Acute kidney injury (AKI) is considered largely reversible based on the capacity of surviving tubular cells to dedifferentiate and replace lost cells via cell division. Here we show by tracking individual tubular cells in conditional Pax8/Confetti mice that kidney function is  recovered after AKI despite substantial tubular cell loss. Cell cycle and ploidy analysis upon AKI in conditional Pax8/FUCCI2aR mice and human biopsies identify endocycle-mediated hypertrophy of tubular cells. By contrast, a small subset of Pax2+ tubular progenitors enriches via higher stress resistance and clonal expansion and regenerates necrotic tubule segments, a process that can be enhanced by suitable drugs. Thus,  renal functional recovery upon AKI involves remnant tubular cell hypertrophy via endocycle and limited progenitor-driven regeneration that can be pharmacologically enhanced.

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.

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.

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.

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 tubularspecific 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.

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.

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...

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.