Renal Progenitors Derived from Urine for Personalized Diagnosis of Kidney Diseases.

BACKGROUND: Chronic kidney disease affects 10% of the world population, and it is associated with progression to end-stage kidney disease and increased morbidity and mortality. The advent of multi-omics technologies has expanded our knowledge on the complexity of kidney diseases, revealing their frequent genetic etiology, particularly in children and young subjects. Genetic heterogeneity and drug screening require patient-derived disease models to establish a correct diagnosis and evaluate new potential treatments and outcomes. SUMMARY: Patient-derived renal progenitors can be isolated from urine to set up proper disease modeling. This strategy allows to make diagnosis of genetic kidney disease in patients carrying unknown significance variants or uncover variants missed from peripheral blood analysis. Furthermore, urinary-derived tubuloids obtained from renal progenitors of patients appear to be potentially valuable for modeling kidney diseases to test ex vivo treatment efficacy or to develop new therapeutic approaches. Finally, renal progenitors derived from urine can provide insights into acute kidney injury and predict kidney function recovery and outcome. KEY MESSAGES: Renal progenitors derived from urine are a promising new noninvasive and easy-to-handle tool, which improves the rate of diagnosis and the therapeutic choice, paving the way toward a personalized healthcare.

Polyploid tubular cells initiate a TGF-ß1 controlled loop that sustains polyploidization and fibrosis after acute kidney injury.

Polyploidization of tubular cells (TC) is triggered by acute kidney injury (AKI) to allow survival in the early phase after AKI, but in the long run promotes fibrosis and AKI-chronic kidney disease (CKD) transition. The molecular mechanism governing the link between polyploid TC and kidney fibrosis remains to be clarified. In this study, we demonstrate that immediately after AKI, expression of cell cycle markers mostly identifies a population of DNA-damaged polyploid TC. Using transgenic mouse models and single-cell RNA sequencing we show that, unlike diploid TC, polyploid TC accumulate DNA damage and survive, eventually resting in the G1 phase of the cell cycle. In vivo and in vitro single-cell RNA sequencing along with sorting of polyploid TC shows that these cells acquire a profibrotic phenotype culminating in transforming growth factor (TGF)-ß1 expression and that TGF-ß1 directly promotes polyploidization. This demonstrates that TC polyploidization is a self-sustained mechanism. Interactome analysis by single-cell RNA sequencing revealed that TGF-ß1 signaling fosters a reciprocal activation loop among polyploid TC, macrophages, and fibroblasts to sustain kidney fibrosis and promote CKD progression. Collectively, this study contributes to the ongoing revision of the paradigm of kidney tubule response to AKI, supporting the existence of a tubulointerstitial cross talk mediated by TGF-ß1 signaling produced by polyploid TC following DNA damage.NEW & NOTEWORTHY Polyploidization in tubular epithelial cells has been neglected until recently. Here, we showed that polyploidization is a self-sustained mechanism that plays an important role during chronic kidney disease development, proving the existence of a cross talk between infiltrating cells and polyploid tubular cells. This study contributes to the ongoing revision of kidney adaptation to injury, posing polyploid tubular cells at the center of the process.

Role of Sex Hormones in Prevalent Kidney Diseases.

Chronic kidney disease (CKD) is a constantly growing global health burden, with more than 840 million people affected worldwide. CKD presents sex disparities in the pathophysiology of the disease, as well as in the epidemiology, clinical manifestations, and disease progression. Overall, while CKD is more frequent in females, males have a higher risk to progress to end-stage kidney disease. In recent years, numerous studies have highlighted the role of sex hormones in the health and diseases of several organs, including the kidney. In this review, we present a clinical overview of the sex-differences in CKD and a selection of prominent kidney diseases causing CKD: lupus nephritis, diabetic kidney disease, IgA nephropathy, and autosomal dominant polycystic kidney disease. We report clinical and experimental findings on the role of sex hormones in the development of the disease and its progression to end-stage kidney disease.

Crystal Clots as Therapeutic Target in Cholesterol Crystal Embolism.

