Poor outcomes in proteinuric kidney diseases are challenging to successfully manage therapeutically due to the heterogeneity of underlying disease pathogenesis and associated risk for progression. The role of cytoskeleton-associated proteins, including the scaffolding protein Anillin (ANLN), are of specific interest in kidney disease given the importance of actin dynamics in the kidney’s specialized epithelial cell types. In this study, we identify the prevalence of genetic variants in ANLN, the gene encoding ANLN, in a cohort of deeply phenotyped individuals with non-diabetic proteinuric kidney disease. Thirty-one individuals (of 864 genotyped) harbor heterozygously expressed variants in ANLN; 7 unrelated individuals shared the same variant (I1109V) in the C-terminal pleckstrin homology (PH) domain, a region necessary for interaction with the plasma membrane. Kidney organoids generated from I1109V induced pluripotent stem cells from 1 of these individuals showed increased epithelial cell mitogen-activated protein kinase 8 network activity and apoptosis, which was enhanced by tumor necrosis factor alpha (TNF-α) and phenocopied by actin polymerization inhibition. TNF-α-treated I1109V organoids also exhibited tubular lumen expansion. Knockdown and re-expression of the analogous ANLN variant in Xenopus laevis embryonic epithelia resulted in defects in cell-cell junction dynamics including wavy cell membranes exhibiting increased transverse movements as well as abnormal junctional F-actin remodeling in response to mechanical stress and leaky barrier function. Taken together, these results indicate that enhanced tubular epithelial cell death, perturbed cell-cell contacts and barrier function defects are associated with a novel ANLN variant discovered in individuals with non-diabetic proteinuric kidney disease.

ONE SENTENCE SUMMARY Enhanced tubular epithelial cell death and perturbed cell-cell junction integrity and barrier function are associated with a novel Anillin coding variant discovered in a cohort of individuals with proteinuric kidney disease.

The authors have declared no competing interest.

All NEPTUNE study related funding is in a pre-competitive framework in the context of an NIH funded grant and all data is owned by NEPTUNE; WGS was funded by NIH, Goldfinch Bio and Maze Therapeutics. National Institutes of Health grants P30DK081943 (ALM, JLH), UH3TR003288 (MK, JLH), R01GM112794 (ALM), R35GM153204 (ALM)(JAS), T32HD007505 (ZC), T32GM14547 (S. Wheeler) Startup funds from the Whiting School of Engineering at Johns Hopkins University (S. Weng)

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

IRB of the University of Michigan gave ethical approval for this work.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data associated with this study are present in the paper or the Supplementary Materials. Next-generation sequencing data have been uploaded to the NCBI GEO repository: scRNAseq (GSE316404) and bulk RNAseq (GSE314915). Previously published data sets from the NCBI GEO repository were used for comparison as noted in the Supplementary Materials and Methods (GSE213972 and GSE230848). NEPTUNE RNA-seq and ERCB RNA-seq data are available at Nephroseq.org (8, 68). Cell cluster marker lists for scRNAseq data sets and lists of DEGs used for the analysis of scRNAseq and bulk RNAseq data are detailed in Data File S1. Figure illustrations generated using BioRender are publicly viewable at the following unique figure URLs: Fig. 2B (https://BioRender.com/duaoc2w), Fig. 2D (https://BioRender.com/2d4z5ir), Fig. 3B (https://BioRender.com/uwe4l1d), Fig. 3C (https://BioRender.com/sy0qv70), Fig. 8 (https://BioRender.com/yxdgg5p). Cell lines are available upon request under a material transfer agreement. All other materials used or generated in this study are commercially available or will be supplied upon reasonable request.