Adult Renal Progenitor Cells Research Articles

#1805 Renal progenitor cells isolated from urine of IgA nephropathy patients formed renal spheroids that can recapitulate the typical IgA1 deposition

Abstract Background and Aims Recent developments in stem cell biology have made it possible to create spheroids and kidney organoids from human and mouse pluripotent stem cells and renal stem cells. Spheroids are a suitable in vitro model for studies of drug studies and can transform into organoids whose rapidly developing field has proven useful in simulating nephrotoxicity, kidney disorders, and kidney development. However, to date in all studies the generation of organoids and tubuloids required the use of sophisticated and complex differentiation protocols involving external administration of several growth factors and needs compliance with a precise and specific timescale. Here we showed for the first time that human adult renal progenitor cells (ARPCs) can be isolated from urines of healthy subjects (HS) or IgA Nephropathy (IgAN) patients and can form spheroids generating very long tubule-like structures naturally, without the external use of chemokines or growth factors to artificially induce the process and recapitulating the IgA1 deposition that is the typical characteristic of the disease. Method Spheroids and tubule-like structures were characterized by whole-mounting and frozen section immunofluorescence (IF) assays and by Flow Cytometry Analysis using CD133, NanoG, Oct3/4, GATA-3, SSEA4, CD249, Aminopepidase N, ZO-1, Uromodulin, and Lotus Tetragonolobus Lectin (LTL) antibodies. ELISA was used to determine the renin secretion and CAM assays to determine ARPCs and ARPC-derived Spheroids angiogenic properties. PKH-26 labeling was used for their in vitro and in vivo tracking. Serum of HS and IgAN patients in culture with spheroids was used to recapitulate the disease and IgA1 deposits were measured by IF. Results ARPC-derived spheroids reflected the renal progenitor properties and recapitulated organoids generating very long tubule-like structures that expressed structural and functional renal tubule markers such as CD249, Aminopepidase N, ZO-1, Uromodulin, and LTL and in some cases shared many structural similarities with some regions of nephrons, such as the distal convoluted tubule, the loop of Henle and proximal convoluted tubules. Moreover, ARPC-derived spheroids secreted elevated levels of renin and, as the cellular counterpart, showed angiogenic properties. To study the proof of concept that ARPC-derived spheroids can be used to establish in vitro models recapitulating renal diseases, we generated IgAN patient-specific renal spheroids using the ARPC mixed cell population method. ARPC-derived spheroids of IgAN patients showed deposits of IgA1 already after four days of culture with patient serum (p = 0.004) and, with a different pattern, after 8 and 15 days (p = 0.0029 and p = 0.0012, respectively). The IgA1 deposition resembled that in the glomeruli of patients diagnosticated with IgAN. In contrast, no positive signal was detected in either IgAN spheroids in culture with FBS, or with inactivated IgAN serum, as well as in spheroids derived from urine of HS in culture with IgAN serum. Conclusion The ability of ARPCs to form spheroids and differentiate into tubular-like structures without the need for external chemokines or growth factors is a significant advancement in the field, even more considering that these cells can be isolated from the urine of patients. These findings open up new possibilities for the regenerative medicine in studying kidney development, disease mechanisms, and regenerative therapy development for renal disorders.

#2723 10-hydroxy-trans-2-decenoic and nonanoic acid delayed senescence in adult renal progenitor cells and increased the anti-aging Klotho protein secretion

