Generation of two isogenic knockout PKD2 iPS cell lines, IRFMNi003-A-1 and IRFMNi003-A-2, using CRISPR/Cas9 technology.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent inherited renal disease, characterized by multiple cysts that can lead to kidney failure resulting in end-stage renal disease. ADPKD is mainly caused by mutations in either the PKD1 and PKD2 genes, encoding for polycystin-1 and polycystin-2, respectively. In order to clarify the disease mechanisms, here we describe the generation of two isogenic induced pluripotent stem cell (iPSC) lines in which the PKD2 gene was deleted using CRISPR/Cas9 technology. The PKD2−/− iPSCs expressed the main pluripotency markers, were able to differentiate into the three germ layers and had a normal karyotype

Generation of PKD1 mono-allelic and bi-allelic knockout iPS cell lines using CRISPR-Cas9 system.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, characterised by the development of multiple fluid-filled cysts in the kidneys and other organs. PKD1 and PKD2 are the two major causative genes encoding for polycystin-1 and polycystin-2, respectively. Here, we report the generation of two isogenic induced pluripotent stem cell (iPSC) lines with either heterozygous or compound heterozygous mutations in the PKD1 gene using CRISPR-Cas9 technology. The PKD1+/- and PKD1-/- iPSCs maintain stem cell-like morphology, normal karyotype, pluripotency and differentiation capacity in the three germ layers

MicroRNA-184 is a downstream effector of albuminuria driving renal fibrosis in rats with diabetic nephropathy

Renal fibrosis is a common complication of diabetic nephropathy and is a major cause of end-stage renal disease. Despite the suggested link between renal fibrosis and microRNA (miRNA) dysregulation in diabetic nephropathy, the identification of the specific miRNAs involved is still incomplete. The aim of this study was to investigate miRNA profiles in the diabetic kidney and to identify potential downstream targets implicated in renal fibrosis. miRNA expression profiling was investigated in the kidneys of 8-month-old Zucker diabetic fatty (ZDF) rats during overt nephropathy. Localisation of the most upregulated miRNA was established by in situ hybridisation. The candidate miRNA target was identified by in silico analysis and its expression documented in the diabetic kidney associated with fibrotic markers. Cultured tubule cells served to assess which of the profibrogenic stimuli acted as a trigger for the overexpressed miRNA, and to investigate underlying epigenetic mechanisms. In ZDF rats, miR-184 showed the strongest differential upregulation compared with lean rats (18-fold). Tubular localisation of miR-184 was associated with reduced expression of lipid phosphate phosphatase 3 (LPP3) and collagen accumulation. Transfection of NRK-52E cells with miR-184 mimic reduced LPP3, promoting a profibrotic phenotype. Albumin was a major trigger of miR-184 expression. Anti-miR-184 counteracted albumin-induced LPP3 downregulation and overexpression of plasminogen activator inhibitor-1. In ZDF rats, ACE-inhibitor treatment limited albuminuria and reduced miR-184, with tubular LPP3 preservation and tubulointerstitial fibrosis amelioration. Albumin-induced miR-184 expression in tubule cells was epigenetically regulated through DNA demethylation and histone lysine acetylation and was accompanied by binding of NF-kappa B p65 subunit to miR-184 promoter. These results suggest that miR-184 may act as a downstream effector of albuminuria through LPP3 to promote tubulointerstitial fibrosis, and offer the rationale to investigate whether targeting miR-184 in association with albuminuria-lowering drugs may be a new strategy to achieve fully anti-fibrotic effects in diabetic nephropathy

Generation of two isogenic iPS cell lines (IRFMNi002-A and IRFMNi002-B) from a patient affected by Focal Segmental Glomerulosclerosis carrying a heterozygous c.565G>A mutation in PAX2 gene

Focal Segmental Glomerulosclerosis (FSGS) is the typical renal histologic lesion in familial steroid-resistant nephrotic syndrome, for which there is currently no treatment. Dysfunction of the glomerular podocyte, a specialized cell that forms the glomerular filtration barrier, is central in the pathogenesis of FSGS. Here, we reported the generation of two isogenic iPS cell lines from a patient affected by FSGS, carrying the c.565G > A mutation in the PAX2 gene. The iPS cell lines we generated expressed pluripotency markers at the mRNA and protein levels and differentiated into all three germ layers. These iPSCs will be instrumental in understanding FSGS pathogenesis

