CELLULAR PRION PROTEIN ATTENUATES RENAL FIBROSIS BY INTERACTING WITH TSPAN7 TO INDUCE FORMATION OF MIGRASOME

CELLULAR PRION PROTEIN ATTENUATES RENAL FIBROSIS BY INTERACTING WITH TSPAN7 TO INDUCE FORMATION OF MIGRASOME

Migrasome is a new type of organelle that was initially discovered in renal tubular epithelial cells. The amount of migrasome increases in urine after kidney injury. Cellular prion protein (Prpc) is a highly conserved soluble secretory protein that is mainly present in the extracellular matrix and functions as a "molecular messenger" between cells. In the kidneys, Prpc is the molecular chaperone of the tetraspanin 7 (TSPAN7), which is the main component of migrasome. The aim of this study is to test the hypothesis whether Prpc acts on the TSPAN7 protein in renal tubular epithelial cells to mediate the formation of migrasome, thereby promoting the repair of renal tubular epithelial cells and alleviating AKI.

Wild-type FVB mice, Prnp-/- mice were used for in vivo studies. Renal I/R injury model was induced by bilateral renal pedicle clamping for 35 minutes. Changes in the expression levels of renal Prpc and coding gene Prnp were observed by Westernblotting and PCR. Localization and source of Prpc in the kidney were clarified by immunofluorescence. Overexpressing Prnp fibroblasts (PrnphiFib) and HK-2 cells (PrnpkiHK-2) were construct by CRISPR-Cas9. HK-2 cells were transwell co cultured with PrnphiFib or treatment with Prpc recombinant protein, followed by H/R treatment, to observe the number of migrasome, the content of mitochondrial markers migrasome (KIF5B, Drp1, Myosin19, etc.), and morphology of migrasome. And immunoprecipitation and immunofluorescence experiments were used to observe the interaction between Prpc and TSPAN7, as well as their co-localization in migrasome.

The expression of Prpc gradually increased from 1 hour after I/R injury and reached its peak at 48 hours, as a fibroblast marker α-SMA and Vimentin significantly increased in the early stages of injury (12 hours after I/R). Further research has found that compared with wild mice, Prnp-/- mice have more severe kidney damage, higher blood creatinine levels, and more damaged mitochondria. The expression level of Prnp in isolated fibroblasts is about 25 times higher than that in renal tubular epithelial cells. PrnphiFib can significantly inhibit the apoptosis of HK-2 cells, and its effect is superior to the anti-apoptotic effect of overexpressing Prnp in HK-2 cells themselves. HK-2 cells form a large number of migrasome under the induction of fibroblast active substance FN. After H/R damage, the number and volume of migrasome as well as the expression levels of Prpc and TSPAN7 were increased. In I/R model, knocking out Prnp significantly inhibited I/R induced TSPAN7 overexpression. Additionally, immunoprecipitation experiments indicated that Prpc can bind to TSPAN7 directly, and they are co located in the migrasome; Knocking out of TSPAN7 inhibits H/R induced migrasome formation and exacerbates damage to renal tubular epithelial cells; The Prion recombinant protein can promote the formation of migrasome and alleviate H/R induced damage to renal tubular epithelial cells.

Fibroblasts are the main source of endogenous Prpc in the kidney. After injury, rapidly activated fibroblasts secrete Prpc, which acts on the TSPAN7 protein in renal tubular epithelial cells, mediates the formation of migrasome, promotes the repair of renal tubular epithelial cells, and reduces AKI.