A priori clinical diagnosis of CKD is defined as pre-WES clinical diagnosis per referral by primary nephrologist. CAKUT, congenital anomalies of the kidney and urinary tract; CKD, chronic kidney disease; ESKD, end-stage kidney disease; GN, glomerulonephritis; SRNS, steroid-resistant nephrotic syndrome; TIKD, tubulointerstitial kidney disease; WES, whole exome sequencing. a Age at first presentation to medical services with evidence of CKD. b Age at start of renal replacement therapy, i.e., dialysis or kidney transplantation. DM Connaughton et al.: Monogenic causation of chronic kidney disease in Ireland c l i n i c a l i n v e s t i g a t i o n Kidney International (2019) 95, 914-928 DM Connaughton et al.: Monogenic causation of chronic kidney disease in Ireland c l i n i c a l i n v e s t i g a t i o n

We identified monogenic mutations in a known human CAKUT gene or CAKUT phenocopy gene as the cause of disease in 14% of the CAKUT families in this study. Whole-exome sequencing provides an etiologic diagnosis in a high fraction of patients with CAKUT and will provide a new basis for the mechanistic understanding of CAKUT.

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CA-KUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.

Nephrotic syndrome (NS) is a leading cause of chronic kidney disease. We found recessive NOS1AP variants in two families with early-onset NS by exome sequencing. Overexpression of wild-type (WT) NOS1AP, but not cDNA constructs bearing patient variants, increased active CDC42 and promoted filopodia and podosome formation. Pharmacologic inhibition of CDC42 or its effectors, formin proteins, reduced NOS1AP-induced filopodia formation. NOS1AP knockdown reduced podocyte migration rate (PMR), which was rescued by overexpression of WT Nos1ap but not by constructs bearing patient variants. PMR in NOS1AP knockdown podocytes was also rescued by constitutively active CDC42Q61L or the formin DIAPH3. Modeling a NOS1AP patient variant in knock-in human kidney organoids revealed malformed glomeruli with increased apoptosis. Nos1apEx3−/Ex3− mice recapitulated the human phenotype, exhibiting proteinuria, foot process effacement, and glomerulosclerosis. These findings demonstrate that recessive NOS1AP variants impair CDC42/DIAPH-dependent actin remodeling, cause aberrant organoid glomerulogenesis, and lead to a glomerulopathy in humans and mice.

BackgroundPodocyte detachment and apoptosis are two risk factors causing podocyte loss, F-actin rearrangement is involved in detachment and apoptosis. However, the nature of events that promote detachment and apoptosis of podocytes and whether detachment occurred simultaneously with apoptosis are still unclear. Previously, it was found that angiopoietin-like3 (Angptl3) induces F-actin rearrangement in podocytes. In this study we investigate whether Angptl3 influences podocyte loss (detachment and apoptosis) and the process through which Angptl3 exactly influenced the podocyte loss.MethodsIn conditionally immortalized mice podocytes, recombinant mice Angptl3 protein (rm-Angptl3) was used to mimic Angptl3 overexpression model and transfection with small interfering RNA (siRNA) to knockdown the expression of Angptl3. Both flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay were used to detect apoptosis. Rearrangement of F-actin was assessed using confocal microscopy. Western blot assay was used to measure levels of Angptl3, integrin α3β1, integrin-linked kinase (ILK), p53, caspase 3, and phosphorylation of integrin β1.ResultsIn a puromycin aminonucleoside (PAN)-induced podocyte injury model, rm-Angptl3 accelerated the loss of podocytes, both detachment and apoptosis occurred, and F-actin rearrangement is involved in the process. However, knockdown of Angptl3 by siRNA markedly ameliorated these injuries. Observed effects were partially correlated with the altered integrin α3β1, ILK and p53, rather than caspase 3.ConclusionsAngptl3 is a novel factor involved in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro. This study helps to deepen the understanding of the mechanisms of podocyte loss and lays the foundation for developing a new successful therapy for podocyte injury via lower expression of Angptl3.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life, and in utero obstruction to urine flow is a frequent cause of secondary upper urinary tract malformations. Here, using whole-exome sequencing, we identified three different biallelic mutations in CHRNA3, which encodes the a3 subunit of the nicotinic acetylcholine receptor, in five affected individuals from three unrelated families with functional lower urinary tract obstruction and secondary CAKUT. Four individuals from two families have additional dysautonomic features, including impaired pupillary light reflexes. Functional studies in vitro demonstrated that the mutant nicotinic acetylcholine receptors were unable to generate current following stimulation with acetylcholine. Moreover, the truncating mutations p.Thr337Asnfs*81 and p.Ser340* led to impaired plasma membrane localization of CHRNA3. Although the importance of acetylcholine signaling in normal bladder function has been recognized, we demonstrate for the first time that mutations in CHRNA3 can cause bladder dysfunction, urinary tract malformations, and dysautonomia. These data point to a pathophysiologic sequence by which monogenic mutations in genes that regulate bladder innervation may secondarily cause CAKUT.

Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the leading cause of chronic kidney disease in children. In total, 174 monogenic causes of isolated or syndromic CAKUT are known. However, syndromic features may be overlooked when the initial clinical diagnosis of CAKUT is made. We hypothesized that the yield of a molecular genetic diagnosis by exome sequencing (ES) can be increased by applying reverse phenotyping, by re-examining the case for signs/symptoms of the suspected clinical syndrome that results from the genetic variant detected by ES. Methods: We conducted ES in an international cohort of 731 unrelated families with CAKUT. We evaluated ES data for variants in 174 genes, in which variants are known to cause isolated or syndromic CAKUT. In cases in which ES suggested a previously unreported syndromic phenotype, we conducted reverse phenotyping. Results: In 83 of 731 (11.4%) families, we detected a likely CAKUT-causing genetic variant consistent with an isolated or syndromic CAKUT phenotype. In 19 of these 83 families (22.9%), reverse phenotyping yielded syndromic clinical findings, thereby strengthening the genotype-phenotype correlation. Conclusion:We conclude that employing reverse phenotyping in the evaluation of syndromic CAKUT genes by ES provides an important tool to facilitate molecular genetic diagnostics in CAKUT.

Background Angiopoietin-like-3 (Angptl3) knockout is known for its protective effects on podocyte injury and proteinuria in the early stage of adriamycin (ADR) nephropathy. The current study re-evaluated the renoprotective effect of Angptl3 knockout in chronic ADR nephropathy and attempted to explore the mechanism underlying the effect associated with Angptl3 knockout in glomerulosclerosis. Methods B6; 129S5 mice were injected with ADR to induce nephropathy. Kidney structure and serum and urine parameters were observed during long-term follow-up. Cultured primary mouse podocytes were exposed to ADR and analyzed for the expression of some relative proteins. Podocyte loss was analyzed in both in vivo and in vitro experiments. Results Angptl3 knockout attenuated proteinuria and hypoproteinemia, protected renal structure and function, and improved the survival of mice over the whole process of ADR nephropathy. Furthermore, Angptl3 knockout reduced the numbers of the detached and apoptotic cells in the renal tissue and alleviated podocyte loss in mice with ADR chronic nephropathy, thereby, delaying the glomerulosclerosis formation. Additional results in vitro showed that Angptl3 knockout attenuated ADR-induced primary podocyte loss, including podocyte detachment and apoptosis. Conclusion In addition to serving a renoprotective role in the early stage of ADR nephropathy, Angptl3 knockout contributed to disease amelioration throughout the ADR nephropathy process. Angptl3 knockout effectively delayed glomerulosclerosis formation by attenuating podocyte loss through rescuing podocytes from detachment and apoptosis. Angptl3 antagonists or inhibitors might have therapeutic potential in the occurrence and progression of nephropathy. Electronic supplementary material The online version of this article (10.1186/s12882-019-1383-1) contains supplementary material, which is available to authorized users.