Abstract Background and Aims Recent investigations have elucidated the complex system involving human adult renal stem/progenitor cells (ARPCs) and their pivotal role in maintaining homeostasis and in facilitating regenerative processes within the adult kidneys. CD133+/CD24+ ARPCs can repair several kinds of renal damage but their reparative properties can be compromised by the cell senescence, contributing to renal aging and impaired damage repair. The functional marker CD133 inversely correlates with cellular senescence and plays a crucial role in the ARPC regulation. Decreased protective factors, hypoxia, and microenvironmental stress drive increasingly cell senescence, disordered inflammation and renal fibrosis. The resulting fibrosis, senescence, and microvascular rarefaction exacerbate damage and promote chronic kidney disease (CKD) progression. In this study, we explore the impact of natural molecular compounds and drugs on slowing down and delaying ARPC senescence. Method ARPCs were treated with the following compounds for 48 hours: nonanoic acid (1 mM), 3-decenoic acid (1 mM), 10-hydroxy-trans-2-decenoic acid (10-HDA, 25uM), and Valproic acid (VPA, 1 mM), either individually or in combination. Lipid microvesicles were used to transmit the bioactive molecules. FACS was used to determine the CD133 levels. the β-galactosidase assays were used to determine cell senescence status. Western blots were performed to assess p16, p53 and p21 markers. Results Cytofluorimetry results demonstrated an increase in the expression of CD133, the inverse cell senescence marker, both following stimulation with decenoic (fold change = 1.8, p = 0.0053) and nonanoic acid (FC = 1.3, p = 0.0056), and with VPA (FC 2, p = 0.0036). Simultaneously, the number of senescent cells decreased, as confirmed by the β-galactosidase assay (FC = 0.13, p = 0.0005). The treatment with decenoic and nonanoic acid also restored high levels of the lncRNA HOTAIR (FC = 14.6 for nonanoic acid and 12.7 for 10-HDA; p = 0.05), that in turn promoted the secretion of the α-Klotho anti-aging protein (FC = 3.1; p = 0.01). Additionally, the western blot analysis revealed that the 10-HDA decreased the expression of cell senescence markers, p16 (FC = 1.6, p = 0.05), p53 (FC = 1.8, p = 0.05) and p21 (FC= 3.7, p = 0.05). Moreover, the nonanoic acid reduced the expression of p16 (FC = 1.8, p = 0.05), p53 (FC = 2, p = 0.05) and p21 (FC= 2.6, p = 0.0007). Also, their combination contributes to decrease p16 (FC = 1.9, p = 0.05), p53 (FC = 1.3, p = 0.05) and p21 (FC= 2.5, p = 0.05). Conclusion This study identifies specific molecules capable of reducing or delaying ARPC senescence. In particular, the two natural compounds 10-HDA and nonanoic acid can also enhance the capacity of renal progenitors to secrete the α-Klotho protein whose decline prevention by several mechanisms can attenuate renal injuries, retard CKD progression, ameliorate extrarenal complications, and improve renal function. These findings present potential therapeutic avenues for delaying or mitigating the progression of CKD.

Human Adult Renal Progenitor Cells Prevent Cisplatin-Nephrotoxicity by Inducing CYP1B1 Overexpression and miR-27b-3p Down-Regulation through Extracellular Vesicles.

Cisplatin is one of the most effective chemotherapeutic agents strongly associated with nephrotoxicity. Tubular adult renal progenitor cells (tARPC) can regenerate functional tubules and participate in the repair processes after cisplatin exposition. This study investigated the molecular mechanisms underlying the protective effect of tARPC on renal epithelium during cisplatin nephrotoxicity. By performing a whole-genome transcriptomic analysis, we found that tARPC, in presence of cisplatin, can strongly influence the gene expression of renal proximal tubular cell [RPTEC] by inducing overexpression of CYP1B1, a member of the cytochrome P450 superfamily capable of metabolizing cisplatin and of hypoxia/cancer-related lncRNAs as MIR210HG and LINC00511. Particularly, tARPC exerted renoprotection and regeneration effects via extracellular vesicles (EV) enriched with CYP1B1 and miR-27b-3p, a well-known CYP1B1 regulatory miRNA. The expression of CYP1B1 by tARPC was confirmed by analyzing biopsies of cisplatin-treated renal carcinoma patients that showed the colocalization of CYP1B1 with the tARPC marker CD133. CYP1B1 was also overexpressed in urinary EV purified from oncologic patients that presented nephrotoxicity episodes after cisplatin treatment. Interestingly CYP1B1 expression significantly correlated with creatinine and eGFR levels. Taken together, our results show that tARPC are able to counteract cisplatin-induced nephrotoxicity via CYP1B1 release through EV. These findings provide a promising therapeutic strategy for nephrotoxicity risk assessment that could be related to abundance of renal progenitors.