Imaging the Kidney with an Unconventional Scanning Electron Microscopy Technique: Analysis of the Subpodocyte Space in Diabetic Mice

Transmission electron microscopy (TEM) remains the gold standard for renal histopathological diagnoses, given its higher resolving power, compared with light microscopy. However, it imposes several limitations on pathologists, including longer sample preparation time and a small observation area. To overcome these, we introduced a scanning electron microscopy (SEM) technique for imaging resin-embedded semi-thin sections of renal tissue. We developed a rapid tissue preparation protocol for experimental models and human biopsies which, alongside SEM digital imaging acquisition of secondary electrons (SE–SEM), enables fast electron microscopy examination, with a resolution similar to that achieved by TEM. We used this unconventional SEM imaging approach to investigate the subpodocyte space (SPS) in BTBR ob/ob mice with type 2 diabetes. Analysis of semi-thin sections with secondary electrons revealed that the SPS had expanded in volume and covered large areas of the glomerular basement membrane, forming wide spaces between the podocyte body and the underlying filtering membrane. Our results show that SE–SEM is a valuable tool for imaging the kidney at the ultrastructural level, filling the magnification gap between light microscopy and TEM, and reveal that in diabetic mice, the SPS is larger than in normal controls, which is associated with podocyte damage and impaired kidney function

Engineered Kidney Tubules for Modeling Patient-Specific Diseases and Drug Discovery

The lack of engineering systems able to faithfully reproduce complex kidney structures in vitro has made it difficult to efficiently model kidney diseases and development. Using polydimethylsiloxane (PDMS) scaffolds and a kidney-derived cell line we developed a system to rapidly engineer custom-made 3D tubules with typical renal epithelial properties. This system was successfully employed to engineer patient-specific tubules, to model polycystic kidney disease (PKD) and test drug efficacy, and to identify a potential new pharmacological treatment. By optimizing our system we constructed functional ureteric bud (UB)-like tubules from human induced pluripotent stem cells (iPSCs), and identified a combination of growth factors that induces budding morphogenesis like embryonic kidneys do. Finally, we applied this assay to investigate budding defects in UB-like tubules derived from a patient with a PAX2 mutation. Our system enables the modeling of human kidney disease and development, drug testing and discovery, and lays the groundwork for engineering anatomically correct kidney tissues in vitro and developing personalized medicine applications

Endothelial Glycocalyx of Peritubular Capillaries in Experimental Diabetic Nephropathy: A Target of ACE Inhibitor-Induced Kidney Microvascular Protection

Peritubular capillary rarefaction is a recurrent aspect of progressive nephropathies. We previously found that peritubular capillary density was reduced in BTBR ob/ob mice with type 2 diabetic nephropathy. In this model, we searched for abnormalities in the ultrastructure of peritubular capillaries, with a specific focus on the endothelial glycocalyx, and evaluated the impact of treatment with an angiotensin-converting enzyme inhibitor (ACEi). Mice were intracardially perfused with lanthanum to visualise the glycocalyx. Transmission electron microscopy analysis revealed endothelial cell abnormalities and basement membrane thickening in the peritubular capillaries of BTBR ob/ob mice compared to wild-type mice. Remodelling and focal loss of glycocalyx was observed in lanthanum-stained diabetic kidneys, associated with a reduction in glycocalyx components, including sialic acids, as detected through specific lectins. ACEi treatment preserved the endothelial glycocalyx and attenuated the ultrastructural abnormalities of peritubular capillaries. In diabetic mice, peritubular capillary damage was associated with an enhanced tubular expression of heparanase, which degrades heparan sulfate residues of the glycocalyx. Heparanase was also detected in renal interstitial macrophages that expressed tumor necrosis factor-α. All these abnormalities were mitigated by ACEi. Our findings suggest that, in experimental diabetic nephropathy, preserving the endothelial glycocalyx is important in order to protect peritubular capillaries from damage and loss