RATIONALE: Cholesterol crystal embolism can be a life-threatening complication of advanced atherosclerosis. Pathophysiology and molecular targets for treatment are largely unknown. OBJECTIVE: We aimed to develop a new animal model of cholesterol crystal embolism to dissect the molecular mechanisms of cholesterol crystal (CC)-driven arterial occlusion, tissue infarction, and organ failure. METHODS AND RESULTS: C57BL/6J mice were injected with CC into the left kidney artery. Primary end point was glomerular filtration rate (GFR). CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR, followed by GFR recovery within 4 weeks, that is, acute kidney disease. In contrast, the extent of kidney infarction was more variable. Blocking necroptosis using mixed lineage kinase domain-like deficient mice or necrostatin-1s treatment protected from kidney infarction but not from GFR loss because arterial obstructions persisted, identifying crystal clots as a primary target to prevent organ failure. CC involved platelets, neutrophils, fibrin, and extracellular DNA. Neutrophil depletion or inhibition of the release of neutrophil extracellular traps had little effects, but platelet P2Y12 receptor antagonism with clopidogrel, fibrinolysis with urokinase, or DNA digestion with recombinant DNase I all prevented arterial occlusions, GFR loss, and kidney infarction. The window-of-opportunity was <3 hours after CC injection. However, combining Nec-1s (necrostatin-1s) prophylaxis given 1 hour before and DNase I 3 hours after CC injection completely prevented kidney failure and infarcts. In vitro, CC did not directly induce plasmatic coagulation but induced neutrophil extracellular trap formation and DNA release mainly from kidney endothelial cells, neutrophils, and few from platelets. CC induced ATP release from aggregating platelets, which increased fibrin formation in a DNase-dependent manner. CONCLUSIONS: CC embolism causes arterial obstructions and organ failure via the formation of crystal clots with fibrin, platelets, and extracellular DNA as critical components. Therefore, our model enables to unravel the pathogenesis of the CC embolism syndrome as a basis for both prophylaxis and targeted therapy.

Radiosensitizing Effect of Trabectedin on Human Soft Tissue Sarcoma Cells.

Trabectedin is used for the treatment of advanced soft tissue sarcomas (STSs). In this study, we evaluated if trabectedin could enhance the efficacy of irradiation (IR) by increasing the intrinsic cell radiosensitivity and modulating tumor micro-environment in fibrosarcoma (HS 93.T), leiomyosarcoma (HS5.T), liposarcoma (SW872), and rhabdomyosarcoma (RD) cell lines. A significant reduction in cell surviving fraction (SF) following trabectedin + IR compared to IR alone was observed in liposarcoma and leiomyosarcoma (enhancement ratio at 50%, ER50: 1.45 and 2.35, respectively), whereas an additive effect was shown in rhabdomyosarcoma and fibrosarcoma. Invasive cells' fraction significantly decreased following trabectedin ± IR compared to IR alone. Differences in cell cycle distribution were observed in leiomyosarcoma and rhabdomyosarcoma treated with trabectedin + IR. In all STS lines, trabectedin + IR resulted in a significantly higher number of γ-H2AX (histone H2AX) foci 30 min compared to the control, trabectedin, or IR alone. Expression of ATM, RAD50, Ang-2, VEGF, and PD-L1 was not significantly altered following trabectedin + IR. In conclusion, trabectedin radiosensitizes STS cells by affecting SF (particularly in leiomyosarcoma and liposarcoma), invasiveness, cell cycle distribution, and γ-H2AX foci formation. Conversely, no synergistic effect was observed on DNA damage repair, neoangiogenesis, and immune system.

CXCL12 blockade preferentially regenerates lost podocytes in cortical nephrons by targeting an intrinsic podocyte-progenitor feedback mechanism.

Insufficient podocyte regeneration after injury is a central pathomechanism of glomerulosclerosis and chronic kidney disease. Podocytes constitutively secrete the chemokine CXCL12, which is known to regulate homing and activation of stem cells; hence we hypothesized a similar effect of CXCL12 on podocyte progenitors. CXCL12 blockade increased podocyte numbers and attenuated proteinuria in mice with Adriamycin-induced nephropathy. Similar studies in lineage-tracing mice revealed enhanced de novo podocyte formation from parietal epithelial cells in the setting of CXCL12 blockade. Super-resolution microscopy documented full integration of these progenitor-derived podocytes into the glomerular filtration barrier, interdigitating with tertiary foot processes of neighboring podocytes. Quantitative 3D analysis revealed that conventional 2D analysis underestimated the numbers of progenitor-derived podocytes. The 3D analysis also demonstrated differences between juxtamedullary and cortical nephrons in both progenitor endowment and Adriamycin-induced podocyte loss, with more robust podocyte regeneration in cortical nephrons with CXCL12 blockade. Finally, we found that delayed CXCL12 inhibition still had protective effects. In vitro studies found that CXCL12 inhibition uncoupled Notch signaling in podocyte progenitors. These data suggest that CXCL12-driven podocyte-progenitor feedback maintains progenitor quiescence during homeostasis, but also limits their intrinsic capacity to regenerate lost podocytes, especially in cortical nephrons. CXCL12 inhibition could be an innovative therapeutic strategy in glomerular disorders.

