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Renin-angiotensin system (RAS) polymorphisms have been studied as candidate risk factors for hypertension with inconsistent results, possibly due to heterogeneity among various environmental factors. We analyzed the association between RAS candidate gene polymorphisms and risk of hypertension among 2722 women and also explored whether these associations varied according to menopausal status, body mass index, and dietary factors. In a main-effects analysis of all 2722 women adjusted for age and race, homozygosity for the AT1R A1166C polymorphism was associated with hypertension (odds ratio, 1.35; 95% confidence interval [CI], 1.03-1.78). We also found that a novel nonsense polymorphism in the aminopeptidase-A gene was associated with hypertension among postmenopausal women (hazard ratio, 1.54; 95% CI, 1.01-2.37), women with inadequate calcium intake (hazard ratio, 2.47; 95% CI, 1.29-4.72) and, marginally, women with inadequate vitamin D intake. In addition, angiotensin-converting enzyme and AT1R A1166C polymorphisms were associated or marginally associated with incident hypertension among postmenopausal women and those with inadequate calcium and vitamin D intakes. These data suggest that demographic and dietary factors may influence the associations between RAS polymorphisms and hypertension and could explain heterogeneity in prior studies. [ABSTRACT FROM AUTHOR]

Nephronophthisis is a form of autosomal recessive hereditary cystic kidney disease that typically progresses to end-stage renal disease by early adulthood. Conversely, focal segmental glomerulosclerosis is a histological glomerular phenotype that can be familial, primary (idiopathic), or secondary to a multitude of pathological processes affecting the kidney, including such tubulointerstitial diseases as nephronophthisis. Mutations in 6 distinct nephronophthisis genes have been described to date. We describe a consanguineous Filipino family with 2 novel sequence variants in the NPHP4 gene. Affected individuals presented with end-stage renal disease and histological features of focal segmental glomerulosclerosis on biopsy. They also had atypical radiological findings, making the clinical diagnosis of the genetic syndrome difficult. Furthermore, although ocular abnormalities and hearing loss were described previously, this is the first report of hepatic disease in patients with mutations in NPHP4. The diagnosis of nephronophthisis was made by means of mutational analysis of the NPHP4 gene after isolation of a region of homozygosity in affected individuals by using whole-genome single-nucleotide polymorphism analysis. Because establishment of the correct diagnosis has implications for therapeutic interventions, prognosis, and, in the case of heritable diseases, appropriate genetic counseling for affected individuals and their families, this report emphasizes the importance of obtaining meticulous clinical information, considering alternative diagnoses, and, when possible, performing genetic evaluation to confirm the diagnosis. We outline an approach to patients with hereditary kidney disease, focusing specifically on the molecular genetic techniques available to evaluate such families and determine a chromosomal region of interest and, subsequently, the diagnosis. Copyright © 2007 by Elsevier Inc. [ABSTRACT FROM AUTHOR]

Background: Understanding the genetic basis of human diseases has become integral to drug development and precision medicine. Recent advancements have enabled the identification of molecular pathways driving diseases, leading to targeted treatment strategies. The increasing investment in rare diseases by the biotech industry underscores the importance of genetic evidence in drug discovery and approval processes. Here we studied a monogenic Mendelian kidney disease, TRPC6-associated podocytopathy (TRPC6-AP), to present its natural history, genetic spectrum, and clinicopathological associations in a large cohort of patients with causal variants in TRPC6, in order to help define the specific features of disease and further facilitate drug development and clinical trials design., Methods: the study involved 64 individuals from 39 families with TRPC6 causal missense variants. Clinical data, including age of onset, laboratory results, response to treatment, kidney biopsy findings, and genetic information, were collected from multiple centers nationally and internationally. Exome or targeted sequencing was performed and variant classification was based on strict criteria. Structural and functional analyses of TRPC6 variants were conducted to understand their impact on protein function. In depth re-analysis of light and electron microscopy specimens for 9 available kidney biopsies was conducted to identify pathological features and correlates of TRPC6-AP., Results: Large-scale sequencing data did not support causality for TRPC6 protein-truncating variants. We identified 21 unique TRPC6 missense variants, clustering in three distinct regions of the protein, and with different effects on TRPC6 3D protein structure. Kidney biopsy analysis revealed FSGS patterns of injury in most cases, along with distinctive podocyte features including diffuse foot process effacement and swollen cell bodies. The majority of patients presented in adolescence or early adulthood but with ample variation (average 22, SD ± 14 years), with frequent progression to kidney failure but with variability in time between presentation and ESKD., Conclusions: This study provides insights into the genetic spectrum, clinicopathological associations, and natural history of TRPC6-AP., (Copyright © 2024 by the American Society of Nephrology.)

Apolipoprotein L1 (APOL1) variants G1 and G2 contribute to the excess risk of kidney disease in individuals of recent African ancestry. Since disease mechanisms and optimal treatments remain controversial, we study the effect of current standard-of-care drugs in mouse models of APOL1 kidney disease. Experiments were performed in APOL1 BAC-transgenic mice, which develop proteinuria and glomerulosclerosis following injection with a pCpG-free IFN-γ plasmid. Proteinuric, plasmid injected G1/G1 and G2/G2 mice were randomized to drug treatment or no treatment. Lisinopril, dapagliflozin, and hydralazine were administered in drinking water starting day seven. The urine albumin/creatinine ratio was measured twice weekly, and the kidneys examined histologically with the focal segmental glomerulosclerosis score computed from periodic acid-Shiff-stained sections. The angiotensin converting enzyme inhibitor lisinopril, at standard dose, reduced proteinuria by approximately 90-fold and reduced glomerulosclerosis in the APOL1 G1/G1 BAC-transgenic mice. These effects were independent of blood pressure. Dapagliflozin did not alter disease progression in either G1/G1 or G2/G2 mice. Proteinuria reduction and glomerulosclerosis in G2/G2 BAC-transgenic mice required lisinopril doses two times higher than were effective in G1/G1 mice but achieved a much smaller benefit. Therefore, in these BAC-transgenic mouse models of APOL1 disease, the anti-proteinuric and anti-glomerulosclerotic effects of standard dose lisinopril were markedly effective in G1/G1 compared with G2/G2 APOL1 mice. Comparable reduction in blood pressure by hydralazine treatment provided no such protection. Neither G1/G1 nor G2/G2 mice showed improvement with the sodium-glucose cotransporter-2 inhibition dapagliflozin. Thus, it remains to be determined if similar differences in ACE inhibitor responsiveness are observed in patients., (Published by Elsevier Inc.)

