Use of mouse hematopoietic stem and progenitor cells to treat acute kidney injury

New and effective treatment for acute kidney injury remains a challenge. Here, we induced mouse hematopoietic stem and progenitor cells (HSPC) to differentiate into cells that partially resemble a renal cell phenotype and tested their therapeutic potential. We sequentially treated HSPC with a combination of protein factors for 1 wk to generate a large number of cells that expressed renal developmentally regulated genes and protein. Cell fate conversion was associated with increased histone acetylation on promoters of renal-related genes. Further treatment of the cells with a histone deacetylase inhibitor improved the efficiency of cell conversion by sixfold. Treated cells formed tubular structures in three-dimensional cultures and were integrated into tubules of embryonic kidney organ cultures. When injected under the renal capsule, they integrated into renal tubules of postischemic kidneys and expressed the epithelial marker E-cadherin. No teratoma formation was detected 2 and 6 mo after cell injection, supporting the safety of using these cells. Furthermore, intravenous injection of the cells into mice with renal ischemic injury improved kidney function and morphology by increasing endogenous renal repair and decreasing tubular cell death. The cells produced biologically effective concentrations of renotrophic factors including VEGF, IGF-1, and HGF to stimulate epithelial proliferation and tubular repair. Our study indicates that hematopoietic stem and progenitor cells can be converted to a large number of renal-like cells within a short period for potential treatment of acute kidney injury.     

Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney

In the kidney, a unique plasticity exists between epithelial and mesenchymal cells. During kidney development, the metanephric mesenchyme contributes to emerging epithelium of the nephron via mesenchymal to epithelial transition (MET). In the injured adult kidney, renal epithelia contribute to the generation of fibroblasts via epithelial-mesenchymal transition, facilitating renal fibrosis. Recombinant human bone morphogenic protein (BMP)-7, a morphogen that is essential for the conversion of epithelia from condensing mesenchyme during kidney development, enhances the repair of tubular structures in the kidney. In this setting, BMP-7 inhibits epithelial-mesenchymal transition involving adult renal epithelial tubular cells and decreases secretion of type I collagen by adult renal fibroblasts. In search of a mechanism behind the ability of BMP-7 to repair damaged renal tubules, we hypothesized that systemic treatment with BMP-7 might induce MET involving adult renal fibroblasts in the injured kidney, generating functional epithelial cells. Here we report that BMP-7 induces formation of epithelial cell aggregates in adult renal fibroblasts associated with reacquisition of E-cadherin expression and decreased motility, mimicking the effect of BMP-7 on embryonic metanephric mesenchyme to generate epithelium. In addition, we provide evidence that BMP-7-mediated repair of renal injury is associated with MET involving adult renal interstitial fibroblasts in mouse models for renal fibrosis. Collectively, these findings suggest that adult renal fibroblasts might retain parts of their original embryonic imprint and plasticity, which can be re-engaged by systemic administration of BMP-7 to mediate repair of tubular injury in a fibrotic kidney.     

Integration potential of mouse and human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a multipotent cell population which has been described to exert renoprotective and regenerative effects in experimental models of kidney injury. Several lines of evidence indicate that MSCs also have the ability to contribute to nephrogenesis, suggesting that the cells can be employed in stem cell-based applications aimed at de novo renal tissue generation. In this study we re-evaluate the capacity of mouse and human bone marrow-derived MSCs to contribute to the development of renal tissue using a novel method of embryonic kidney culture. Although MSCs show expression of some genes involved in renal development, their contribution to nephrogenesis is very limited in comparison to other stem cell types tested. Furthermore, we found that both mouse and human MSCs have a detrimental effect on the ex vivo development of mouse embryonic kidney, this effect being mediated through a paracrine action. Stimulation with conditioned medium from a mouse renal progenitor population increases the ability of mouse MSCs to integrate into developing renal tissue and prevents the negative effects on kidney development, but does not appear to enhance their ability to undergo nephrogenesis.     

Adult human CD133/1(+) kidney cells isolated from papilla integrate into developing kidney tubules

Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin(+) and CD133/1(+) cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1(+) cells. Isolated CD133/1(+) papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1(+) progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay

Renal stem or progenitor cells with a multilineage differentiation potential remain to be isolated, and the differentiation mechanism of these cell types in kidney development or regeneration processes is unknown. In an attempt to resolve this issue, we set up an in vitro culture system using NIH3T3 cells stably expressing Wnt4 (3T3Wnt4) as a feeder layer, in which a single renal progenitor in the metanephric mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP(high) cells), a zinc-finger nuclear factor essential for kidney development, form colonies, and that they reconstitute a three-dimensional kidney structure in an organ culture setting. We also found that Rac- and JNK-dependent planar cell polarity (PCP) pathways downstream of Wnt4 positively regulate the colony size, and that the JNK pathway is also involved in mesenchymal-to-epithelial transformation of colony-forming progenitors. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation.     