#4708 RENAL ORGANOID FROM URINE-DERIVED HUMAN ADULT RENAL PROGENITOR CELLS CAN SPONTANEOUSLY GENERATE LONG RENAL TUBULES

Abstract Background and Aims Organoids are self-organizing 3D aggregations of cells that represent the structure and function of organs. Kidney organoids have the potential to advance the field of nephrology by providing a tool for the study of human kidney development and disease, by providing a tool for in vitro drug screening, and ultimately, for regenerative therapy. They can be derived from embryonic stem cells or induced pluripotent stem cells. We aimed to explore for the first time, the potentiality of human adult renal progenitor cells (ARPCs), isolated from urine of healthy subjects and patients, to generate spheroids and organoids for regenerative purpose. Method ARPCs were isolated from urine by immunolabeling. Optical microscopy, immunofluorescence experiments and cytofluorimetric analysis were used to characterize spheroids and organoids. CD133, NANOG, SSA4, OCT 3-4 and GATA-3, SOX2 stemness marker antibodies were used to study spheroids and CD13, Lotus tetragonolobus lectin, ZO-1, uromodulin, and aminopeptidase antibodies were used to investigate renal tubular markers. PKH26 was used to study cell migration from organoids to tubules. Real-time PCR was used to generate gene expression data of aquaporin profile in renal tubules. Chick embryo chorioallantoic membrane (CAM) assays were used as in vivo model for angiogenesis. Results We isolated ARPCs from urine of healthy subjects or CKD patients. Usually, organoids were generated using complicate induction protocols with several chemical factors. However, for the first time we generated renal spheroids and organoids under 3D culture conditions without any stimulation, such as chemokines or growth factors, starting from ARPCs isolated from subject urine. We showed that the spheroids express high levels of stem cell markers as CD133 and NANOG, functional and constitutional marker of ARPCs, and high levels of stem cell markers that normally are low or not at all expressed in ARPCs and are typical of embryonic stem cells: GATA-3, SSEA4, and Sox2. Moreover, the spheroids were able to induce angiogenesis when implanted in chick embryo chorioallantoic membrane. The urine-derived CD133+ organoids can spontaneously generate long renal tubules in 6–12 days, as showed by PKH26 tagging. The renal tubules were positive for the proximal tubule cell markers CD13 (aminopeptidase N), Lotus tetragonolobus lectin and ZO-1. Moreover, renal tubules expressed the aminopeptidase A (CD249) that is normally present in proximal tubules and glomeruli of the kidneys and that catalyzed the conversion of the Angiotensin II in Angiotensin III witch is important in the local regulation of blood pressure. Also uromodulin was expressed by tubules. It is normally present in epithelial cells of the thick ascending limb of Henle's loop, and after proteolytical cleavage, is secreted into urine. In addition, the renal tubules express several genes typical of renal tubules as AQP1 channel protein that facilitates the flow of water molecules into the cells of the proximal tubule and descending limb of the loop of Henle; γGlut enzyme used for the transport of amino acids across cell membranes; Na/H exchanger 1 (SLC9A1), transporter involved in the regulation of pH homeostasis, cell migration and cell volume; Na/Gluc-1 co-transporter SGLUT1, which promotes the passage of glucose across the membrane in renal tubular cells. In some cases, tubule structures were similar to that found in nephrons, including proximal convoluted tubule, Loop of Henle, Distal Convoluted tubule and collecting duct, as shown by optical microscopy. Conclusion For the first time we demonstrated that kidney organoids can be generated starting by urine-derived human adult renal progenitor cells from patients and for the first time we showed that these organoids can generate, spontaneously, without specific stimulation, long portions of renal tubules. These results open new perspectives in the field of the regenerative medicine as ARPCs can be isolated from urine of patients and used to generate spheroids for the study of human kidney development and disease, for in vitro drug screening, and ultimately, for regenerative therapy.

The Long Non-coding RNA HOTAIR Controls the Self-renewal, Cell Senescence, and Secretion of Anti-aging Protein α-Klotho in Human Adult Renal Progenitor Cells.

