Defining the molecular correlate of arteriolar hyalinosis in kidney disease progression by integration of single cell transcriptomic analysis and pathology scoring

Arteriolar hyalinosis in kidneys is an independent predictor of cardiovascular disease, the main cause of mortality in chronic kidney disease (CKD). The underlying molecular mechanisms of protein accumulation in the subendothelial space are not well understood. Using single cell transcriptomic data and whole slide images from kidney biopsies of patients with CKD and acute kidney injury in the Kidney Precision Medicine Project, the molecular signals associated with arteriolar hyalinosis were evaluated. Co-expression network analysis of the endothelial genes yielded three gene set modules as significantly associated with arteriolar hyalinosis. Pathway analysis of these modules showed enrichment of transforming growth factor beta / bone morphogenetic protein (TGFβ / BMP) and vascular endothelial growth factor (VEGF) signaling pathways in the endothelial cell signatures. Ligand-receptor analysis identified multiple integrins and cell adhesion receptors as over-expressed in arteriolar hyalinosis, suggesting a potential role of integrin-mediated TGFβ signaling. Further analysis of arteriolar hyalinosis associated endothelial module genes identified focal segmental glomerular sclerosis as an enriched term. On validation in gene expression profiles from the Nephrotic Syndrome Study Network cohort, one of the three modules was significantly associated with the composite endpoint (> 40% reduction in estimated glomerular filtration rate (eGFR) or kidney failure) independent of age, sex, race, and baseline eGFR, suggesting poor prognosis with elevated expression of genes in this module. Thus, integration of structural and single cell molecular features yielded biologically relevant gene sets, signaling pathways and ligand-receptor interactions, underlying arteriolar hyalinosis and putative targets for therapeutic intervention. ### Competing Interest Statement Dr. Hodgin reports grants outside of this work from NIH, Department of Defense, Gilead, Astra Zeneca, Moderna, Novo Nordisk, Janssen and Eli Lilly. Dr. Kretzler reports grants and contracts through the University of Michigan with the National Institutes of Health, Chan Zuckerberg Initiative, AstraZeneca, NovoNordisk, Eli Lilly, Gilead, Goldfinch Bio, Janssen, Boehringer-Ingelheim, Moderna, European Union Innovative Medicine Initiative, Certa, Chinook, amfAR, Angion, RenalytixAI, Travere, Regeneron, IONIS and Maze Therapeutics. He has received consulting fees through the University of Michigan from Astellas, Poxel, Janssen and UCB. Dr. Kretzler serves on the NIH-NCATS council and is on the board of Nephcure Kidney International. In addition, M.K. has a patent PCT/EP2014/073413, Biomarkers and methods for progression prediction for chronic kidney disease licensed. Dr. Rosas has funding support from Bayer, AstraZeneca and NIH/NIDDK. She is a scientific advisory board member for Bayer, AstraZeneca and Travere. Dr. DAgati receives royalties from UpToDate and editorial board member of Kidney International. Dr. Vazquez is on the Advisory Committee of the UT Southwestern Health Systems- DaVita Dialysis Joint Venture. Dr. Randhawa has received consulting fees from Allovir Inc, is a member on the Board of Directors of the Renal Pathology Scoiety, Chair of th Banff Foundation of Allograft Pathology TCMR Working Group and Editor of Clinical Transplantation. Dr. Barisoni serves on the advisory boards of Vertex, International Society of Glomerular Diseases and Nephcure- Scientific Advisory Board. Dr. Henderson has received grants outside of current study from Pfizer, Q32 Bio, Visterra. He is on the advisory board and has received consulting fees from Novartis. JMH has a patent pending entitled, Methods for identifying and treating antibody-mediated acquired primary or recurrent idiopathic nephrotic syndrome. Dr. Palevsky reports grant support from the NIH/NIDDK for the current work and consulting for Janssen Research & Development, LLC unrelated to the current work. Outside of current work, Dr. Rosas has funding from NIH/NIDDK, Bayer, Astrazeneca, has participated in the data monitoring board for the NAD+ Augmentation in Cardiac Surgery Associated Myocardial Injury Trial (NACAM) and was a President of the National Kidney Foundation. ### Funding Statement The KPMP data presented here is funded by the following grants from the NIDDK: U2C DK114886, UH3DK114861, UH3DK114866, 2U01DK114866-07, UH3DK114870, UH3DK114908, UH3DK114915, UH3DK114926, UH3DK114907, UH3DK114923 and UH3DK114933. This work was supported in part by the Intramural Research Program at the National Institute of Diabetes and Digestive and Kidney Diseases (DK069062) to RGN, the American Diabetes Association (Clinical Science Award 1-08-CR-42) to RGN, and (DK083912, DK082841, DK020572, DK092926) to RGN, by the extramural research program of the National Institute of Diabetes and Digestive and Kidney Diseases R24 DK082841 and P30 DK081943 to MK. The Nephrotic Syndrome Study Network Consortium (NEPTUNE), U54-DK-083912, is a part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network (RDCRN), supported through collaboration between the Office of Rare Diseases Research, National Center for Advancing Translational Sciences and the National Institute of Diabetes, Digestive, and Kidney Diseases. Additional funding and/or programmatic support for this project has also been provided by the University of Michigan, the NephCure Kidney International and the Halpin Foundation. A.S.N. receives salary and research support from NIDDK (K23 DK125529). Christine Limonte was supported by the Clinical Scientist in Nephrology Award from the American Kidney Fund. Dr. Vazquez was supported by grants UH3DK114870-03 and UO1DK133091-01 from the National Institute of Diabetes, Digestive and Kidney Diseases. ### Author Declarations 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: IRBMED, University of Michigan, approved HUM00150968 University of Washington Institutional Review Board approved IRB#20190213 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 The KPMP single cell data used in this study is available from . Other datasets used include transcriptomic data from NEPTUNE, an American Indian cohort and the Human Kidney Transplant Transcriptomic Atlas. Neptune mRNA sequencing and clinical data are controlled access data and will be available to researchers upon request to NEPTUNE-STUDY@umich.edu. Owing to ethical considerations and privacy protection concerns, and to avoid identifying individual study participants in Pima Indian population, the Institutional Review Board of the National Institute of Diabetes and Digestive and Kidney Diseases has stipulated that individual-level gene expression and genotype data from this study cannot be made publicly available for the American Indian cohort. The single cell count matrices for the Human Kidney Transplant Transcriptomic Atlas are available from the Gene Expression Omnibus under accession number GSE 169285.