Modeled microgravity unravels the roles of mechanical forces in renal progenitor cell physiology.

BACKGROUND: The glomerulus is a highly complex system, composed of different interdependent cell types that are subjected to various mechanical stimuli. These stimuli regulate multiple cellular functions, and changes in these functions may contribute to tissue damage and disease progression. To date, our understanding of the mechanobiology of glomerular cells is limited, with most research focused on the adaptive response of podocytes. However, it is crucial to recognize the interdependence between podocytes and parietal epithelial cells, in particular with the progenitor subset, as it plays a critical role in various manifestations of glomerular diseases. This highlights the necessity to implement the analysis of the effects of mechanical stress on renal progenitor cells. METHODS: Microgravity, modeled by Rotary Cell Culture System, has been employed as a system to investigate how renal progenitor cells respond to alterations in the mechanical cues within their microenvironment. Changes in cell phenotype, cytoskeleton organization, cell proliferation, cell adhesion and cell capacity for differentiation into podocytes were analyzed. RESULTS: In modeled microgravity conditions, renal progenitor cells showed altered cytoskeleton and focal adhesion organization associated with a reduction in cell proliferation, cell adhesion and spreading capacity. Moreover, mechanical forces appeared to be essential for renal progenitor differentiation into podocytes. Indeed, when renal progenitors were exposed to a differentiative agent in modeled microgravity conditions, it impaired the acquisition of a complex podocyte-like F-actin cytoskeleton and the expression of specific podocyte markers, such as nephrin and nestin. Importantly, the stabilization of the cytoskeleton with a calcineurin inhibitor, cyclosporine A, rescued the differentiation of renal progenitor cells into podocytes in modeled microgravity conditions. CONCLUSIONS: Alterations in the organization of the renal progenitor cytoskeleton due to unloading conditions negatively affect the regenerative capacity of these cells. These findings strengthen the concept that changes in mechanical cues can initiate a pathophysiological process in the glomerulus, not only altering podocyte actin cytoskeleton, but also extending the detrimental effect to the renal progenitor population. This underscores the significance of the cytoskeleton as a druggable target for kidney diseases.

A "Kidney-on-the-Chip" Model Composed of Primary Human Tubular, Endothelial, and White Blood Cells.

State-of-the-art cell culture systems may enlist a variety of features to push the significance of in vitro models beyond classical 2D single cell culture; among them are the 3D scaffolds of organic or artificial materials, multi-cell setups, and the use of primary cells as source materials. Obviously, operational complexity increases with each additional feature and feasibility, whereas reproducibility may suffer.We report a multicellular setup using primary human cells and the Mimetas scaffold that aims to increase pathophysiological significance of in vitro culture and simultaneously allows for relatively high-throughput and easy handling.

Preparation of Human Kidney Progenitor Cultures and Their Differentiation into Podocytes.

Kidney diseases are a global health concern. Modeling of kidney disease for translational research is often challenging because of species specificities or the postmitotic status of kidney epithelial cells that make primary cultures, for example podocytes. Here, we report a protocol for preparing primary cultures of podocytes based on the isolation and in vitro propagation of immature kidney progenitor cells subsequently differentiated into mature podocytes. This protocol can be useful for studying physiology and pathophysiology of human kidney progenitors and to obtain differentiated podocytes for modeling podocytopathies and other kidney disorders involving podocytes.

No NLRP3 inflammasome activity in kidney epithelial cells, not even when the NLRP3-A350V Muckle-Wells variant is expressed in podocytes of diabetic mice.