The long non-coding RNAs (lncRNA) play an important role in several biological processes, including some renal diseases. Nevertheless, little is known about lncRNA that are expressed in the healthy kidneys and involved in renal cell homeostasis and development, and even less is known about lncRNA involved in the maintenance of human adult renal stem/progenitor cells (ARPCs) that have been shown to be very important for renal homeostasis and repair processes. Through a whole-genome transcriptome screening, we found that the HOTAIR lncRNA is highly expressed in renal progenitors and potentially involved in cell cycle and senescence biological processes. By CRISPR/Cas9 genome editing, we generated HOTAIR knockout ARPC lines and established a key role of this lncRNA in ARPC self-renewal properties by sustaining their proliferative capacity and limiting the apoptotic process. Intriguingly, the HOTAIR knockout led to the ARPC senescence and to a significant decrease in the CD133 stem cell marker expression which is an inverse marker of ARPC senescence and can regulate renal tubular repair after the damage. Furthermore, we found that ARPCs expressed high levels of the α-Klotho anti-aging protein and especially 2.6-fold higher levels compared to that secreted by renal proximal tubular cells (RPTECs). Finally, we showed that HOTAIR exerts its function through the epigenetic silencing of the cell cycle inhibitor p15 inducing the trimethylation of the histone H3K27. Altogether, these results shed new light on the mechanisms of regulation of these important renal cells and may support the future development of precision therapies for kidney diseases.

FC023: Human Adult Renal Progenitor Cells Secrete in the Kidney Very High Levels of the Anti-Ageing Protein Klotho Sustained by the Long No-Coding RNA Hotair

Abstract BACKGROUND AND AIMS The complex system of human adult renal stem/progenitor cells (ARPCs) and their crucial role in the renewal of epithelial renal cells and regenerative processes in the adult kidneys have been recently discovered. In the human kidney, CD133+/CD24+ ARPCs are present within different segments of the nephron as a population with epithelial expansion, self-renewal and differentiation capabilities. Understanding the factors that regulate the ARPCs behaviour is fundamental to completely take advantage of their potential and to develop treatments for renal injury. Recent studies showed that long no-coding RNA (lncRNAs) are essential to establish developmental patterning and maintain the stem cell pluripotency network, further underscoring their important role in stem cell biology/technology and cellular reprogramming. lncRNAs can be dysregulated in different types of diseases and can also modulate the cellular senescence processes. For the first time, we studied their function in ARPCs showing that they express high levels of a particular lncRNA, HOTAIR, influencing cell senescence. METHOD Whole-genome lncRNA expression was performed by Agilent microarray. lncRNA expression was validated by real-time PCR. CRISPR/Cas9 system has been used to knock-down HOTAIR lncRNA. SA-β-Gal experiments were used to evaluate cellular senescence in normal ARPCs and ARPCs knock-out for HOTAIR. By ELISA, it was evaluated the expression of secreted anti-ageing protein Klotho. Fluorescence-activated cell sorting (FACS) was applied to measure CD133 and protein p15 expression in normal and transfected cells. Chromatin immunoprecipitation assay (ChIP) was used to evaluate H3K27me3 in the promoter of p15. RESULTS We studied the lncRNA profile of renal proximal tubular cells (RPTEC) and tubular ARPCs. We found 611 lncRNAs specifically expressed in ARPCs compared with RPTECs (Fold Change > 2; FDR < 0.05). Among the most significantly modulated lncRNAs, we demonstrated that the lncRNA HOX Transcript Antisense RNA (HOTAIR) is highly expressed in ARPCs (Fold change = 15; P < 0.001). To study the functions related to the ARPCs properties, knock-out cell lines for HOTAIR were created by the CRISPR/Cas9 technique. The silenced lines for HOTAIR immediately assumed a senescent phenotype confirmed by the beta-galactosidase assay and decreased proliferation (proliferation rate of ˂60%, P < 0.001). Moreover, we found that the constitutional, functional and inverse-senescence marker CD133+ was downregulated in knock-out cells (Fold change = 15; P < 0.01). We then studied the secreted levels of the anti-ageing protein α-Klotho which is essential to the maintenance of normal renal function and has potential clinical applications in the diagnosis of acute renal failure (AKI) and chronic kidney disease (CKD). We discovered that ARPCs produce α-Klotho at much higher levels compared to RPTEC, at a mean concentration of 200 pg/mL versus 33 pg/mL (Fold increase = 6.6; P < 0.001). Interestingly, following the HOTAIR knock-out, the levels of α-Klotho secreted by the ARPCs dropped sharply, with a 4-fold decrease (P < .001). In addition, we found that HOTAIR was responsible for the ARPC self-renewal through the epigenetic induction of the cell cycle inhibitor p15 through the trimethylation of histone H3K27 associated with the promoter region of p15. In ARPCs knock-out for HOTAIR p15 increased significantly (Fold increase = 3; P < 0.005) and the H3K27me3 in the promoter of p15 was less enriched (Fold decrease = 1.5; P < 0.05), leading to the increase of the cell cycle inhibitor and cell senescence. CONCLUSION These data demonstrated that HOTAIR regulates the self-renewal capacity of ARPCs and prevents them from becoming senescent in the short term. Moreover, HOTAIR influences ARPC ability to secrete high levels of α-Klotho, and those renal progenitors, through α-Klotho secretion, are able to influence its levels in surrounding tissues and to modulate, therefore, kidney ageing.