About 37 million people in the United States have chronic kidney disease, a disease that encompasses multiple causes. About 10% or more of kidney diseases in adults and as many as 70% of selected chronic kidney diseases in children are expected to be explained by genetic causes. Despite the advances in genetic testing and an increasing understanding of the genetic bases of certain kidney diseases, genetic testing in nephrology lags behind other medical fields. More understanding of the benefits and logistics of genetic testing is needed to advance the implementation of genetic testing in chronic kidney diseases. Accordingly, the National Kidney Foundation convened a Working Group of experts with diverse expertise in genetics, nephrology, and allied fields to develop recommendations for genetic testing for monogenic disorders and to identify genetic risk factors for oligogenic and polygenic causes of kidney diseases. Algorithms for clinical decision making on genetic testing and a road map for advancing genetic testing in kidney diseases were generated. An important aspect of this initiative was the use of a modified Delphi process to reach group consensus on the recommendations. The recommendations and resources described herein provide support to nephrologists and allied health professionals to advance the use of genetic testing for diagnosis and screening of kidney diseases., (Copyright © 2024. Published by Elsevier Inc.)

Inverted formin-2 (INF2) gene mutations are among the most common causes of genetic focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth (CMT) disease. Recent studies suggest that INF2, through its effects on actin and microtubule arrangement, can regulate processes including vesicle trafficking, cell adhesion, mitochondrial calcium uptake, mitochondrial fission, and T-cell polarization. Despite roles for INF2 in multiple cellular processes, neither the human pathogenic R218Q INF2 point mutation nor the INF2 knock-out allele is sufficient to cause disease in mice. This discrepancy challenges our efforts to explain the disease mechanism, as the link between INF2-related processes, podocyte structure, disease inheritance pattern, and their clinical presentation remains enigmatic. Here, we compared the kidney responses to puromycin aminonucleoside (PAN) induced injury between R218Q INF2 point mutant knock-in and INF2 knock-out mouse models and show that R218Q INF2 mice are susceptible to developing proteinuria and FSGS. This contrasts with INF2 knock-out mice, which show only a minimal kidney phenotype. Co-localization and co-immunoprecipitation analysis of wild-type and mutant INF2 coupled with measurements of cellular actin content revealed that the R218Q INF2 point mutation confers a gain-of-function effect by altering the actin cytoskeleton, facilitated in part by alterations in INF2 localization. Differential analysis of RNA expression in PAN-stressed heterozygous R218Q INF2 point-mutant and heterozygous INF2 knock-out mouse glomeruli showed that the adhesion and mitochondria-related pathways were significantly enriched in the disease condition. Mouse podocytes with R218Q INF2, and an INF2-mutant human patient's kidney organoid-derived podocytes with an S186P INF2 mutation, recapitulate the defective adhesion and mitochondria phenotypes. These results link INF2-regulated cellular processes to the onset and progression of glomerular disease. Thus, our data demonstrate that gain-of-function mechanisms drive INF2-related FSGS and explain the autosomal dominant inheritance pattern of this disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs., Competing Interests: Declaration of interests H.L.R. and M.E.T. have received support from Illumina and Microsoft to support rare disease gene discovery and diagnosis. A.O’D.-L. is on the scientific advisory board for Congenica Inc. D.G.M. is a paid advisor to GlaxoSmithKline, Insitro, Variant Bio, and Overtone Therapeutics and has received research support from AbbVie, Astellas, Biogen, BioMarin, Eisai, Google, Merck, Microsoft, Pfizer, and Sanofi-Genzyme. C.A.W. is a paid advisor to Maze Therapeutics. M.E.T. has also received reagents and/or research support from Levo Therapeutics, Pacific Biosciences, Oxford Nanopore, and Ionis Pharmaceuticals., (Copyright © 2024 American Society of Human Genetics. All rights reserved.)

Competing Interests: M.R.P. and D.J.F. report research support from Vertex. The remaining authors declare no competing interests.

African Americans have a significantly higher risk of developing chronic kidney disease, especially focal segmental glomerulosclerosis -, than European Americans. Two coding variants (G1 and G2) in the APOL1 gene play a major role in this disparity. While 13% of African Americans carry the high-risk recessive genotypes, only a fraction of these individuals develops FSGS or kidney failure, indicating the involvement of additional disease modifiers. Here, we show that the presence of the APOL1 p.N264K missense variant, when co-inherited with the G2 APOL1 risk allele, substantially reduces the penetrance of the G1G2 and G2G2 high-risk genotypes by rendering these genotypes low-risk. These results align with prior functional evidence showing that the p.N264K variant reduces the toxicity of the APOL1 high-risk alleles. These findings have important implications for our understanding of the mechanisms of APOL1-associated nephropathy, as well as for the clinical management of individuals with high-risk genotypes that include the G2 allele., (© 2023. The Author(s).)

Pediatric steroid-sensitive nephrotic syndrome (pSSNS) is the most common childhood glomerular disease. Previous genome-wide association studies (GWAS) identified a risk locus in the HLA Class II region and three additional independent risk loci. But the genetic architecture of pSSNS, and its genetically driven pathobiology, is largely unknown. Here, we conduct a multi-population GWAS meta-analysis in 38,463 participants (2440 cases). We then conduct conditional analyses and population specific GWAS. We discover twelve significant associations-eight from the multi-population meta-analysis (four novel), two from the multi-population conditional analysis (one novel), and two additional novel loci from the European meta-analysis. Fine-mapping implicates specific amino acid haplotypes in HLA-DQA1 and HLA-DQB1 driving the HLA Class II risk locus. Non-HLA loci colocalize with eQTLs of monocytes and numerous T-cell subsets in independent datasets. Colocalization with kidney eQTLs is lacking but overlap with kidney cell open chromatin suggests an uncharacterized disease mechanism in kidney cells. A polygenic risk score (PRS) associates with earlier disease onset. Altogether, these discoveries expand our knowledge of pSSNS genetic architecture across populations and provide cell-specific insights into its molecular drivers. Evaluating these associations in additional cohorts will refine our understanding of population specificity, heterogeneity, and clinical and molecular associations., (© 2023. The Author(s).)