Background: The NLRP3 inflammasome integrates several danger signals into the activation of innate immunity and inflammation by secreting IL-1ß and IL-18. Most published data relate to the NLRP3 inflammasome in immune cells, but some reports claim similar roles in parenchymal, namely epithelial, cells. For example, podocytes, epithelial cells critical for the maintenance of kidney filtration, have been reported to express NLRP3 and to release IL-ß in diabetic kidney disease, contributing to filtration barrier dysfunction and kidney injury. We questioned this and hence performed independent verification experiments. Methods: We studied the expression of inflammasome components in human and mouse kidneys and human podocytes using single-cell transcriptome analysis. Human podocytes were exposed to NLRP3 inflammasome agonists in vitro and we induced diabetes in mice with a podocyte-specific expression of the Muckle-Wells variant of NLRP3, leading to overactivation of the Nlrp3 inflammasome (Nphs2Cre;Nlrp3A350V) versus wildtype controls. Phenotype analysis included deep learning-based glomerular and podocyte morphometry, tissue clearing, and STED microscopy of the glomerular filtration barrier. The Nlrp3 inflammasome was blocked by feeding ß-hydroxy-butyrate. Results: Single-cell transcriptome analysis did not support relevant NLRP3 expression in parenchymal cells of the kidney. The same applied to primary human podocytes in which NLRP3 agonists did not induce IL-1ß or IL-18 secretion. Diabetes induced identical glomerulomegaly in wildtype and Nphs2Cre;Nlrp3A350V mice but hyperfiltration-induced podocyte loss was attenuated and podocytes were larger in Nphs2Cre;Nlrp3A350V mice, an effect reversible with feeding the NLRP3 inflammasome antagonist ß-hydroxy-butyrate. Ultrastructural analysis of the slit diaphragm was genotype-independent hence albuminuria was identical. Conclusion: Podocytes express low amounts of the NLRP3 inflammasome, if at all, and do not produce IL-1ß and IL-18, not even upon introduction of the A350V Muckle-Wells NLRP3 variant and upon induction of podocyte stress. NLRP3-mediated glomerular inflammation is limited to immune cells.

The Pathology Lesion Patterns of Podocytopathies: How and why?

Podocytopathies are a group of proteinuric glomerular disorders driven by primary podocyte injury that are associated with a set of lesion patterns observed on kidney biopsy, i.e., minimal changes, focal segmental glomerulosclerosis, diffuse mesangial sclerosis and collapsing glomerulopathy. These unspecific lesion patterns have long been considered as independent disease entities. By contrast, recent evidence from genetics and experimental studies demonstrated that they represent signs of repeated injury and repair attempts. These ongoing processes depend on the type, length, and severity of podocyte injury, as well as on the ability of parietal epithelial cells to drive repair. In this review, we discuss the main pathology patterns of podocytopathies with a focus on the cellular and molecular response of podocytes and parietal epithelial cells.

Differentiation of crescent-forming kidney progenitor cells into podocytes attenuates severe glomerulonephritis in mice.

Crescentic glomerulonephritis is characterized by vascular necrosis and parietal epithelial cell hyperplasia in the space surrounding the glomerulus, resulting in the formation of crescents. Little is known about the molecular mechanisms driving this process. Inducing crescentic glomerulonephritis in two Pax2Cre reporter mouse models revealed that crescents derive from clonal expansion of single immature parietal epithelial cells. Preemptive and delayed histone deacetylase inhibition with panobinostat, a drug used to treat hematopoietic stem cell disorders, attenuated crescentic glomerulonephritis with recovery of kidney function in the two mouse models. Three-dimensional confocal microscopy and stimulated emission depletion superresolution imaging of mouse glomeruli showed that, in addition to exerting an anti-inflammatory and immunosuppressive effect, panobinostat induced differentiation of an immature hyperplastic parietal epithelial cell subset into podocytes, thereby restoring the glomerular filtration barrier. Single-cell RNA sequencing of human renal progenitor cells in vitro identified an immature stratifin-positive cell subset and revealed that expansion of this stratifin-expressing progenitor cell subset was associated with a poor outcome in human crescentic glomerulonephritis. Treatment of human parietal epithelial cells in vitro with panobinostat attenuated stratifin expression in renal progenitor cells, reduced their proliferation, and promoted their differentiation into podocytes. These results offer mechanistic insights into the formation of glomerular crescents and demonstrate that selective targeting of renal progenitor cells can attenuate crescent formation and the deterioration of kidney function in crescentic glomerulonephritis in mice.

