Comprehensive Search for Novel Circulating miRNAs and Axon Guidance Pathway Proteins Associated with Risk of ESKD in Diabetes
Authors: Satake Eiichiro , Saulnier Pierre-Jean , Kobayashi Hiroki , Gupta Manoj K. , Looker Helen C. , Wilson Jonathan M. , Md Dom Zaipul I. , Ihara Katsuhito , O’Neil Kristina , Krolewski Bozena , Pipino Caterina , Pavkov Meda E. , Nair Viji , Bitzer Markus , Niewczas Monika A. , Kretzler Matthias , Mauer Michael , Doria Alessandro , Najafian Behzad , Kulkarni Rohit N. , Duffin Kevin L. , Pezzolesi Marcus G. , Kahn C. Ronald , Nelson Robert G. , Krolewski Andrzej S. 
Published: June 2021

Significance Statement
Mechanisms underlying the progression of diabetic kidney disease to ESKD are not fully understood. Through profiling of circulating microRNAs (miRNAs) and proteins in individuals with type 1 and type 2 diabetes from four independent cohorts, the authors identified a signature of 17 miRNAs and six axon guidance pathway proteins that were robustly associated with severity of early structural lesions in kidney biopsy specimens and with an increased 10-year risk of ESKD. The study reveals novel mechanisms and proteins that govern progression to ESKD and point to the importance of systemic factors in the development of diabetic kidney disease. Some of the circulating miRNAs and axon guidance pathway proteins represent potential targets for new therapies to prevent and treat this condition.

Background
Mechanisms underlying the pro gression of diabetic kidney disease to ESKD are not fully understood.

Methods
We performed global microRNA (miRNA) analysis on plasma from two cohorts consisting of 375 individuals with type 1 and type 2 diabetes with late diabetic kidney disease, and targeted proteomics analysis on plasma from four cohorts consisting of 746 individuals with late and early diabetic kidney disease. We examined structural lesions in kidney biopsy specimens from the 105 individuals with early diabetic kidney disease. Human umbilical vein endothelial cells were used to assess the effects of miRNA mimics or inhibitors on regulation of candidate proteins.

Results
In the late diabetic kidney disease cohorts, we identified 17 circulating miRNAs, represented by four exemplars (miR-1287-5p, miR-197-5p, miR-339-5p, and miR-328-3p), that were strongly associated with 10-year risk of ESKD. These miRNAs targeted proteins in the axon guidance pathway. Circulating levels of six of these proteins—most notably, EFNA4 and EPHA2—were strongly associated with 10-year risk of ESKD in all cohorts. Furthermore, circulating levels of these proteins correlated with severity of structural lesions in kidney biopsy specimens. In contrast, expression levels of genes encoding these proteins had no apparent effects on the lesions. In in vitro experiments, mimics of miR-1287-5p and miR-197-5p and inhibitors of miR-339-5p and miR-328-3p upregulated concentrations of EPHA2 in either cell lysate, supernatant, or both.

Conclusions
This study reveals novel mechanisms involved in progression to ESKD and points to the importance of systemic factors in the development of diabetic kidney disease. Some circulating miRNAs and axon guidance pathway proteins represent potential targets for new therapies to prevent and treat this condition.