Adult human CD133/1 kidney cells isolated from papilla integrate into developing kidney tubules

Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin⁺ and CD133/1⁺ cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1⁺ cells. Isolated CD133/1⁺ papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1⁺ progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Kidney Specific Protein-Positive Cells Derived from Embryonic Stem Cells Reproduce Tubular Structures In Vitro and Differentiate into Renal Tubular Cells

Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.     

In vitro generation of three-dimensional renal structures

End-stage renal disease is currently being treated effectively by transplantation. However, increasing demand and donor shortage make this treatment challenging. Recent advances in cell-based therapies have provided potential opportunities to alleviate the current challenges of donor shortage. In this study we developed a system to generate renal structures in vitro using primary kidney cells. This system involves the cultivation of expanded primary renal cells in a three-dimensional collagen-based culture system. After one week of growth, individual renal cells began to form renal structures resembling tubules and glomeruli. Histologically, these structures show phenotypic resemblance to native kidney structures. The reconstituted tubules stained positively for Tamm-Horsfall protein, which is expressed in the thick ascending limb of Henle's Loop and distal convoluted tubules. These results show that renal structures can be reconstituted in a three-dimensional culture system, which may eventually be used for renal cell therapy applications.     

In vitro regeneration of kidney from pluripotent stem cells

Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitro is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.     

Chemically defined medium environment for the development of renal stem cells into tubules

The use of stem cells is a valuable therapeutical option for the regeneration of diseased tissues and organs. However, the involved cellular processes are hardly known. To gain detailed information about their development, a new culture technology was developed. Embryonic renal tissue containing stem/progenitor cells was mounted within a perfusion culture container at the interface of an artificial interstitium made of polyester. Using this innovative approach we show that renal tubules develop in chemically defined Iscove's modified Dulbecco's medium without serum addition and without coating by extracellular matrix proteins. The development of tubules depends on the administration of aldosterone, and can be visualized by immunohistochemical labeling. The presented technology makes the exact analysis of developmental steps now possible, and provides a new powerful tool to optimize growth and differentiation of renal stem cells. It may also enable many other kinds of stem cells to steer their development into functional tissues under clearly defined in vitro conditions.     

Species Diversity Regarding the Presence of Proximal Tubular Progenitor Cells of the Kidney

The cellular source for tubular regeneration following kidney injury is a matter of dispute, with reports suggesting a stem or progenitor cells as the regeneration source while linage tracing studies in mice seemingly favor the classical theory, where regeneration is performed by randomly surviving cells. We, and others have previously described a scattered cell population localized to the tubules of human kidney, which increases in number following injury. Here we have characterized the species distribution of these proximal tubular progenitor cells (PTPCs) in kidney tissue from chimpanzee, pig, rat and mouse using a set of human PTPC markers. We detected PTPCs in chimpanzee and pig kidneys, but not in mouse tissue. Also, subjecting mice to the unilateral urethral obstruction model, caused clear signs of tubular injury, but failed to induce the PTPC phenotype in renal tubules.Key words: Acute tubular necrosis, tubular regeneration, species diversity, proximal tubules     

Protective Effect of Human Amniotic Fluid Stem Cells in an Immunodeficient Mouse Model of Acute Tubular Necrosis

Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kit^pos stem cells, derived from human amniotic fluid (hAFSC) can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN.     

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 °C and 5% CO₂. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 µM resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.     

Tracing the life of the kidney tubule- re-establishing dogma and redirecting the options

Renal pathology suggests that tubular repair results from tubular proliferation. In contrast, recent studies propose that postnatal kidney repair may involve renal stem cells. In this issue of Cell Stem Cell, Humphreys et al. (2008) use lineage tracing to genetically assess contribution of adult nontubular cells (potentially stem cells) to repair of damaged renal tubules.     

In search of adult renal stem cells

The therapeutic potential of adult stem cells in the treatment of chronic degenerative diseases has becoming increasingly evident over the last few years. Significant attention is currently being paid to the development of novel treatments for acute and chronic kidney diseases too. To date, promising sources of stem cells for renal therapies include adult bone marrow stem cells and the kidney precursors present in the early embryo. Both cells have clearly demonstrated their ability to differentiate into the kidney's specialized structures. Adult renal stem cells have yet to be identified, but the papilla is where the stem cell niche is probably located. Now we need to isolate and characterize the fraction of papillary cells that constitute the putative renal stem cells. Our growing understanding of the cellular and molecular mechanisms behind kidney regeneration and repair processes - together with a knowledge of the embryonic origin of renal cells - should induce us, however, to bear in mind that in the kidney, as in other mesenchymal tissues, the need for a real stem cell compartment might be less important than the phenotypic flexibility of tubular cells. Thus, by displaying their plasticity during kidney maintenance and repair, terminally differentiated cells may well function as multipotent stem cells despite being at a later stage of maturation than adult stem cells. One of the major tasks of Regenerative Medicine will be to disclose the molecular mechanisms underlying renal tubular plasticity and to exploit its biological and therapeutic potential.     