Tubular cell polyploidy protects from lethal acute kidney injury but promotes consequent chronic kidney disease.

Acute kidney injury (AKI) is frequent, often fatal and, for lack of specific therapies, can leave survivors with chronic kidney disease (CKD). We characterize the distribution of tubular cells (TC) undergoing polyploidy along AKI by DNA content analysis and single cell RNA-sequencing. Furthermore, we study the functional roles of polyploidization using transgenic models and drug interventions. We identify YAP1-driven TC polyploidization outside the site of injury as a rapid way to sustain residual kidney function early during AKI. This survival mechanism comes at the cost of senescence of polyploid TC promoting interstitial fibrosis and CKD in AKI survivors. However, targeting TC polyploidization after the early AKI phase can prevent AKI-CKD transition without influencing AKI lethality. Senolytic treatment prevents CKD by blocking repeated TC polyploidization cycles. These results revise the current pathophysiological concept of how the kidney responds to acute injury and identify a novel druggable target to improve prognosis in AKI survivors.

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.

Molecular Mechanisms of Renal Progenitor Regulation: How Many Pieces in the Puzzle?

Kidneys of mice, rats and humans possess progenitors that maintain daily homeostasis and take part in endogenous regenerative processes following injury, owing to their capacity to proliferate and differentiate. In the glomerular and tubular compartments of the nephron, consistent studies demonstrated that well-characterized, distinct populations of progenitor cells, localized in the parietal epithelium of Bowman capsule and scattered in the proximal and distal tubules, could generate segment-specific cells in physiological conditions and following tissue injury. However, defective or abnormal regenerative responses of these progenitors can contribute to pathologic conditions. The molecular characteristics of renal progenitors have been extensively studied, revealing that numerous classical and evolutionarily conserved pathways, such as Notch or Wnt/ß-catenin, play a major role in cell regulation. Others, such as retinoic acid, renin-angiotensin-aldosterone system, TLR2 (Toll-like receptor 2) and leptin, are also important in this process. In this review, we summarize the plethora of molecular mechanisms directing renal progenitor responses during homeostasis and following kidney injury. Finally, we will explore how single-cell RNA sequencing could bring the characterization of renal progenitors to the next level, while knowing their molecular signature is gaining relevance in the clinic.

Particulate kidney extracellular matrix: bioactivity and proteomic analysis of a novel scaffold from porcine origin.

Decellularized matrices are attractive substrates, being able to retain growth factors and proteins present in the native tissue. Several biomaterials can be produced by processing these matrices. However, new substrates capable of being injected that reverse local kidney injuries are currently scarce. Herein, we hypothesized that the decellularized particulate kidney porcine ECM (pKECM) could support renal progenitor cell cultures for posterior implantation. Briefly, kidneys are cut into pieces, decellularized by immersion on detergent solutions, lyophilized and reduced into particles. Then, ECM particles are analyzed for nuclear material remaining by DNA quantification and histological examination, molecular conformation by FITR and structural morphology by SEM. Protein extraction is also optimized for posterior identification and quantification by mass spectrometry. The results obtained confirm the collagenous structure and composition of the ECM, the effective removal of nucleic material and the preservation of ECM proteins with great similarity to human kidneys. Human renal progenitor cells (hRPCs) are seeded in different ratios with pKECM, on 3D suspensions. The conducted assays for cell viability, proliferation and distribution over 7 days of culture suggest that these matrices as biocompatible and bioactive substrates for hRPCs. Also, by analyzing CD133 expression, an optimal ratio for specific phenotypic expression is revealed, demonstrating the potential of these substrates to modulate cellular behavior. The initial hypothesis of developing and characterizing a particulate ECM biomaterial as a consistent substrate for 3D cultures is successfully validated. The findings in this manuscript suggest these particles as valuable tools for regenerative nephrology by minimizing surgeries and locally reversing small injuries which can lead to chronic renal disfunction.

Co-cultures of renal progenitors and endothelial cells on kidney decellularized matrices replicate the renal tubular environment in vitro.