Background: Persons with toxic gain-of-function variants in the gene encoding apolipoprotein L1 ( APOL1 ) are at greater risk for the development of rapidly progressive, proteinuric nephropathy. Despite the known genetic cause, therapies targeting proteinuric kidney disease in persons with two APOL1 variants (G1 or G2) are lacking., Methods: We used tetracycline-inducible APOL1 human embryonic kidney (HEK293) cells to assess the ability of a small-molecule compound, inaxaplin, to inhibit APOL1 channel function. An APOL1 G2-homologous transgenic mouse model of proteinuric kidney disease was used to assess inaxaplin treatment for proteinuria. We then conducted a single-group, open-label, phase 2a clinical study in which inaxaplin was administered to participants who had two APOL1 variants, biopsy-proven focal segmental glomerulosclerosis, and proteinuria (urinary protein-to-creatinine ratio of ≥0.7 to <10 [with protein and creatinine both measured in grams] and an estimated glomerular filtration rate of ≥27 ml per minute per 1.73 m 2 of body-surface area). Participants received inaxaplin daily for 13 weeks (15 mg for 2 weeks and 45 mg for 11 weeks) along with standard care. The primary outcome was the percent change from the baseline urinary protein-to-creatinine ratio at week 13 in participants who had at least 80% adherence to inaxaplin therapy. Safety was also assessed., Results: In preclinical studies, inaxaplin selectively inhibited APOL1 channel function in vitro and reduced proteinuria in the mouse model. Sixteen participants were enrolled in the phase 2a study. Among the 13 participants who were treated with inaxaplin and met the adherence threshold, the mean change from the baseline urinary protein-to-creatinine ratio at week 13 was -47.6% (95% confidence interval, -60.0 to -31.3). In an analysis that included all the participants regardless of adherence to inaxaplin therapy, reductions similar to those in the primary analysis were observed in all but 1 participant. Adverse events were mild or moderate in severity; none led to study discontinuation., Conclusions: Targeted inhibition of APOL1 channel function with inaxaplin reduced proteinuria in participants with two APOL1 variants and focal segmental glomerulosclerosis. (Funded by Vertex Pharmaceuticals; VX19-147-101 ClinicalTrials.gov number, NCT04340362.)., (Copyright © 2023 Massachusetts Medical Society.)

Two heterozygous missense variants (G1 and G2) of Apolipoprotein L1 (APOL1) found in individuals of recent African ancestry can attenuate the severity of infection by some forms of Trypanosoma brucei. However, these two variants within a broader African haplotype also increase the risk of kidney disease in Americans of African descent. Although overexpression of either variant G1 or G2 causes multiple pathogenic changes in cultured cells and transgenic mouse models, the mechanism(s) promoting kidney disease remain unclear. Human serum APOL1 kills trypanosomes through its cation channel activity, and cation channel activity of recombinant APOL1 has been reconstituted in lipid bilayers and proteoliposomes. Although APOL1 overexpression increases whole cell cation currents in HEK-293 cells, the ion channel activity of APOL1 has not been assessed in glomerular podocytes, the major site of APOL1-associated kidney diseases. We characterize APOL1-associated whole cell and on-cell cation currents in HEK-293 T-Rex cells and demonstrate partial inhibition of currents by anti-APOL antibodies. We detect in primary human podocytes a similar cation current inducible by interferon-γ (IFNγ) and sensitive to inhibition by anti-APOL antibody as well as by a fragment of T. brucei Serum Resistance-Associated protein (SRA). CRISPR knockout of APOL1 in human primary podocytes abrogates the IFNγ-induced, antibody-sensitive current. Our novel characterization in HEK-293 cells of heterologous APOL1-associated cation conductance inhibited by anti-APOL antibody and our documentation in primary human glomerular podocytes of endogenous IFNγ-stimulated, APOL1-mediated, SRA and anti-APOL-sensitive ion channel activity together support APOL1-mediated channel activity as a therapeutic target for treatment of APOL1-associated kidney diseases., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Genetic variants in APOL1 are a major contributor to the increased risk of kidney disease in people of recent African ancestry., Summary: Two alleles in the APOL1 gene, referred to as G1 and G2, confer increased risk of kidney disease under a recessive model of risk inheritance. Disease risk is inherited as a recessive trait: People with genotypes G1/G1, G2/G2, and G1/G2 (i.e., a risk allele from each parent) have increased risk for what we refer to here as APOL1-associated kidney disease. In the USA, about 13% of the self-identified African-American population has a high-risk genotype. As we discuss below, APOL1 is an unusual disease gene. Most studies to date have suggested that the G1 and G2 variants have toxic, gain-of-function effects on the encoded protein., Key Message: In this article, we review key concepts critical to understanding APOL1-associated kidney disease, emphasizing ways in which it is highly atypical for a human disease-causing gene., Competing Interests: D. Friedman and M. Pollak are inventors on patents related to APOL1, own equity in Apolo1bio, and receive research funding from and have consulted for Vertex., (Copyright © 2023 by The Author(s). Published by S. Karger AG, Basel.)

Background: Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear., Methods: We generated a stable deletion in Tbc1d8b and used microhomology-mediated end-joining for genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulation of endocytic regulators and transgenesis of murine Tbc1d8b provided a comprehensive functional analysis of Tbc1d8b., Results: A null allele of Drosophila TBC1D8B exhibited a nephrocyte-restricted phenotype of nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from the Tbc1d8b locus bearing the edited tag predominantly localized to mature early and late endosomes. Tbc1d8b was required for endocytic cargo processing and degradation. Silencing Hrs , a regulator of endosomal maturation, phenocopied loss of Tbc1d8b . Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, we discovered four novel TBC1D8B variants within a cohort of 363 patients with FSGS and validated a functional effect of two variants in Drosophila, suggesting a personalized platform for TBC1D8B -associated FSGS., Conclusions: Variants in TBC1D8B are not infrequent among patients with FSGS. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking., (Copyright © 2022 by the American Society of Nephrology.)

APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-γ (IFN-γ)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-γ stimuli, supporting the idea that ADAR regulates APOL1 levels in vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.

Introduction: The discovery of apolipoprotein L1 (ApoL1) has raised important ethical and clinical questions about genetic testing in the context of living and deceased kidney donation. Largely missing from this discussion are the perspectives of those African Americans (AA) most likely to be impacted by ApoL1 testing., Methods: We surveyed 331 AA potential and former living kidney donors (LKDs), kidney transplant candidates and recipients, and nonpatients at three United States transplant programs about their ApoL1 testing attitudes., Results: Overall, 72% felt that transplant programs should offer ApoL1 testing to AA potential LKDs. If a potential LKD has the high-risk genotype, 79% felt that the LKD should be allowed to make their own donation decision or participate in shared decision-making with transplant doctors. More than half of the potential LKDs (58%) would undergo ApoL1 testing and 81% of former LKDs would take the test now if offered. Most transplant candidates expressed a low likelihood of accepting a kidney from a LKD (79%) or a deceased donor (67%) with the high-risk genotype., Conclusions: There is strong support among LKDs and transplant patients for ApoL1 testing when evaluating potential kidney donors of African ancestry. Inclusion of AA stakeholders in developing guidelines and educational programs for ApoL1 testing is critical., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Kidney diseases often lack optimal treatments, causing millions of deaths each year. Thus, developing appropriate model systems to study human kidney disease is of utmost importance. Some of the most promising human kidney models are organoids or small organ-resembling tissue collectives, derived from human-induced pluripotent stem cells (hiPSCs). However, they are more akin to a first-trimester fetal kidney than an adult kidney. Therefore, new strategies are needed to advance their maturity. They have great potential for disease modeling and eventually auxiliary therapy if they can reach the maturity of an adult kidney. In this review, we will discuss the current state of kidney organoids in terms of their similarity to the human kidney and use as a disease modeling system thus far. We will then discuss potential pathways to advance the maturity of kidney organoids to match an adult kidney for more accurate human disease modeling.