Retinoic Acid Benefits Glomerular Organotypic Differentiation from Adult Renal Progenitor Cells In Vitro.

When in certain culture conditions, organotypic cultures are able to mimic developmental stages of an organ, generating higher-order structures containing functional subunits and progenitor niches. Despite the major advances in the area, researchers have not been able to fully recapitulate the complexity of kidney tissue. Pluripotent stem cells are extensively used in the field, but very few studies make use of adult stem cells. Herein, we describe a simple and feasible method for achieving glomerular epithelial differentiation on an organotypic model comprising human renal progenitor cells from adult kidney (hRPCs). Their glomerular differentiative potential was studied using retinoic acid (RA), a fundamental molecule for intermediate mesoderm induction on early embryogenesis. Immunofluorescence, specific cell surface markers expression and gene expression analysis confirm the glomerular differentiative potential of RA in a short-term culture. We also compared the potential of RA with a potent WNT agonist, CHIR99021, on the differentiative capacity of hRPCs. Gene expression and immunofluorescence analysis confirmed that hRPCs are more sensitive to RA stimulation when compared to CHIR9901. Endothelial cells were also included on the spheroids, resulting in a higher organizational level. The assembly potential of these cells and their selective stimulation will give new insights on adult organotypic cell culture studies and will hopefully guide more works in this important area of research.

Current State of Renal Regenerative Therapies.

The worldwide increase in the number of patients with end-stage renal disease leads to a growing waiting list for kidney transplantation resulting from the scarcity of kidney donors. Therefore, alternative treatment options for patients with end-stage renal disease are being sought. In vitro differentiation of stem cells into renal tissue is a promising approach to repair nonfunctional kidney tissue. Impressive headway has been made in the use of stem cells with the use of adult renal progenitor cells, embryonic stem cells, and induced pluripotent stem cells for the development toward primitive kidney structures. Currently, efforts are directed at improving long-term maintenance and stability of the cells. This review aims to provide a comprehensive overview of the cell sources used for the generation of kidney cells and strategies used for transplantation in in vivo models. Furthermore, it provides a perspective on stability and safety during future clinical application of in vitro generated kidney cells.

Inhibin-A and Decorin Secreted by Human Adult Renal Stem/Progenitor Cells Through the TLR2 Engagement Induce Renal Tubular Cell Regeneration

Acute kidney injury (AKI) is a public health problem worldwide. Several therapeutic strategies have been made to accelerate recovery and improve renal survival. Recent studies have shown that human adult renal progenitor cells (ARPCs) participate in kidney repair processes, and may be used as a possible treatment to promote regeneration in acute kidney injury. Here, we show that human tubular ARPCs (tARPCs) protect physically injured or chemically damaged renal proximal tubular epithelial cells (RPTECs) by preventing cisplatin-induced apoptosis and enhancing proliferation of survived cells. tARPCs without toll-like receptor 2 (TLR2) expression or TLR2 blocking completely abrogated this regenerative effect. Only tARPCs, and not glomerular ARPCs, were able to induce tubular cell regeneration process and it occurred only after damage detection. Moreover, we have found that ARPCs secreted inhibin-A and decorin following the RPTEC damage and that these secreted factors were directly involved in cell regeneration process. Polysaccharide synthetic vesicles containing these molecules were constructed and co-cultured with cisplatin damaged RPTECs. These synthetic vesicles were not only incorporated into the cells, but they were also able to induce a substantial increase in cell number and viability. The findings of this study increase the knowledge of renal repair processes and may be the first step in the development of new specific therapeutic strategies for renal repair.

