Imputation-powered whole-exome analysis identifies genes associated with kidney function and disease in the UK Biobank.

Genome-wide association studies have discovered hundreds of associations between common genotypes and kidney function but cannot comprehensively investigate rare coding variants. Here, we apply a genotype imputation approach to whole exome sequencing data from the UK Biobank to increase sample size from 166,891 to 408,511. We detect 158 rare variants and 105 genes significantly associated with one or more of five kidney function traits, including genes not previously linked to kidney disease in humans. The imputation-powered findings derive support from clinical record-based kidney disease information, such as for a previously unreported splice allele in PKD2, and from functional studies of a previously unreported frameshift allele in CLDN10. This cost-efficient approach boosts statistical power to detect and characterize both known and novel disease susceptibility variants and genes, can be generalized to larger future studies, and generates a comprehensive resource ( https://ckdgen-ukbb.gm.eurac.edu/ ) to direct experimental and clinical studies of kidney disease.

A slit-diaphragm-associated protein network for dynamic control of renal filtration.

The filtration of blood in the kidney which is crucial for mammalian life is determined by the slit-diaphragm, a cell-cell junction between the foot processes of renal podocytes. The slit-diaphragm is thought to operate as final barrier or as molecular sensor of renal filtration. Using high-resolution proteomic analysis of slit-diaphragms affinity-isolated from rodent kidney, we show that the native slit-diaphragm is built from the junction-forming components Nephrin, Neph1 and Podocin and a co-assembled high-molecular weight network of proteins. The network constituents cover distinct classes of proteins including signaling-receptors, kinases/phosphatases, transporters and scaffolds. Knockout or knock-down of either the core components or the selected network constituents tyrosine kinase MER (MERTK), atrial natriuretic peptide-receptor C (ANPRC), integral membrane protein 2B (ITM2B), membrane-associated guanylate-kinase, WW and PDZ-domain-containing protein1 (MAGI1) and amyloid protein A4 resulted in target-specific impairment or disruption of the filtration process. Our results identify the slit-diaphragm as a multi-component system that is endowed with context-dependent dynamics via a co-assembled protein network.

Wildtype heterogeneity contributes to clonal variability in genome edited cells.

Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype-phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts (477 up- and 306 downregulated) when comparing two populations of wild-type cells. Furthermore, we show a variety of cellular and biochemical alterations in different wild-type clones in a range that is commonly interpreted as biologically relevant in genome-edited cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. We therefore propose to generate matched isogenic control cells prior to genome editing to increase reproducibility.

Clock genes rescue nphp mutations in zebrafish.

The zebrafish pronephros model, using morpholino oligonucleotides (MO) to deplete target genes, has been extensively used to characterize human ciliopathy phenotypes. Recently, discrepancies between MO and genetically defined mutants have questioned this approach. We analyzed zebrafish with mutations in the nphp1-4-8 module to determine the validity of MO-based results. While MO-mediated depletion resulted in glomerular cyst and cloaca malformation, these ciliopathy-typical manifestations were observed at a much lower frequency in zebrafish embryos with defined nphp mutations. All nphp1-4-8 mutant zebrafish were viable and displayed decreased manifestations in the next (F2) generation, lacking maternal RNA contribution. While genetic compensation was further supported by the observation that nphp4-deficient mutants became partially refractory to MO-based nphp4 depletion, zebrafish embryos, lacking one nphp gene, became more sensitive to MO-based depletion of additional nphp genes. Transcriptome analysis of nphp8 mutant embryos revealed an upregulation of the circadian clock genes cry1a and cry5. MO-mediated depletion of cry1a and cry5 caused ciliopathy phenotypes in wild-type embryos, while cry1a and cry5 depletion in maternal zygotic nphp8 mutant embryos increased the frequency of glomerular cysts compared to controls. Importantly, cry1a and cry5 rescued the nephropathy-related phenotypes in nphp1, nphp4 or nphp8-depleted zebrafish embryos. Our results reveal that nphp mutant zebrafish resemble the MO-based phenotypes, albeit at a much lower frequency. Rapid adaption through upregulation of circadian clock genes seems to ameliorate the loss of nphp genes, contributing to phenotypic differences.

Genome-wide studies reveal factors associated with circulating uromodulin and its relationships to complex diseases.

Uromodulin (UMOD) is a major risk gene for monogenic and complex forms of kidney disease. The encoded kidney-specific protein uromodulin is highly abundant in urine and related to chronic kidney disease, hypertension, and pathogen defense. To gain insights into potential systemic roles, we performed genome-wide screens of circulating uromodulin using complementary antibody-based and aptamer-based assays. We detected 3 and 10 distinct significant loci, respectively. Integration of antibody-based results at the UMOD locus with functional genomics data (RNA-Seq, ATAC-Seq, Hi-C) of primary human kidney tissue highlighted an upstream variant with differential accessibility and transcription in uromodulin-synthesizing kidney cells as underlying the observed cis effect. Shared association patterns with complex traits, including chronic kidney disease and blood pressure, placed the PRKAG2 locus in the same pathway as UMOD. Experimental validation of the third antibody-based locus, B4GALNT2, showed that the p.Cys466Arg variant of the encoded N-acetylgalactosaminyltransferase had a loss-of-function effect leading to higher serum uromodulin levels. Aptamer-based results pointed to enzymes writing glycan marks present on uromodulin and to their receptors in the circulation, suggesting that this assay permits investigating uromodulin's complex glycosylation rather than its quantitative levels. Overall, our study provides insights into circulating uromodulin and its emerging functions.

Therapeutic Effects of Inhibition of Sphingosine-1-Phosphate Signaling in HIF-2α Inhibitor-Resistant Clear Cell Renal Cell Carcinoma.

Specific inhibitors of HIF-2α have recently been approved for the treatment of ccRCC in VHL disease patients and have shown encouraging results in clinical trials for metastatic sporadic ccRCC. However, not all patients respond to therapy and pre-clinical and clinical studies indicate that intrinsic as well as acquired resistance mechanisms to HIF-2α inhibitors are likely to represent upcoming clinical challenges. It would be desirable to have additional therapeutic options for the treatment of HIF-2α inhibitor resistant ccRCCs. Here we investigated the effects on tumor growth and on the tumor microenvironment of three different direct and indirect HIF-α inhibitors, namely the HIF-2α-specific inhibitor PT2399, the dual HIF-1α/HIF-2α inhibitor Acriflavine, and the S1P signaling pathway inhibitor FTY720, in the autochthonous Vhl/Trp53/Rb1 mutant ccRCC mouse model and validated these findings in human ccRCC cell culture models. We show that FTY720 and Acriflavine exhibit therapeutic activity in several different settings of HIF-2α inhibitor resistance. We also identify that HIF-2α inhibition strongly suppresses T cell activation in ccRCC. These findings suggest prioritization of sphingosine pathway inhibitors for clinical testing in ccRCC patients and also suggest that HIF-2α inhibitors may inhibit anti-tumor immunity and might therefore be contraindicated for combination therapies with immune checkpoint inhibitors.