Unraveling the complex behavior of healthy and disease podocytes by analyzing the changes in their unique arrangement of foot processes, slit diaphragm, and the three-dimensional (3D) morphology is a long-standing goal in kidney-glomerular research. The complexities surrounding the podocytes' accessibility in animal models and growing evidence of differences between humans and animal systems have compelled researchers to look for alternate approaches to study podocyte behaviors. With the advent of bioengineered models, an increasingly powerful and diverse set of tools is available to develop novel podocyte culture systems. This review discusses the pertinence of various culture models of podocytes to study podocyte mechanisms in both normal physiology and disease conditions. While no one in vitro system comprehensively recapitulates podocytes' in vivo architecture, we emphasize how the existing systems can be exploited to answer targeted questions on podocyte structure and function. We highlight the distinct advantages and limitations of using these models to study podocyte behaviors and screen therapeutics. Finally, we discuss various considerations and potential engineering strategies for developing next-generation complex 3D culture models for studying podocyte behaviors in vitro . Impact Statement In various glomerular kidney diseases, there are numerous alterations in podocyte structure and function. Yet, many of these disease events and the required targeted therapies remain unknown, resulting in nonspecific treatments. The scientific and clinical communities actively search for new modes to develop structurally and functionally relevant podocyte culture systems to gain insights into various diseases and develop therapeutics. Current in vitro systems help in some ways but are not sufficient. A deeper understanding of these previous approaches is essential to advance the field, and importantly, bioengineering strategies can contribute a unique toolbox to establish next-generation podocyte systems.

Apolipoprotein L1 (APOL1)-associated focal segmental glomerulosclerosis (FSGS) is the dominant form of FSGS in Black individuals. There are no targeted therapies for this condition, in part because the molecular mechanisms underlying APOL1's pathogenic contribution to FSGS are incompletely understood. Studying the transcriptomic landscape of APOL1 FSGS in patient kidneys is an important way to discover genes and molecular behaviors that are unique or most relevant to the human disease. With the hypothesis that the pathology driven by the high-risk APOL1 genotype is reflected in alteration of gene expression across the glomerular transcriptome, we compared expression and co-expression profiles of 15,703 genes in 16 Black patients with FSGS at high-risk vs 14 Black patients with a low-risk APOL1 genotype. Expression data from APOL1-inducible HEK293 cells and normal human glomeruli were used to pursue genes and molecular pathways uncovered in these studies. We discovered increased expression of APOL1 and nine other significant differentially expressed genes in high-risk patients. This included stanniocalcin, which has a role in mitochondrial and calcium-related processes along with differential correlations between high- and low-risk APOL1 and metabolism pathway genes. There were similar correlations with extracellular matrix- and immune-related genes, but significant loss of co-expression of mitochondrial genes in high-risk FSGS, and an NF-κB-down regulating gene, NKIRAS1, as the most significant hub gene with strong differential correlations with NDUF family (mitochondrial respiratory genes) and immune-related (JAK-STAT) genes. Thus, differences in mitochondrial gene regulation appear to underlie many differences observed between high- and low-risk Black patients with FSGS., (Copyright © 2021 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Two variants in the gene encoding apolipoprotein L1 (APOL1) that are highly associated with African ancestry are major contributors to the large racial disparity in rates of human kidney disease. We previously demonstrated that recruitment of APOL1 risk variants G1 and G2 from the endoplasmic reticulum to lipid droplets leads to reduced APOL1-mediated cytotoxicity in human podocytes., Methods: We used CRISPR-Cas9 gene editing of induced pluripotent stem cells to develop human-derived APOL1 G0/G0 and APOL1 G2/G2 kidney organoids on an isogenic background, and performed bulk RNA sequencing of organoids before and after treatment with IFN- γ . We examined the number and distribution of lipid droplets in response to treatment with inhibitors of diacylglycerol O-acyltransferases 1 and 2 (DGAT1 and DGAT2) in kidney cells and organoids., Results: APOL1 was highly upregulated in response to IFN- γ in human kidney organoids, with greater increases in organoids of high-risk G1 and G2 genotypes compared with wild-type (G0) organoids. RNA sequencing of organoids revealed that high-risk APOL1 G2/G2 organoids exhibited downregulation of a number of genes involved in lipogenesis and lipid droplet biogenesis, as well as upregulation of genes involved in fatty acid oxidation. There were fewer lipid droplets in unstimulated high-risk APOL1 G2/G2 kidney organoids than in wild-type APOL1 G0/G0 organoids. Whereas DGAT1 inhibition reduced kidney organoid lipid droplet number, DGAT2 inhibition unexpectedly increased organoid lipid droplet number. DGAT2 inhibition promoted the recruitment of APOL1 to lipid droplets, with associated reduction in cytotoxicity., Conclusions: Lipogenesis and lipid droplet formation are important modulators of APOL1-associated cytotoxicity. Inhibition of DGAT2 may offer a potential therapeutic strategy to attenuate cytotoxic effects of APOL1 risk variants., (Copyright © 2022 by the American Society of Nephrology.)

Background: Despite increasing recognition that CKD may have underlyi ng genetic causes, genetic testing remains limited. This study evaluated the diagnostic yield and phenotypic spectrum of CKD in individuals tested through the KIDNEYCODE sponsored genetic testing program., Methods: Unrelated individuals who received panel testing (17 genes) through the KIDNEYCODE sponsored genetic testing program were included. Individuals had to meet at least one of the following eligibility criteria: eGFR ≤90 ml/min per 1.73m 2 and hematuria or a family history of kidney disease; or suspected/biopsy-confirmed Alport syndrome or FSGS in tested individuals or relatives., Results: Among 859 individuals, 234 (27%) had molecular diagnoses in genes associated with Alport syndrome ( n =209), FSGS ( n =12), polycystic kidney disease ( n =6), and other disorders ( n =8). Among those with positive findings in a COL4A gene, the majority were in COL4A5 ( n =157, 72 hemizygous male and 85 heterozygous female individuals). A positive family history of CKD, regardless of whether clinical features were reported, was more predictive of a positive finding than was the presence of clinical features alone. For the 248 individuals who had kidney biopsies, a molecular diagnosis was returned for 49 individuals (20%). Most ( n =41) individuals had a molecular diagnosis in a COL4A gene, 25 of whom had a previous Alport syndrome clinical diagnosis, and the remaining 16 had previous clinical diagnoses including FSGS ( n =2), thin basement membrane disease ( n =9), and hematuria ( n =1). In total, 491 individuals had a previous clinical diagnosis, 148 (30%) of whom received a molecular diagnosis, the majority (89%, n =131) of which were concordant., Conclusions: Although skewed to identify individuals with Alport syndrome, these findings support the need to improve access to genetic testing for patients with CKD-particularly in the context of family history of kidney disease, hematuria, and hearing loss., Competing Interests: A.R. Chang reports being a scientific advisor to, and receiving an honorarium from, Reata Pharmaceuticals, Inc. A. Mitchell, A. Morales, B.A. Johnson, K. Robinson, S.L. Bristow, and S. Aradhya report being employed by and being shareholders in Invitae. B.A. Warady reports being an advisor to, and receiving honoraria from, Reata Pharmaceuticals, Inc. G.A. Block reports being a former employee of Reata Pharmaceuticals, Inc., and reports being employed by US Renal Care. J.A. McKay and S. McCalley report being employees of Reata Pharmaceuticals, Inc. K. Gibson reports serving as a consultant for and receiving research support from Aurinia and Travere, Inc. (formerly Retrophin, Inc.); and reports serving as a consultant for Reata Pharmaceuticals, Inc. K.V. Lieberman reports receiving honoraria from Reata Pharmaceuticals, Inc. P. Devrajan reports being an advisor to Alnylam Pharmaceuticals, Inc., BioPorto Diagnostics, Inc., Dicerna Pharmaceuticals, Inc., Natera, Inc., Reata Pharmaceuticals, Inc., and UpToDate, Inc.; and reports receiving research support from the National Institutes of Health (P50DK096418). KIDNEYCODE was offered through a sponsored genetic program by Reata Pharmaceuticals and Invitae., (Copyright © 2022 by the American Society of Nephrology.)

