Sandeep Gupta

Isolation and Characterization of Kidney-Derived Stem Cells

Acute kidney injury is followed by regeneration of damaged renal tubular epithelial cells. The purpose of this study was to test the hypothesis that renal stem cells exist in the adult kidney and participate in the repair process. A unique population of cells that behave in a manner that is consistent with a renal stem cell were isolated from rat kidneys and were termed multipotent renal progenitor cells (MRPC). Features of these cells include spindle-shaped morphology; self-renewal for >200 population doublings without evidence for senescence; normal karyotype and DNA analysis; and expression of vimentin, CD90 (thy1.1), Pax-2, and Oct4 but not cytokeratin, MHC class I or II, or other markers of more differentiated cells. MRPC exhibit plasticity that is demonstrated by the ability of the cells to be induced to express endothelial, hepatocyte, and neural markers by reverse transcriptase-PCR and immunohistochemistry. The cells can differentiate into renal tubules when injected under the capsule of an uninjured kidney or intra-arterially after renal ischemia-reperfusion injury. Oct4 expression was seen in some tubular cells in the adult kidney, suggesting these cells may be candidate renal stem cells. It is proposed that MRPC participate in the regenerative response of the kidney to acute injury.

Effect of Notch activation on the regenerative response to acute renal failure

Episodes of acute renal failure (ARF) are not always fully reversible and may lead to chronic disease, due in part to an inadequate regenerative response. The Notch signaling pathway is involved in determining cell fate during development, and tissue maintenance and repair in adult organs. The purpose of this study was to examine the role of the Notch pathway in renal regeneration following ARF. Kidney injury, induced by ischemia-reperfusion, resulted in early activation of the Notch pathway, as evidenced by increased expression of Notch1 and Notch2 intracellular domain (cleaved Notch). The effect of exogenous administration of the Notch ligand Delta-like-4 (DLL4) on recovery from ARF was then studied. Rats were pretreated by intraperitoneal injection of DLL4 or vehicle control. Two days following the last DLL4 dose, ARF was induced by bilateral renal artery clamping for 45 min followed by reperfusion. The severity of renal injury was similar in DLL4 and control rats. Renal recovery was facilitated by DLL4 treatment, as evidenced by faster return of serum creatinine to baseline by 48 h in DLL4-treated rats as against 5 days in vehicle-treated control rats. Cell proliferation was higher in the DLL4-treated group. In conclusion, activation of the Notch pathway occurs following ARF. Pretreatment with the Notch ligand DLL4 enhanced recovery from ARF and represents a potential novel therapeutic option for regenerating the injured kidney.

Do stem cells exist in the adult kidney

Adult stem cells exist in many organs and play a critical role in normal cell turnover and the response to injury. The existence of adult stem cells in the mammalian kidney remains controversial. Kidney stem cells have been isolated and characterized by many groups, often with discrepant results. This article will review the current state of knowledge regarding adult kidney stem cells and discuss future directions for kidney stem cell research.

Brief Report: Analysis of Endogenous Oct4 Activation during Induced Pluripotent Stem Cell Reprogramming Using an Inducible Oct4 Lineage Label

The activation of endogenous Oct4 transcription is a key step in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells but until now it has been difficult to analyze this critical event in the reprogramming process. We have generated a transgenic mouse that expresses the tamoxifen‐inducible Cre recombinase MerCreMer under the control of the endogenous Oct4 locus, enabling lineage tracing of Oct4 expression in cells in vivo or in vitro, during either reprogramming or differentiation. Using this novel resource, we have determined the timing and outcome of endogenous Oct4 induction during fibroblast reprogramming. We show that both the initiation of this key reprogramming step and the ability of cells activating endogenous Oct4 expression to complete reprogramming are not influenced by the presence of exogenous c‐Myc, although the overall efficiency of the process is increased by c‐Myc. Oct4 lineage tracing reveals that new reprogramming events continue to initiate over a period of 3 weeks. Furthermore, the analysis of mixed colonies, where only a subset of daughter cells induce endogenous Oct4 expression, indicates the role of unknown, stochastic events in the progression of reprogramming from the initial events to a pluripotent state. Our transgenic mouse model and cells derived from it provide powerful and precise new tools for the study of iPS cell reprogramming mechanisms and have wider implications for the investigation of the role of Oct4 during development. STEM CELLS2012;30:2596–2601

