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

BackgroundThe NLRP3 inflammasome integrates several danger signals into the activation of innate immunity and inflammation by secreting IL-1β 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-β in diabetic kidney disease, contributing to filtration barrier dysfunction and kidney injury. We questioned this and hence performed independent verification experiments.MethodsWe 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.ResultsSingle-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-1β 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.ConclusionPodocytes express low amounts of the NLRP3 inflammasome, if at all, and do not produce IL-1β 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

Studio del ruolo delle cellule tubulari nel processo di rigenerazione del rene dopo danno ischemico mediante stategia di "Lineage tracing".

L’origine delle cellule responsabili della risposta rigenerativa del tubulo renale a seguito di un danno è ancora poco conosciuta e oggetto di intenso dibattito. Finora, nel rene dei mammiferi, si è ritenuto che le cellule tubulari differenziate sopravvissute al danno riacquisiscono la capacità di proliferare per rimpiazzare le cellule morte. Questi risultati sono stati però ottenuti impiegando modelli e tecniche di studio non del tutto idonei a valutare la reale capacità rigenerativa delle cellule tubulari differenziate. Le strategie di “lineage tracing” rappresentano lo strumento ideale per valutare e quantificare l’entità dei processi proliferativi e rigenerativi. Pertanto, in questo lavoro di tesi è stato utilizzato e ottimizzato un modello inducibile di topo transgenico. I processi rigenerativi sono stati quindi monitorati in conseguenza di danno renale acuto e l’eventuale rigenerazione è stata valutata a distanza di 30 giorni dall’ischemia

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

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

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.This work was supported by the Portuguese Foundation of Technology (FCT) with the PhD Grant on the Doctoral Program on Advanced Therapies for Health (PATH) (PD/BD/128102/2016) and the the project Cells4_IDs (PTDC/BTM-SAL/28882/2017), under the Compete2020 Funding Program

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

"Epub ahead of print"Aim Herein we propose creating a bilayer tubular kidney inâ 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 coâ 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 tubularâ 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 epitheliumâ 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 modeling renal function and disease inâ 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.The authors want to acknowledge the financial support obtained by the European Regional Development Fund (ERDF) on the project FROnTHERA (NORTE-01-0145-FEDER-000023) and the FCT PhD Grant on the Doctoral Program on Advanced Therapies for Health (PATH) (PD/BD/128102/2016)

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