A multi-step computational strategy identifies human podocyte markers.

NPHS1, NPHS2, PTPRO, and PLA2R1 were correlated with all other transcripts in kidney cortices from 85 individuals. A: overlay of 585 transcripts obtained by cross-referencing the top-ranking 1124 transcripts (2%) from each analysis. B—D: correlations between NPHS1 and WIPF3, between NPHS2 and PCOLCE2, and between PLA2R1 and ARMH4. P values, Spearman Rho coefficients, and n values are shown in the panels. E: immunohistochemical staining (brown) for 15 gene products with evidence of a glomerular staining pattern in the Human Protein Atlas [22]. F: fold enrichment of the mean number of reads (in transcripts per million) in the cortex (n = 85) versus medulla (n = 4) for each of the 15 transcripts. G: the nine transcripts enriched in cortex > 1.5-fold in order of correlation strength vs. the four podocyte markers in panel A. H: overlay of the nine markers with top-ranking (1%) transcripts from correlations with mesangial cell (PDGFRB), endothelial cell (PECAM1), and blood (ABO) markers. I: the identified transcripts (brown circle) were cross-referenced with hits for albuminuria (green circle) and lupus nephritis (pink circle) in genome-wide association studies. MYL3, WIPF3, and ARMH4 were found in the respective overlaps.</p

ARMH4 is enriched in isolated human glomeruli, increases in differentiated podocytes, and, like WIPF3, responds to LMX1B.

A: representative Western blots from cortices (CTXs) and glomeruli (GLOs) isolated from three patients show higher levels of ARMH4 and NPHS2 in glomeruli compared to cortex. Total protein is shown below. To avoid overlap between lanes in the total protein stain, samples were loaded in every second lane (black lanes = empty wells). B: quantification of ARMH4 protein expression shows fold change (FC) enrichment in the glomeruli relative to cortex. Data was normalized to total protein in the same lane. C: Western blot of protein from tubular epithelial cells (hPTCs) overexpressing ARMH4 shows a major band at 130 kDa that increases in a virus concentration-dependent manner. This result was confirmed in three independent experiments. D: representative Western blots from subcellular fractionation show ARMH4 antibody-reactive bands (green bands) in the cytosolic (Cyt), membrane (Mem) and nuclear (Nucl) fractions in hPTCs overexpressing ARMH4 (OE) compared to Null. Overexpression of ARMH4 increases a 130 kDa band in the membrane fraction. A 100 kDa band in the cytosolic fraction does not respond to overexpression, suggesting unspecific binding. This analysis was repeated twice using the same samples, and representative blots were combined. E: immunolabeling of ARMH4 (green) showing widely distributed fluorescence in podocytes that are fully differentiated (DIF, bottom row), whereas a small fraction of cells among the undifferentiated population stain for ARMH4 (NOD, top row). Within the dashed rectangle to the right, a large and arborized podocyte is shown at higher magnification. Its cell body and major processes stains for ARMH4. Scale bars: 100 μm. Magnification: 10x (rectangle 40x). F: ARMH4 is upregulated at the RNA level in differentiated podocytes (DIF) vs. non-differentiated podocytes (NOD). G: quantification of endogenous protein levels shows that ARMH4, but not WIPF3, is spontaneously upregulated in differentiated podocytes (DIF) compared to non-differentiated podocytes (NOD), similar to the podocyte marker N-WASP. Representative bands are shown in H. I: ARMH4 and WIPF3 transcripts increase in response to LMX1B overexpression in hPTCs.</p

Confirmation of podocyte expression by immunostaining.