AIM: Herein we propose creating a bilayer tubular kidney in-vitro model. It is hypothesized that membranes composed of decellularized porcine kidney extracellular matrix are valid substitutes of the tubular basement membrane by mimicking the physiological relevance of the in vivo environment and disease phenotypes. METHODS: Extracellular matrix was obtained from decellularized porcine kidneys. After processing by lyophilization and milling, it was dissolved in an organic solvent and blended with poly(caprolactone). Porous membranes were obtained by electrospinning and seeded with human primary renal progenitor cells to evaluate phenotypic alterations. To create a bilayer model of the in vivo tubule, the same cells were differentiated into epithelial tubular cells and co-cultured with endothelial cells in opposite sites. RESULTS: Our results demonstrate increasing metabolic activity, proliferation and total protein content of renal progenitors over time. We confirmed the expression of several genes encoding epithelial transport proteins and we could also detect tubular-specific proteins by immunofluorescence stainings. Functional and transport assays were performed trough the bilayer by quantifying both human serum albumin uptake and inulin leakage. Furthermore, we validated the chemical modulation of nephrotoxicity on this epithelium-endothelium model by cisplatin exposure. CONCLUSION: The use of decellularized matrices in combination with primary renal cells was shown to be a valuable tool for modelling renal function and disease in vitro. We successfully validated our hypothesis by replicating the physiological conditions of an in vitro tubular bilayer model. The developed system may contribute significantly for the future investigation of advanced therapies for kidney diseases.

Acute kidney injury promotes development of papillary renal cell adenoma and carcinoma from renal progenitor cells.

Acute tissue injury causes DNA damage and repair processes involving increased cell mitosis and polyploidization, leading to cell function alterations that may potentially drive cancer development. Here, we show that acute kidney injury (AKI) increased the risk for papillary renal cell carcinoma (pRCC) development and tumor relapse in humans as confirmed by data collected from several single-center and multicentric studies. Lineage tracing of tubular epithelial cells (TECs) after AKI induction and long-term follow-up in mice showed time-dependent onset of clonal papillary tumors in an adenoma-carcinoma sequence. Among AKI-related pathways, NOTCH1 overexpression in human pRCC associated with worse outcome and was specific for type 2 pRCC. Mice overexpressing NOTCH1 in TECs developed papillary adenomas and type 2 pRCCs, and AKI accelerated this process. Lineage tracing in mice identified single renal progenitors as the cell of origin of papillary tumors. Single-cell RNA sequencing showed that human renal progenitor transcriptome showed similarities to PT1, the putative cell of origin of human pRCC. Furthermore, NOTCH1 overexpression in cultured human renal progenitor cells induced tumor-like 3D growth. Thus, AKI can drive tumorigenesis from local tissue progenitor cells. In particular, we find that AKI promotes the development of pRCC from single progenitors through a classical adenoma-carcinoma sequence.

Substrate Stiffness Modulates Renal Progenitor Cell Properties via a ROCK-Mediated Mechanotransduction Mechanism.

Stem cell (SC)-based tissue engineering and regenerative medicine (RM) approaches may provide alternative therapeutic strategies for the rising number of patients suffering from chronic kidney disease. Embryonic SCs and inducible pluripotent SCs are the most frequently used cell types, but autologous patient-derived renal SCs, such as human CD133+CD24+ renal progenitor cells (RPCs), represent a preferable option. RPCs are of interest also for the RM approaches based on the pharmacological encouragement of in situ regeneration by endogenous SCs. An understanding of the biochemical and biophysical factors that influence RPC behavior is essential for improving their applicability. We investigated how the mechanical properties of the substrate modulate RPC behavior in vitro. We employed collagen I-coated hydrogels with variable stiffness to modulate the mechanical environment of RPCs and found that their morphology, proliferation, migration, and differentiation toward the podocyte lineage were highly dependent on mechanical stiffness. Indeed, a stiff matrix induced cell spreading and focal adhesion assembly trough a Rho kinase (ROCK)-mediated mechanism. Similarly, the proliferative and migratory capacity of RPCs increased as stiffness increased and ROCK inhibition, by either Y27632 or antisense LNA-GapmeRs, abolished these effects. The acquisition of podocyte markers was also modulated, in a narrow range, by the elastic modulus and involved ROCK activity. Our findings may aid in 1) the optimization of RPC culture conditions to favor cell expansion or to induce efficient differentiation with important implication for RPC bioprocessing, and in 2) understanding how alterations of the physical properties of the renal tissue associated with diseases could influenced the regenerative response of RPCs.