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.     

The tubulogenic effect of aldosterone is attributed to intact binding and intracellular response of the mineralocorticoid receptor

Little is known about the extra- and intracellular stimuli inducing renal stem/progenitor cells to develop into three-dimensionally structured tubules. To study this specific development in a controlled environment, we used an advanced culture technique. Embryonic tissue derived from neonatal rabbit kidney was placed in a perfusion culture container at the interface of an artificial interstitium made of a polyester fleece. Culture was carried out in chemically defined Iscove’s Modified Dulbecco’s Medium (IMDM) for 13 days. Development of tubules was histochemically detected on cryosections labeled with Soybean Agglutinin (SBA). The experiments showed that aldosterone exerts a specific tubulogenic effect. Application of aldosterone (1 × 10⁻⁷ M) raised numerous SBA-labeled tubules, while in the absence of the steroid hormone the development of tubules was lacking. Specificity of hormone action was analyzed by the use of aldosterone antagonists. Administration of spironolactone (1 × 10⁻⁴ M) and canrenoate (1 × 10⁻⁵ M) completely inhibited the development of tubules. Finally, disrupting the intracellular molecular complex of the mineralocorticoid receptor (MR) and heat shock proteins by geldanamycin (2 μg/ml) prevented the development of tubules. Our results suggest that the tubulogenic effect induced by aldosterone is attributed to both hormone binding and an undisturbed intracellular response of the MR.     

Phagocytosis of E. coli by renal tubular epithelia

Despite significant advances in our understanding or renal tubular cell function, the in vivo handling of E. coli by renal tubules has not been previously investigated. The present studies were, therefore, designed to study this aspect of nephron function. Live and dead E. coli and vehicle alone were microinjected into the proximal tubular lumen of a single nephron of rats, and the microinjected tubules were morphologically studied at one-half, two, four, and six hours after. The bacteria initially contacted the luminal cell membrane. The luminal cell membrane adjacent to the bacteria subsequently invaginated, and both live and dead E. coli eventually became internalized into the tubular epithelial cytoplasm. Since dead E. coli are unlikely to invade the cells, their intracytoplasmic localization is a result of tubular epithelial phagocytosis. Similar microinjections of dead E. coli together with rat erythrocytes revealed a preferential phagocytosis of dead E. coli. Examination of the microinjected nephron with dead E. coli 48 hours after also demonstrated a development of microscopic interstitial nephritis surrounding the microinjected tubule. In conclusion, the renal tubular epithelia of the proximal and distal segments of rat nephron have phagocytic potential for E. coli which are further capable of inducing an inflammatory reaction around the microinjected tubule.     

Contrasting expression patterns of three members of the myc family of protooncogenes in the developing and adult mouse kidney

The myc family of protooncogenes encode similar but distinct nuclear proteins. Since N-myc, c-myc, and L-myc have been found to be expressed in the newborn kidney, we studied their expression during murine kidney development. By organ culture studies and in situ hybridization of tissue sections, we found that each of the three members of the myc gene family shows a remarkably distinct expression pattern during kidney development. It is known that mesenchymal stem cells of the embryonic kidney convert into epithelium if properly induced. We demonstrate the N-myc expression increases during the first 24 h of in vitro culture as an early response to induction. Moreover, the upregulation was transient and expression levels were already low during the first stages of overt epithelial cell polarization. In contrast, neither c-myc nor L-myc were upregulated by induction of epithelial differentiation. c-myc was expressed in the uninduced mesenchyme but subsequently became restricted to the newly formed epithelium and was not expressed in the surrounding loose mesenchyme. At onset of terminal differentiation c-myc expression was turned off also from the epithelial tubules. We conclude that N-myc is a marker for induction and early epithelial differentiation states. That the undifferentiated mesenchyme, unlike stromal cells of later developmental stages, express c-myc demonstrates that the undifferentiated mesenchymal stem cells are distinct from the stromal cells. The most astonishing finding, however, was the high level of L-myc mRNA in the ureter, ureter-derived renal pelvis, papilla, and collecting ducts. In the ureter, expression increased, rather than decreased, with advancing maturation and was highest in adult tissue. Our results suggest that each of the three members of the myc gene family are involved in quite disparate differentiation processes, even within one tissue.