Cellular therapy of kidney diseases

The understanding of cellular sources of kidney regeneration has rapidly evolved in the last decade. It is now believed that regeneration occurs predominantly from cells that reside within the injured kidney, with minimal contribution from extra‐renal cells. We now know that improved kidney regeneration seen following exogenous administration of stem cells occur predominantly by noncellular paracrine mechanisms. Of all extra‐renal stem cells, mesenchymal stem cells (MSC) are the most promising stem cell type for treating kidney diseases. There is an ongoing clinical trial evaluating safety and efficacy of MSC in treating acute kidney injury (AKI). Results of this trial are expected to bring use of MSC closer to the clinical realm. An improved understanding of the small molecules that facilitate kidney regeneration and are secreted by MSC will likely result in the development of new therapies for treating AKI. Identification of adult stem cell markers will result in improved understanding of pathophysiology of kidney diseases and could lead to the development of new cellular therapies. Directed differentiation of stem cells into desired cell types such as erythropoietin producing cells will allow selective replacement of lost kidney function. Cell‐based therapies for patients with chronic kidney disease are presently in proof‐of‐principle stage and are expected to evolve in the coming years with improved understanding of stem cell biology. Technological advancement in cellular therapy is expected to provide improved therapeutic options for patients with kidney diseases in the near future.

Kidney regeneration and resident stem cells

Given its complexity, high metabolic activity and excretory functions, the kidney is particularly susceptible to acute ischemic and toxin-mediated injury. Current therapies do not facilitate kidney regeneration, and there is an increasing interest in newer therapies that are based on cellular sources of kidney regeneration, such as stem cell therapy. Our understanding of cellular sources for kidney regeneration and stem cells present in the adult kidney has dramatically evolved over the recent years. Herein, we discuss the current understanding of kidney stem cells present in the adult mammalian kidney and their role in kidney regeneration. We have also summarized the best available evidence supporting the role of stem cells in kidney regeneration.

Prevention of Acute Kidney Injury by Tauroursodeoxycholic Acid in Rat and Cell Culture Models

Background Acute kidney injury (AKI) has grave short- and long-term consequences. Often the onset of AKI is predictable, such as following surgery that compromises blood flow to the kidney. Even in such situations, present therapies cannot prevent AKI. As apoptosis is a major form of cell death following AKI, we determined the efficacy and mechanisms of action of tauroursodeoxycholic acid (TUDCA), a molecule with potent anti-apoptotic and pro-survival properties, in prevention of AKI in rat and cell culture models. TUDCA is particularly attractive from a translational standpoint, as it has a proven safety record in animals and humans. Methodology/Principal Findings We chose an ischemia-reperfusion model in rats to simulate AKI in native kidneys, and a human kidney cell culture model to simulate AKI associated with cryopreservation in transplanted kidneys. TUDCA significantly ameliorated AKI in the test models due to inhibition of the mitochondrial pathway of apoptosis and upregulation of survival pathways. Conclusions This study sets the stage for testing TUDCA in future clinical trials for prevention of AKI, an area that needs urgent attention due to lack of effective therapies.

Identification and Characterization of Sall1-Expressing Cells Present in the Adult Mouse Kidney

Background: Sall1 is a transcription factor that best identifies stem cells present in the mouse embryonic kidney. Mutations in Sall1 gene in mice can lead to dysgenesis of kidney, while in humans it results in the Townes-Brocks syndrome, which is associated with the kidney agenesis. Unlike the embryonic kidney, Sall1 expression in the adult kidney is largely unknown. We hypothesized that similar to the embryonic kidney, Sall1 expression can identify stem cells present in the adult kidney. Accordingly in this study, we identified Sall1-expressing cells in the adult mouse kidney, determined their role in kidney regeneration following ischemia-reperfusion injury (IRI), and sought the effect of age on Sall1 expression. Methods and Results: By immunofluorescence Sall1-expressing cells were identified in the proximal tubule at the cortico-medullary junction and constituted 0.5% of all tubular cells. Rare Sall1-positive cells were also identified in the outer cortex and distal tubules. Sall1 expression was not seen in the glomerular, interstitial, or vascular compartments. Following IRI, 90% of Sall1-expressing cells proliferated and 5% of Sall1-positive cells showed asymmetrical cell division with one of the two adjacent Sall1-positive cells incorporating chlorodeoxyuridine (CldU). Following IRI, there was an increase in Sall1 expression at 4 and 12 h, a decrease at 5 and 10 days, and baseline expression at day 30 by quantitative polymerase chain reaction (qRT-PCR) and Western blot analysis. There was no age-related change in Sall1 expression as determined by qRT-PCR, Western blot analysis, and immunofluorescence. Conclusions: We conclude that Sall1-expressing cells are present in the adult mouse kidney, predominantly in the proximal tubules. Sall1-expressing cells proliferate following IRI and some of the Sall1-positive cells undergo asymmetrical cell division. Therefore, Sall1 is a promising marker for identification of stem cells present in the adult mouse kidney.

Stem cells and the kidney: where do we go from here?

The kidney has a remarkable capacity to regenerate and restore its structure and function after acute injury. However, kidney regeneration is frequently delayed or inadequate, resulting in significant morbidity and mortality. Incomplete recovery of the injured kidney may lead to progressive loss of