Correction: miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

Adult renal progenitor cells (ARPCs) were recently identified in the cortex of the renal parenchyma and it was demonstrated that they were positive for PAX2, CD133, CD24 and exhibited multipotent differentiation ability. Recent studies on stem cells indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Distinct sets of miRNAs are specifically expressed in pluripotent stem cells but not in adult tissues, suggesting a role for miRNAs in stem cell self-renewal. We compared miRNA expression profiles of ARPCs with that of mesenchymal stem cells (MSCs) and renal proximal tubular cells (RPTECs) finding distinct sets of miRNAs that were specifically expressed in ARPCs. In particular, miR-1915 and miR-1225-5p regulated the expression of important markers of renal progenitors, such as CD133 and PAX2, and important genes involved in the repair mechanisms of ARPCs, such as TLR2. We demonstrated that the expression of both the renal stem cell markers CD133 and PAX2 depends on lower miR-1915 levels and that the increase of miR-1915 levels improved capacity of ARPCs to differentiate into adipocyte-like and epithelial-like cells. Finally, we found that the low levels of miR-1225-5p were responsible for high TLR2 expression in ARPCs. Therefore, together, miR-1915 and miR-1225-5p seem to regulate important traits of renal progenitors: the stemness and the repair capacity.

CTR2 Identifies a Population of Cancer Cells with Stem Cell-like Features in Patients with Clear Cell Renal Cell Carcinoma

In clear cell renal cell carcinoma tissue samples we identified and characterized a population of renal cell carcinoma derived CD133+/CD24+ cancer cells. We studied differences between these cells and their nonneoplastic counterpart, tubular adult renal progenitor cells. CD133+/CD24+ renal cell carcinoma derived cells were isolated from 40 patients. The mesenchymal phenotype and stemness proteomic profile of these renal cell carcinoma derived cells were characterized. Colony forming efficiency and self-renewal ability were tested by limiting dilution. Tumorigenic properties were evaluated invitro by soft agar assay. The angiogenic response was evaluated invivo by the chorioallantoic membrane angiogenic assay. Microarray analysis was performed on 6 tubular adult renal progenitor cell and 6 renal cell carcinoma derived cell clones. Membrane protein expression was evaluated by flow cytometry and immunofluorescence staining. CD133+/CD24+ cells were isolated from normal and tumor kidney tissue. Fluorescence activated cell sorting revealed that renal cell carcinoma derived cells did not express mesenchymal stem cell markers. CD133+/CD24+ tumor cells were more undifferentiated than tubular adult renal progenitor cells. Renal cell carcinoma derived cells were clonigenic and could differentiate into adipocytes, epithelial and osteogenic cells. They could also regenerate tumor cells invitro and induce angiogenesis invivo. Gene expression profile identified CTR2 as a membrane marker for this neoplastic population. CTR2 was involved in renal cell carcinoma derived cell cisplatin resistance. Our results indicate the presence of a CD133+/CD24+/CTR2+ cancer cell population in clear cell renal cell carcinoma. These cells have some stem cell-like features, including invitro self-maintenance and differentiating capabilities, and they can induce an angiogenic response invivo.

Mouse adult renal progenitor cells in combination with erythropoietin or suramin - a potential new strategy for the treatment of acute kidney injury

Experimental evidence has indicated a role of adult renal progenitor cells in kidney regeneration and a protective role of the kidney by erythropoietin (EPO) and suramin in animal models and in humans after acute kidney injury (AKI). Han and colleagues analyzed different therapeutic effects between mouse renal progenitor cells (MRPCs), MRPC/EPO, or MRPC/suramin on the regeneration and protection of renal function after AKI. Their results revealed that MRPCs in combination with EPO or suramin are able to attenuate renal damage and promote renal recovery after ischemia/reperfusion injury in a mouse model. The researchers concluded that the combined approach with MRPCs and EPO or suramin could be a new therapeutic strategy for AKI.

MiR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

Adult renal progenitor cells (ARPCs) were recently identified in the cortex of the renal parenchyma and it was demonstrated that they were positive for PAX2, CD133, CD24 and exhibited multipotent differentiation ability. Recent studies on stem cells indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Distinct sets of miRNAs are specifically expressed in pluripotent stem cells but not in adult tissues, suggesting a role for miRNAs in stem cell self-renewal. We compared miRNA expression profiles of ARPCs with that of mesenchymal stem cells (MSCs) and renal proximal tubular cells (RPTECs) finding distinct sets of miRNAs that were specifically expressed in ARPCs. In particular, miR-1915 and miR-1225-5p regulated the expression of important markers of renal progenitors, such as CD133 and PAX2, and important genes involved in the repair mechanisms of ARPCs, such as TLR2. We demonstrated that the expression of both the renal stem cell markers CD133 and PAX2 depends on lower miR-1915 levels and that the increase of miR-1915 levels improved capacity of ARPCs to differentiate into adipocyte-like and epithelial-like cells. Finally, we found that the low levels of miR-1225-5p were responsible for high TLR2 expression in ARPCs. Therefore, together, miR-1915 and miR-1225-5p seem to regulate important traits of renal progenitors: the stemness and the repair capacity.