People of recent sub-Saharan African ancestry develop kidney failure much more frequently than other groups. A large fraction of this disparity is due to two coding sequence variants in the APOL1 gene. Inheriting two copies of these APOL1 risk variants, known as G1 and G2, causes high rates of focal segmental glomerulosclerosis (FSGS), HIV-associated nephropathy and hypertension-associated end-stage kidney disease. Disease risk follows a recessive mode of inheritance, which is puzzling given the considerable data that G1 and G2 are toxic gain-of-function variants. We developed coisogenic bacterial artificial chromosome (BAC) transgenic mice harboring either the wild-type (G0), G1 or G2 forms of human APOL1. Expression of interferon gamma (IFN-γ) via plasmid tail vein injection results in upregulation of APOL1 protein levels together with robust induction of heavy proteinuria and glomerulosclerosis in G1/G1 and G2/G2 but not G0/G0 mice. The disease phenotype was greater in G2/G2 mice. Neither heterozygous (G1/G0 or G2/G0) risk variant mice nor hemizygous (G1/-, G2/-) mice had significant kidney injury in response to IFN-γ, although the heterozygous mice had a greater proteinuric response than the hemizygous mice, suggesting that the lack of significant disease in humans heterozygous for G1 or G2 is not due to G0 rescue of G1 or G2 toxicity. Studies using additional mice (multicopy G2 and a non-isogenic G0 mouse) supported the notion that disease is largely a function of the level of risk variant APOL1 expression. Together, these findings shed light on the recessive nature of APOL1-nephropathy and present an important model for future studies., Competing Interests: Competing interests D.F. and M.P. are co-inventors of patents related to APOL1 diagnostics and therapeutics, own equity in Apolo1Bio, and have research funding from and consulted for Vertex., (© 2021. Published by The Company of Biologists Ltd.)

Purpose: The etiology of calcium-oxalate kidney stone formation remains elusive. Biallelic mutations in HOGA1 are responsible for primary hyperoxaluria type 3 and result in oxalate overproduction and kidney stone disease. Our previous study showed that carriers of HOGA1 mutations have elevated urinary levels of oxalate precursors. In this study we explored the possibility that mutations in HOGA1 confer a dominant phenotype in the form of kidney stone disease or hyperoxaluria., Materials and Methods: An observational analytic case control study was designed to determine the prevalence of pathogenic HOGA1 mutations among adults with calcium-oxalate kidney stone disease. Given the high prevalence of HOGA1 mutations among Ashkenazi Jews, this group was evaluated separately. Carrier frequency of any of the 52 reported pathogenic mutations was compared to data derived from gnomAD for the corresponding ethnic group. Sanger sequencing of HOGA1 gene was performed on DNA samples from the following groups: 60 Ashkenazi Jews and 86 nonAshkenazi calcium-oxalate stone formers, 150 subjects with low and 150 with high urinary oxalate levels., Results: The carrier prevalence of pathogenic mutations among the Ashkenazi Jews was 1.7% compared to 2.8% in the corresponding control group (p=0.9 OR=0.6 95% CI 0.01-3.51). We did not detect any mutation among the nonAshkenazi study group. No correlation was detected between hyperoxaluria and HOGA1 variants., Conclusions: This study shows that mutations in HOGA1 do not confer a dominant phenotype in the form of calcium-oxalate kidney stone disease or hyperoxaluria.

Apolipoprotein L1 (ApoL1) predictive genetic testing for kidney disease, and its emerging role in transplantation, remains controversial as it may exacerbate underlying disparities among African Americans (AAs) at increased risk. We conducted an online simulation among AAs (N = 585) about interest in ApoL1 testing and its cofactors, under 2 scenarios: as a potential living donor (PLD), and as a patient awaiting transplantation. Most respondents (61%) expressed high interest in genetic testing as a PLD: age ≥35 years (adjusted odds ratio [aOR], 1.75; 95% confidence interval [CI], 1.18, 2.60, P = .01), AA identity (aOR, 1.67; 95% CI, 1.02, 2.72, P = .04), perceived kidney disease risk following donation (aOR, 1.68; 95% CI, 1.03, 2.73, P = .03), interest in genetics (aOR, 2.89; 95% CI, 1.95, 4.29, P = .001), and genetics self-efficacy (aOR, 2.38; 95% CI, 1.54, 3.67, P = .001) were positively associated with ApoL1 test interest. If awaiting transplantation, most (89%) believed that ApoL1 testing should be done on AA deceased donors, and older age (aOR, 1.85; 95% CI, 1.03, 3.32, P = .04) and greater interest in genetics (aOR, 2.61; 95% CI, 1.41, 4.81, P = .002) were associated with interest in testing deceased donors. Findings highlight strong support for ApoL1 testing in AAs and the need to examine such opinions among PLDs and transplant patients to enhance patient education efforts., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Rates of many types of severe kidney disease are much higher in Black individuals than most other ethnic groups. Much of this disparity can now be attributed to genetic variants in the apoL1 (APOL1) gene found only in individuals with recent African ancestry. These variants greatly increase rates of hypertension-associated ESKD, FSGS, HIV-associated nephropathy, and other forms of nondiabetic kidney disease. We discuss the population genetics of APOL1 risk variants and the clinical spectrum of APOL1 nephropathy. We then consider clinical issues that arise for the practicing nephrologist caring for the patient who may have APOL1 kidney disease., (Copyright © 2021 by the American Society of Nephrology.)

