Functionally Essential Tubular Proteins Are Lost to Urine-Excreted, Large Extracellular Vesicles during Chronic Renal Insufficiency.

BACKGROUND: The 5/6 nephrectomy (5/6Nx) rat model recapitulates many elements of human CKD. Within weeks of surgery, 5/6Nx rats spontaneously exhibit proximal tubular damage, including the production of very large extracellular vesicles and brush border shedding. We hypothesized that production and elimination of these structures, termed large renal tubular extracellular vesicles (LRT-EVs), into the urine represents a pathologic mechanism by which essential tubule proteins are lost. METHODS: LRT-EVs were isolated from 5/6Nx rat urine 10 weeks after surgery. LRT-EV diameters were measured. LRT-EV proteomic analysis was performed by tandem mass spectrometry. Data are available via the ProteomeXchange Consortium with identifier PXD019207. Kidney tissue pathology was evaluated by trichrome staining, TUNEL staining, and immunohistochemistry. RESULTS: LRT-EV size and a lack of TUNEL staining in 5/6Nx rats suggest LRT-EVs to be distinct from exosomes, microvesicles, and apoptotic bodies. LRT-EVs contained many proximal tubule proteins that, upon disruption, are known to contribute to CKD pathologic hallmarks. Select proteins included aquaporin 1, 16 members of the solute carrier family, basolateral Na+/K+-ATPase subunit ATP1A1, megalin, cubilin, and sodium-glucose cotransporters (SLC5A1 and SLC5A2). Histologic analysis confirmed the presence of apical membrane proteins in LRT-EVs and brush border loss in 5/6Nx rats. CONCLUSIONS: This study provides comprehensive proteomic analysis of a previously unreported category of extracellular vesicles associated with chronic renal stress. Because LRT-EVs contain proteins responsible for essential renal functions known to be compromised in CKD, their formation and excretion may represent an underappreciated pathogenic mechanism.

miR-146b-5p has a sex-specific role in renal and cardiac pathology in a rat model of chronic kidney disease.

Chronic kidney disease presents a complex and distinct pathological landscape in men and women, yet this difference is poorly understood. microRNAs are powerful molecular regulators of pathophysiology in the kidney and other organs. We previously reported a significant upregulation of miR-146b-5p in the 5/6 nephrectomy rat model of chronic kidney disease. Here we investigated the sex-specific contribution of miR-146b-5p to renocardiac pathology by generating a novel miR-146b-/- rat and characterized the expression of miR-146b-5p in both wild-type and knockout animals. The 5/6 nephrectomy or sham surgery was performed on rats of each genotype and sex. Renal pathology was examined through gross histology, plasma and urinary analysis of electrolytes and metabolites, and by chronic blood pressure monitoring. Cardiac pathology was monitored via echocardiography and pressure-volume analysis. The miR-146b-/- rats show functional knockout of miR-146b-5p in both the kidney and heart. While 5/6 nephrectomy induced tissue hypertrophy, miR-146b-/- female rats displayed exacerbated renal hypertrophy. Additionally, miR-146b-/- female rats exhibited a marked elevation of renal fibrosis and significant renal dysfunction yet lower blood pressure and less pronounced cardiac remodeling. These phenotypic differences were not exhibited in miR-146b-/- male rats. Ovariectomy ameliorated renal pathology and abolished genotypic differences. In vitro examination of transforming growth factor-ß signaling in combination with miR-146b-5p manipulation supports a role for miR-146b-5p in mediating the protective benefit of estrogen from renal pathologies. Thus, our data highlight an important role of miR-146b-5p in modulating kidney disease progression and provide new avenues for the study of sex-specific pathology.

MiR-146a/b: a family with shared seeds and different roots.

MicroRNAs are small, noncoding, RNAs known for their powerful modulation of molecular processes, making them a major focus for studying pathological mechanisms. The human miR-146 family of microRNAs consists of two member genes, MIR146A and MIR146B These two microRNAs are located on different chromosomes and exhibit differential regulation in many cases. However, they are nearly identical in sequence, sharing a seed region, and are thus predicted to target the same set of genes. A large proportion of the microRNA (miR)-146 literature focuses on its role in regulating the innate immune response in the context of various pathologies by modulating two widely studied target genes in the toll-like receptor signaling cascade. A growing subset of the literature reports a role of miR-146 in cardiovascular and renal disease, and data suggest there is exciting potential for miR-146 as a diagnostic and therapeutic target. Nevertheless, the published literature is confounded by unclear and imprecise language concerning the specific effects of the two miR-146 family members. The present review will compare the genomic origin and regulation of miR-146a and miR-146b, discuss some approaches to overcome analytical and experimental challenges, and summarize findings in major areas of miR-146 research. Moving forward, careful evaluation of miR-146a/b specificity in analytical and experimental approaches will aid researchers in elucidating the functional relevance of differential regulation of the miR-146 family members in health and disease.