Improving outcomes of acute kidney injury using mouse renal progenitor cells alone or in combination with erythropoietin or suramin

IntroductionSo far, no effective therapy is available for acute kidney injury (AKI), a common and serious complication with high morbidity and mortality. Interest has recently been focused on the potential therapeutic effect of mouse adult renal progenitor cells (MRPC), erythropoietin (EPO) and suramin in the recovery of ischemia-induced AKI. The aim of the present study is to compare MRPC with MRPC/EPO or MRPC/suramin concomitantly in the treatment of a mouse model of ischemia/reperfusion (I/R) AKI.MethodsMRPC were isolated from adult C57BL/6-gfp mice. Male C57BL/6 mice (eight-weeks old, n = 72) were used for the I/R AKI model. Serum creatinine (Cr), blood urea nitrogen (BUN) and renal histology were detected in MRPC-, MRPC/EPO-, MRPC/suramin- and PBS-treated I/R AKI mice. E-cadherin, CD34 and GFP protein expression was assessed by immunohistochemical assay.ResultsMRPC exhibited characteristics consistent with renal stem cells. The features of MRPC were manifested by Pax-2, Oct-4, vimentin, α-smooth muscle actin positive, and E-cadherin negative, distinguished from mesenchymal stem cells (MSC) by expression of CD34 and Sca-1. The plasticity of MRPC was shown by the ability to differentiate into osteoblasts and lipocytes in vitro. Injection of MRPC, especially MRPC/EPO and MRPC/suramin in I/R AKI mice attenuated renal damage with a decrease of the necrotic injury, peak plasma Cr and BUN. Furthermore, seven days after the injury, MRPC/EPO or MRPC/suramin formed more CD34+ and E-cadherin+ cells than MRPC alone.ConclusionsThese results suggest that MRPC, in particular MRPC/EPO or MRPC/suramin, promote renal repair after injury and may be a promising therapeutic strategy.

BuMPy road of delayed graft function

BuMPy road of delayed graft function

BMP-2 induces a profibrotic phenotype in adult renal progenitor cells through Nox4 activation

Adult renal progenitor cells (ARPCs) isolated from the human kidney may contribute to repair featuring acute kidney injury (AKI). Bone morphogenetic proteins (BMPs) regulate differentiation, modeling, and regeneration processes in several tissues. The aim of this study was to evaluate the biological actions of BMP-2 in ARPCs in vitro and in vivo. BMP-2 was expressed in ARPCs of normal adult human kidneys, and it was upregulated in vivo after delayed graft function (DGF) of renal transplantation, a condition of AKI. ARPCs expressed BMP receptors, suggesting their potential responsiveness to BMP-2. Incubation of ARPCs with this growth factor enhanced reactive oxygen species (ROS) production, NADPH oxidase activity, and Nox4 protein expression. In vivo, Nox4 was localized in BMP-2-expressing CD133+ cells at the tubular level after DGF. BMP-2 incubation induced α-smooth muscle actin (SMA), collagen I, and fibronectin protein expression in ARPCs. Moreover, α-SMA colocalized with CD133 in vivo after DGF. The oxidative stimulus (H(2)O(2)) induced α-SMA expression in ARPCs, while the antioxidant N-acetyl-cysteine inhibited BMP-2-induced α-SMA expression. Nox4 silencing abolished BMP-2-induced NADPH oxidase activation and myofibroblastic induction. We showed that 1) ARPCs express BMP-2, 2) this expression is increased in a model of AKI; 3) BMP-2 may induce the commitment of ARPCs toward a myofibroblastic phenotype in vitro and in vivo; and 4) this profibrotic effect is mediated by Nox4 activation. Our findings suggest a novel mechanism linking AKI with progressive renal damage.