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10 -11 ). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10 -15 ). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: FSGS caused by mutations in INF2 is characterized by a podocytopathy with mistrafficked nephrin, an essential component of the slit diaphragm. Because INF2 is a formin-type actin nucleator, research has focused on its actin-regulating function, providing an important but incomplete insight into how these mutations lead to podocytopathy. A yeast two-hybridization screen identified the interaction between INF2 and the dynein transport complex, suggesting a newly recognized role of INF2 in regulating dynein-mediated vesicular trafficking in podocytes., Methods: Live cell and quantitative imaging, fluorescent and surface biotinylation-based trafficking assays in cultured podocytes, and a new puromycin aminoglycoside nephropathy model of INF2 transgenic mice were used to demonstrate altered dynein-mediated trafficking of nephrin in INF2 associated podocytopathy., Results: Pathogenic INF2 mutations disrupt an interaction of INF2 with dynein light chain 1, a key dynein component. The best-studied mutation, R218Q, diverts dynein-mediated postendocytic sorting of nephrin from recycling endosomes to lysosomes for degradation. Antagonizing dynein-mediated transport can rescue this effect. Augmented dynein-mediated trafficking and degradation of nephrin underlies puromycin aminoglycoside-induced podocytopathy and FSGS in vivo ., Conclusions: INF2 mutations enhance dynein-mediated trafficking of nephrin to proteolytic pathways, diminishing its recycling required for maintaining slit diaphragm integrity. The recognition that dysregulated dynein-mediated transport of nephrin in R218Q knockin podocytes opens an avenue for developing targeted therapy for INF2-mediated FSGS., (Copyright © 2021 by the American Society of Nephrology.)

In addition to their fundamental role in clearance, the kidneys release select molecules into the circulation, but whether any of these anabolic functions provides insight on kidney health is unknown. Using aptamer-based proteomics, we characterized arterial (A)-to-renal venous (V) gradients for >1,300 proteins in 22 individuals who underwent invasive sampling. Although most of the proteins that changed significantly decreased from A to V, consistent with renal clearance, several were found to increase, the most significant of which was testican-2. To assess the clinical implications of these physiologic findings, we examined proteomic data in the Jackson Heart Study (JHS), an African-American cohort ( n = 1,928), with replication in the Framingham Heart Study (FHS), a White cohort ( n = 1,621). In both populations, testican-2 had a strong, positive correlation with estimated glomerular filtration rate (eGFR). In addition, higher baseline testican-2 levels were associated with a lower rate of eGFR decline in models adjusted for age, gender, hypertension, type 2 diabetes, body mass index, baseline eGFR, and albuminuria. Glomerular expression of testican-2 in human kidneys was demonstrated by immunohistochemistry, immunofluorescence, and electron microscopy, while single-cell RNA sequencing of human kidneys showed expression of the cognate gene, SPOCK2 , exclusively in podocytes. In vitro, testican-2 increased glomerular endothelial tube formation and motility, raising the possibility that its secretion has a functional role within the glomerulus. Taken together, our findings identify testican-2 as a podocyte-derived biomarker of kidney health and prognosis., Competing Interests: Competing interest statement: D.J.F. and M.R.P. are equity holders in Apolo1bio, receive research support from Vertex, and are inventors on patents related to APOL1 diagnostics and therapeutics.

Background: Two coding renal risk variants (RRVs) of the APOL1 gene (G1 and G2) are associated with large increases in CKD rates among populations of recent African descent, but the underlying molecular mechanisms are unknown. Mammalian cell culture models are widely used to study cytotoxicity of RRVs, but results have been contradictory. It remains unclear whether cytotoxicity is RRV-dependent or driven solely by variant-independent overexpression. It is also unknown whether expression of the reference APOL1 allele, the wild-type G0, could prevent cytotoxicity of RRVs., Methods: We generated tetracycline-inducible APOL1 expression in human embryonic kidney HEK293 cells and examined the effects of increased expression of APOL1 (G0, G1, G2, G0G0, G0G1, or G0G2) on known cytotoxicity phenotypes, including reduced viability, increased swelling, potassium loss, aberrant protein phosphorylation, and dysregulated energy metabolism. Furthermore, whole-genome transcriptome analysis examined deregulated canonical pathways., Results: At moderate expression, RRVs but not G0 caused cytotoxicity in a dose-dependent manner that coexpression of G0 did not reduce. RRVs also have dominant effects on canonical pathways relevant for the cellular stress response., Conclusions: In HEK293 cells, RRVs exhibit a dominant toxic gain-of-function phenotype that worsens with increasing expression. These observations suggest that high steady-state levels of RRVs may underlie cellular injury in APOL1 nephropathy, and that interventions that reduce RRV expression in kidney compartments may mitigate APOL1 nephropathy., (Copyright © 2020 by the American Society of Nephrology.)

Background: Genetic mutations in α -actinin-4 (ACTN4)-an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes-have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known., Methods: Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo . We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF- β levels increase phosphorylation of ACTN4., Results: Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro . Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF- β stimulates phosphorylation of ACTN4 at S159 in podocytes., Conclusions: These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation., (Copyright © 2020 by the American Society of Nephrology.)

Genetic variants in the APOL1 gene, found only in individuals of recent African ancestry, greatly increase risk of multiple types of kidney disease. These APOL1 kidney risk alleles are a rare example of genetic variants that are common but also have a powerful effect on disease susceptibility. These alleles rose to high frequency in sub-Saharan Africa because they conferred protection against pathogenic trypanosomes that cause African sleeping sickness. We consider the genetic evidence supporting the association between APOL1 and kidney disease across the range of clinical phenotypes in the APOL1 nephropathy spectrum. We then explore the origins of the APOL1 risk variants and evolutionary struggle between humans and trypanosomes at both the molecular and population genetic level. Finally, we survey the rapidly growing literature investigating APOL1 biology as elucidated from experiments in cell-based systems, cell-free systems, mouse and lower organism models of disease, and through illuminating natural experiments in humans.

The kidney is subject to a wide range of abnormalities, many of which have a significant hereditable component. Next generation sequencing is increasingly bringing the genetic drivers of Mendelian disease into focus at the base pair level, whereas inexpensive genotyping arrays have surveyed hundreds of thousands of individuals to identify common variants that predispose to kidney dysfunction. In this first article in a CJASN series on kidney genomics, we review how both rare and common variants contribute to kidney disease, explore how evolution may influence the genetic variants that affect kidney function, consider how genetic information is and will be used in the clinic, and identify some of the most important future directions for kidney disease research. Forthcoming articles in the series will elaborate on many of these themes., (Copyright © 2020 by the American Society of Nephrology.)

