies have demonstrated that kidney tissue can be generated from pluripotent stem cells, but they also identify limitations. The first is that perfusion of cell aggregates is limited, restricting the size to which they can be grown. The second is that aggregates lack the structural integrity required for convenient engraftment and suturing or adhesion to regions of kidney injury. In this study, we evaluated the capacity of silk to serve as a support for the growth and differentiation of kidney tissue from primary cells and from human induced pluripotent stem cells. We find that cells can differentiate to epithelia characteristic of the developing kidney on this material and that these structures are maintained following engraftment under the capsule of the adult kidney. Blood vessel investment can be promoted by the addition of vascular endothelial growth factor to the scaffold, but the proliferation of stromal cells within the graft presents a challenge, which will require some readjustment of cell growth and differentiation conditions. In summary, we find that silk can be used to support growth of stem cell derived kidney tissue.
• Bond, K. H., Duarte, C. W., Congdon, C. B., Hainmhire, E. O., Humphreys, B. D., Oxburgh, L., Karolak, M. J., Gupta, A. K., Fetting, J. L., & Emery, I. F. (2018). Abstract 200: FOXD1 promotes stromal investment in clear cell renal cell carcinoma. Tumor Biology. doi:10.1158/1538-7445.am2018-200
• Garikipati, V. N., Singh, S. P., Mohanram, Y., Gupta, A. K., Kapoor, D., & Nityanand, S. (2018). Isolation and characterization of mesenchymal stem cells from human fetus heart. PloS one, 13(2), e0192244.
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  Mesenchymal stem cells (MSCs) are promising cells for cardiovascular regenerative medicine. However, their potential may be limited, because of their restricted cardiovascular differentiation potential and decline in their number and functional characteristics with increasing donor age. We have previously shown that rat fetus heart harbors primitive MSCs and administration of these cells improved left ventricular (LV) function after ischemia/reperfusion injury in rats. To evaluate their potential as a new cell type for clinical cardiovascular cell therapy, we have undertaken this study on the isolation and characterization of human fetal cardiac MSCs (hfC-MSCs).
• Gupta, A. K., Jadhav, S. H., Tripathy, N. K., & Nityanand, S. (2015). Fetal Kidney Cells Can Ameliorate Ischemic Acute Renal Failure in Rats through Their Anti-Inflammatory, Anti-Apoptotic and Anti-Oxidative Effects. PloS one, 10(6), e0131057.
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  Fetal kidney cells may contain multiple populations of kidney stem cells and thus appear to be a suitable cellular therapy for the treatment of acute renal failure (ARF) but their biological characteristics and therapeutic potential have not been adequately explored. We have culture expanded fetal kidney cells derived from rat fetal kidneys, characterized them and evaluated their therapeutic effect in an ischemia reperfusion (IR) induced rat model of ARF. The fetal kidney cells grew in culture as adherent spindle shaped/polygonal cells and expressed CD29, CD44, CD73, CD90, CD105, CD24 and CD133 markers. Administration of PKH26 labeled fetal kidney cells in ARF rats resulted in a significant decrease in the levels of blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin and decreased tubular necrosis in the kidney tissues (p
• Gupta, A. K., Jadhav, S. H., Tripathy, N. K., & Nityanand, S. (2015). Fetal kidney stem cells ameliorate cisplatin induced acute renal failure and promote renal angiogenesis. World journal of stem cells, 7(4), 776-88.
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  To investigate whether fetal kidney stem cells (fKSC) ameliorate cisplatin induced acute renal failure (ARF) in rats and promote renal angiogenesis.

Proceedings Publications

• Carroll, T. L., Gupta, A. K., Oxburgh, L., Pan, X., & Sarkar, P. (2020).

  Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids

  . In Journal.
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  ABSTRACT Recent years have seen rapid advances in directed differentiation of human pluripotent stem cells (PSCs) to kidney cells. However, a fundamental difficulty in emulating kidney tissue formation is that kidney development is iterative. Recent studies argue that the human nephron forms through gradual contribution of nephron progenitor cells whose differentiation fates depend on the time at which they are recruited. We show that the majority of PSC-derived nephron progenitor cells differentiated in a short wave in organoid formation and to improve fidelity of PSC-derived organoids, we emulated the asynchronous mix found in the fetal kidney by combining cells differentiated at different times in the same organoid. Asynchronous mixing promoted nephrogenesis, and lineage marking data showed that proximal and distal nephron components preferentially derive from cell populations differentiated at distinct times. When engrafted under the kidney capsule these heterochronic organoids were vascularized and displayed essential features of kidney tissue. Micro-CT and injection of a circulating vascular marker demonstrated that engrafted kidney tissue was connected to the systemic circulation by 2 weeks after engraftment. Proximal tubule glucose uptake was confirmed using intravenous injection of fluorescent dextran. Despite these promising measures of graft function, overgrowth of stromal cells prevented long-term study, and we propose that this is a technical feature of the engraftment procedure rather than a specific shortcoming of the directed differentiation because kidney organoids derived from primary cells and whole embryonic kidneys develop the same stromal overgrowth when engrafted under the kidney capsule.