Frozen human kidney sections (5 μm) were double stained for the foot process marker NPHS2 or podocyte marker WT1 together with ARMH4 or WIPF3. A: ARMH4 shows perinuclear staining in cells that are outlined by NPHS2-positive contours, and ARMH4 is present in most of the cells that are positive for WT1. This is highlighted in the insets in the merged images to the right. B: WIPF3 immunoreactivity is observed in the cytosol of cells positive for NPHS2 or WT1. The upper inset in the merged images shows a cell body encircled by podocin that is also positive for WIPF3 and localized in close vicinity to podocyte foot processes. Bottom rows in A and B show negative control images for the primary antibodies used. Scale bars: 50 μm. C: DAB staining (in brown) of 2 μm thick paraffin-embedded kidney sections shows ARMH4 and WIPF3 immunoreactivity in glomerular podocytes. At higher magnification, ARMH4 shows an excentric perinuclear pattern, whereas WIPF3 seems to be distributed through the podocyte cytosol and along the glomerular basal membrane. Scale bars: 100 μm.</p

WIPF3 may contribute to cell movement and cytoskeletal regulation in podocytes.

A: N-WASP protein levels increase in both hPTCs and podocytes after overexpression of WIPF3. B: stabilization of N-WASP increases linearly with WIPF3 overexpression (WIPF3 OE), and representative Western blots are shown in C. D: podocytes overexpressing WIPF3 show increased migration compared to null cells in the wound healing assay. Representative images of the scratch wound at different time points are shown in E (scale bars: 100 μm). F: phalloidin staining, an indication of actin filament formation, was assayed in hPTCs after overexpression of WIPF3 and treatment with the CDC42 activator EGF. The effects of EGF and WIPF3 were tested with a two-way ANOVA (p = 0.06 for EGF and p = 0.17 for WIPF3). Representative images are shown in G (scale bars: 100 μm). Cells from three patients, with two replicates from each, were analyzed (n = 6).</p

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ARMH4 and WIPF3 in the healthy and disease state.

A: the RNA level of ARMH4 is reduced in patients suffering from collapsing (coll.) and classic (class.) FSGS compared to normal kidney (NK), but not in those diagnosed with minimal change disease (MCD). The WIPF3 level, on the other hand, was not significantly different in any of the diseases (B). C: immunohistochemistry of glomeruli in patients diagnosed with MCD (n = 2), FSGS (n = 2), MN (n = 2) and CG (n = 2) suggests lower intensity of ARMH4 immunoreactivity vs. normal kidney (NK). In CG, ARMH4 is not seen in the crescent of a glomerulus while some staining is maintained in the healthy-looking part of the same glomerulus (G1 = crescent vs. G2 = healthy tissue). The glomerular and tubular morphology were supported by H&E staining (CG-H&E). Arrows in CG show ARMH4 in some cells of a swollen proximal tubule. Scale bars: 100 μm.</p

SARS-CoV-2 virus replicates in cultured renal cell carcinoma cells and causes a distinct virus cytopathogenic effect.

(A) Negative strand qPCR analysis of SARS-CoV-2 viral RNA copy number at 24h, 48h and 72h post-infection in primary normal tubular epithelial cells, CCRCC cells, PRCC cells and the renal cell carcinoma cell line RCC4. (B) Representative images of virus cytopathogenic effect (CPE) for normal (NKE863), clear cell renal cell carcinoma (CCRCC) (CCRCC863) and papillary renal cell carcinoma (PRCC) (PRCC545) at 48 hours after infection. The CPE causes rounding up of cells, followed by detachment from the growth surface. Mock is control. Scale bar = 200μm.</p

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Expression of ACE2, TMPRSS2 and NRP1 in clear cell, papillary and chromophobe renal cell carcinoma and across 32 distinct cancer forms.

(A) Bioinformatic analysis of the TCGA data sets for renal cell carcinoma showing mRNA expression levels for the ACE2, TMPRSS2 and NRP1 genes in individual RCC cases for chromophobe renal cell carcinoma (CHRCC), clear cell renal cell carcinoma (CCRCC) and papillary renal cell carcinoma (PRCC). (B) Comparison of the mRNA expression levels for ACE2, TMPRSS2 and NRP1 across 32 different types of malignancies deposited at the TCGA atlas. KIRC denotes CCRCC (red square), KIRP denotes PRCC (blue square) and KICH denotes CHRCC (green square). (C) Immunoblotting results for ACE2, TMPRSS2 and NRP1 proteins in lysates from normal tissue, CCRCC, PRCC and CHRCC. Results from densitometry of the protein bands is shown below the blots.</p

Clinical characteristics of the patients included in the study.

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