Background: Mutations in the gene encoding inverted formin-2 (INF2), a member of the formin family of actin regulatory proteins, are among the most common causes of autosomal dominant FSGS. INF2 is regulated by interaction between its N-terminal diaphanous inhibitory domain (DID) and its C-terminal diaphanous autoregulatory domain (DAD). INF2 also modulates activity of other formins, such as the mDIA subfamily, and promotes stable microtubule assembly. Why the disease-causing mutations are restricted to the N terminus and how they cause human disease has been unclear., Methods: We examined INF2 isoforms present in podocytes and evaluated INF2 cleavage as an explanation for immunoblot findings. We evaluated the expression of INF2 N- and C-terminal fragments in human kidney disease conditions. We also investigated the localization and functions of the DID-containing N-terminal fragment in podocytes and assessed whether the FSGS-associated R218Q mutation impairs INF2 cleavage or the function of the N-fragment., Results: The INF2-CAAX isoform is the predominant isoform in podocytes. INF2 is proteolytically cleaved, a process mediated by cathepsin proteases, liberating the N-terminal DID to function independently. Although the N-terminal region normally localizes to podocyte foot processes, it does not do so in the presence of FSGS-associated INF2 mutations. The C-terminal fragment localizes to the cell body irrespective of INF2 mutations. In podocytes, the N-fragment localizes to the plasma membrane, binds mDIA1, and promotes cell spreading in a cleavage-dependent way. The disease-associated R218Q mutation impairs these N-fragment functions but not INF2 cleavage., Conclusions: INF2 is cleaved into an N-terminal DID-containing fragment and a C-terminal DAD-containing fragment. Cleavage allows the N-terminal fragment to function independently and helps explain the clustering of FSGS-associated mutations., (Copyright © 2020 by the American Society of Nephrology.)

Introduction: Focal segmental glomerulosclerosis (FSGS) is a histopathologically defined kidney lesion. FSGS can be observed with various underlying causes, including highly penetrant monogenic renal disease. We recently identified pathogenic variants of UMOD , a gene encoding the tubular protein uromodulin, in 8 families with suspected glomerular disease., Methods: To validate pathogenic variants of UMOD , we reviewed the clinical and pathology reports of members of 8 families identified to have variants of UMOD . Clinical, laboratory, and pathologic data were collected, and genetic confirmation for UMOD was performed by Sanger sequencing., Results: Biopsy-proven cases of FSGS were verified in 21% (7 of 34) of patients with UMOD variants. The UMOD variants seen in 7 families were mutations previously reported in autosomal dominant tubulointerstitial kidney disease-uromodulin (ADTKD-UMOD). For one family with 3 generations affected, we identified p.R79G in a noncanonical transcript variant of UMOD co-segregating with disease. Consistent with ADTKD, most patients in our study presented with autosomal dominant inheritance, subnephrotic range proteinuria, minimal hematuria, and renal impairment. Kidney biopsies showed histologic features of glomerular injury consistent with secondary FSGS, including focal sclerosis and partial podocyte foot process effacement., Conclusion: Our study demonstrates that with the use of standard clinical testing and kidney biopsy, clinicians were unable to make the diagnosis of ADTKD-UMOD; patients were often labeled with a clinical diagnosis of FSGS. We show that genetic testing can establish the diagnosis of ADTKD-UMOD with secondary FSGS. Genetic testing in individuals with FSGS histology should not be limited to genes that directly impair podocyte function., (© 2020 International Society of Nephrology. Published by Elsevier Inc.)

Background: Genetic Variants in Apolipoprotein L1 (APOL1) are associated with large increases in CKD rates among African Americans. Experiments in cell and mouse models suggest that these risk-related polymorphisms are toxic gain-of-function variants that cause kidney dysfunction, following a recessive mode of inheritance. Recent data in trypanosomes and in human cells indicate that such variants may cause toxicity through their effects on mitochondria., Methods: To examine the molecular mechanisms underlying APOL1 risk variant-induced mitochondrial dysfunction, we generated tetracycline-inducible HEK293 T-REx cells stably expressing the APOL1 nonrisk G0 variant or APOL1 risk variants. Using these cells, we mapped the molecular pathway from mitochondrial import of APOL1 protein to APOL1-induced cell death with small interfering RNA knockdowns, pharmacologic inhibitors, blue native PAGE, mass spectrometry, and assessment of mitochondrial permeability transition pore function., Results: We found that the APOL1 G0 and risk variant proteins shared the same import pathway into the mitochondrial matrix. Once inside, G0 remained monomeric, whereas risk variant proteins were prone to forming higher-order oligomers. Both nonrisk G0 and risk variant proteins bound components of the mitochondrial permeability transition pore, but only risk variant proteins activated pore opening. Blocking mitochondrial import of APOL1 risk variants largely eliminated oligomer formation and also rescued toxicity., Conclusions: Our study illuminates important differences in the molecular behavior of APOL1 nonrisk and risk variants, and our observations suggest a mechanism that may explain the very different functional effects of these variants, despite the lack of consistently observed differences in trafficking patterns, intracellular localization, or binding partners. Variant-dependent differences in oligomerization pattern may underlie APOL1's recessive, gain-of-function biology., (Copyright © 2019 by the American Society of Nephrology.)

The Apolipoprotein L1 (APOL1) risk variants G1 and G2 are associated with high rates of kidney disease in African Americans in genetic studies. However, our understanding of APOL1 biology has lagged far behind. Here we report that engineering G1 and G2 mutations on unnatural haplotype backgrounds instead of on the specific G1 and G2 haplotype backgrounds that occur in nature profoundly alters APOL1-mediated cytotoxicity in experimental systems. Thus, in addition to helping resolve some important controversies in the APOL1 field, our demonstration of the critical influence of haplotype background may apply more generally to the study of other genetic variants that cause or predispose to human disease., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Natural mutations such as lysine 255 to glutamic acid (K to E), threonine 259 to isoleucine (T to I) and serine 262 to proline (S to P) that occur within the actin binding domain of alpha-actinin-4 (ACTN4) cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS) in affected humans. This appears due to elevated actin binding propensity in podocytes resulting in a 'frozen' cytoskeleton. What is challenging is how this cellular behavior would be compatible with other cell functions that rely on cytoskeleton plasticity. Our previous finding revealed that wild type ACTN4 can be phosphorylated at tyrosine 4 and 31 upon stimulation by epidermal growth factor (EGF) to reduce the binding to actin cytoskeleton. We queried whether the elevated actin binding activity of FSGS mutants can be downregulated by EGF-mediated phosphorylation, to discern a mechanism by which the actin-cytoskeleton can be released in FSGS. In this manuscript, we first constructed variants with Y4/31E to mimic the phosphorylation at tyrosines 4 and 31 based on earlier modeling simulations that predicted that this would bury the actin binding domains and lead to a decrease in actin binding activity. We found that Y4/31E significantly reduced the actin binding activity of K255E, T259I and S262P, dramatically preventing them from aggregating in, and inhibiting motility of, podocytes, fibroblasts and melanoma cells. A putative kinase target site at Y265 in the actin binding domain was also generated as a phosphomimetic ACTN4 Y265E that demonstrated even greater binding to actin filaments than K255E and the other FSGS mutants. That the tyrosine kinase regulation of FSGS mutation binding to actin filaments can occur in cells was shown by phosphorylation on Y4 and Y31 of the K225E after extended exposure of cells to EGF, with a decrease in ACTN4 aggregates in fibroblasts. These findings will provide evidence for targeting the N-termini of FSGS ACTN4 mutants to downregulate their actin binding activities for ameliorating the glomerulosclerotic phenotype of patients.

Background: Over the past two decades, the importance of genetic factors in the development of FSGS has become increasingly clear. However, despite many known monogenic causes of FSGS, single gene defects explain only 30% of cases., Methods: To investigate mutations underlying FSGS, we sequenced 662 whole exomes from individuals with sporadic or familial FSGS. After quality control, we analyzed the exome data from 363 unrelated family units with sporadic or familial FSGS and compared this to data from 363 ancestry-matched controls. We used rare variant burden tests to evaluate known disease-associated genes and potential new genes., Results: We validated several FSGS-associated genes that show a marked enrichment of deleterious rare variants among the cases. However, for some genes previously reported as FSGS related, we identified rare variants at similar or higher frequencies in controls. After excluding such genes, 122 of 363 cases (33.6%) had rare variants in known disease-associated genes, but 30 of 363 controls (8.3%) also harbored rare variants that would be classified as "causal" if detected in cases; applying American College of Medical Genetics filtering guidelines (to reduce the rate of false-positive claims that a variant is disease related) yielded rates of 24.2% in cases and 5.5% in controls. Highly ranked new genes include SCAF1 , SETD2 , and LY9 . Network analysis showed that top-ranked new genes were located closer than a random set of genes to known FSGS genes., Conclusions: Although our analysis validated many known FSGS-causing genes, we detected a nontrivial number of purported "disease-causing" variants in controls, implying that filtering is inadequate to allow clinical diagnosis and decision making. Genetic diagnosis in patients with FSGS is complicated by the nontrivial rate of variants in known FSGS genes among people without kidney disease., (Copyright © 2019 by the American Society of Nephrology.)

Introduction: Coding variants in apolipoprotein L-1 (APOL1) are associated with an increased risk of end-stage kidney disease (ESRD) in African American individuals under a recessive model of inheritance. The effect of the APOL1 risk alleles on kidney disease has been observed in studies in African American and African populations. Despite the 130 million individuals of recent African ancestry in South America, the impact of APOL1 has not been explored., Methods: In this case-control study, we tested APOL1 genotype in 106 Brazilian HD (hemodialysis) patients with African ancestry and compared risk allele frequency with 106 healthy first-degree relatives. The association of risk alleles and ESRD was calculated with a linear mixed model and was adjusted for relatedness and additional confounders. In a broader survey, the age of dialysis initiation and APOL1 variants were analyzed in 274 HD patients., Results: Two APOL1 risk alleles were 10 times more common in patients with ESRD than in controls (9.4% vs. 0.9%; odds ratio [OR]: 10.95, SE = 1.49, P  = 0.0017). Carriers of 2 risk alleles initiated dialysis 12 years earlier than patients with zero risk alleles., Conclusion: The APOL1 risk variants were less frequent in dialysis patients of African ancestry in Brazil than in the United States. Nonetheless, carriers of 2 risk variants had 10-fold higher odds of ESRD. Age of dialysis initiation was markedly lower in 2-risk allele carriers, suggesting a more aggressive disease phenotype. The Brazilian population represents an opportunity to identify different sets of genetic modifiers or environmental triggers that might be present in more extensively studied populations.

Two coding variants in the apolipoprotein L1 (APOL1) gene (termed G1 and G2) are strongly associated with increased risk of nondiabetic kidney disease in people of recent African ancestry. The mechanisms by which the risk variants cause kidney damage, although not well-understood, are believed to involve injury to glomerular podocytes. The intracellular localization and function of APOL1 in podocytes remain unclear, with recent studies suggesting possible roles in the endoplasmic reticulum (ER), mitochondria, endosomes, lysosomes, and autophagosomes. Here, we demonstrate that APOL1 also localizes to intracellular lipid droplets (LDs). While a large fraction of risk variant APOL1 (G1 and G2) localizes to the ER, a significant proportion of wild-type APOL1 (G0) localizes to LDs. APOL1 transiently interacts with numerous organelles, including the ER, mitochondria, and endosomes. Treatment of cells that promote LD formation with oleic acid shifted the localization of G1 and G2 from the ER to LDs, with accompanying reduction of autophagic flux and cytotoxicity. Coexpression of G0 APOL1 with risk variant APOL1 enabled recruitment of G1 and G2 from the ER to LDs, accompanied by reduced cell death. The ability of G0 APOL1 to recruit risk variant APOL1 to LDs may help explain the recessive pattern of kidney disease inheritance. These studies establish APOL1 as a bona fide LD-associated protein, and reveal that recruitment of risk variant APOL1 to LDs reduces cell toxicity, autophagic flux, and cell death. Thus, interventions that divert APOL1 risk variants to LDs may serve as a novel therapeutic strategy to alleviate their cytotoxic effects., Competing Interests: Conflict of interest statement: M.R.P. and D.J.F. have filed patents related to APOL1-associated kidney disease, and D.J.F. and M.R.P. own equity in ApoLo1 Bio, LLC.

Alport syndrome is a hereditary disease affecting Type IV collagen characterized by hematuria, progressive renal failure, sensorineural hearing loss, and ocular abnormalities. Most cases are X-linked and involve the COL4A5 gene with a minority of patients having autosomal recessive mutations in the COL4A3 or COL4A4 genes encoding the α3(IV) or α4(IV) chain respectively. Here, we describe the case of a 31-year-old woman who presented during pregnancy with hematuria and proteinuria and was diagnosed with autosomal recessive Alport syndrome (ARAS) post-partum. Her biopsy was notable for findings of segmental glomerulosclerosis with some collapsing features, in addition to thin basement membranes and rare "splitting". Genetic testing identified 2 novel mutations in the COL4A4 gene: a truncating frame shift mutation c.3861delinsCTC and a missense mutation c.4708G>A (p.Glu1570Lys), both of which we assert to be pathogenic. She had normal full-term delivery without complications. This case has several unique features including the relatively mild disease phenotype and the findings of glomerular scarring with collapsing features on renal biopsy. The successful pregnancy outcome and her clinical presentation add to the growing body of evidence that ARAS can have a variable phenotype., (© 2019 S. Karger AG, Basel.)