ABSTRACT
BACKGROUND: Levels of urinary microvesicles, which are increased during various kidney injuries, have diagnostic potential for renal diseases. However, the significance of urinary microvesicles as a renal disease indicator is dampened by the difficulty to ascertain their cell source. OBJECTIVES: The aim of this study was to demonstrate that podocytes can release migrasomes, a unique class of microvesicle with size ranging between 400 and 2,000 nm, and the urine level of migrasomes may serve as novel non-invasive biomarker for early podocyte injury. METHOD: In this study, immunofluorescence labeling, electronic microscopy, nanosite, and sequential centrifugation were used to purify and analyze migrasomes. RESULTS: Migrasomes released by podocytes differ from exosomes as they have different content and mechanism of release. Compared to podocytes, renal tubular cells secrete markedly less migrasomes. Moreover, secretion of migrasomes by human or murine podocytes was strongly augmented during podocyte injuries induced by LPS, puromycin amino nucleoside (PAN), or a high concentration of glucose (HG). LPS, PAN, or HG-induced podocyte migrasome release, however, was blocked by Rac-1 inhibitor. Strikingly, a higher level of podocyte migrasomes in urine was detected in mice with PAN-nephropathy than in control mice. In fact, increased urinary migrasome number was detected earlier than elevated proteinuria during PAN-nephropathy, suggesting that urinary migrasomes are a more sensitive podocyte injury indicator than proteinuria. Increased urinary migrasome number was also detected in diabetic nephropathy patients with proteinuria level <5.5 g/day. CONCLUSIONS: Our findings reveal that podocytes release the "injury-related" migrasomes during migration and provide urinary podocyte migrasome as a potential diagnostic marker for early podocyte injury.
ABSTRACT
Canonical transient receptor potential-6 (TRPC6) channels have been implicated in a variety of chronic kidney diseases including familial and acquired forms of focal and segmental glomerulosclerosis (FSGS) and renal fibrosis following ureteral obstruction. Here we have examined the role of TRPC6 in progression of inflammation and fibrosis in the nephrotoxic serum (NTS) model of crescentic glomerulonephritis. This was assessed in rats with non-functional TRPC6 channels due to genomic disruption of an essential domain in TRPC6 channels (Trpc6 del/del rats) and wild-type littermates (Trpc6 wt/wt rats). Administration of NTS evoked albuminuria and proteinuria observed 4 and 28 days later that was equally severe in Trpc6 wt/wt and Trpc6 del/del rats. By 28 days, there were dense deposits of complement and IgG within glomeruli in both genotypes, accompanied by severe inflammation and fibrosis readily observed by standard histological methods, and also by increases in renal cortical expression of multiple markers (α-smooth muscle actin, vimentin, NLRP3, and CD68). Tubulointerstitial fibrosis appeared equally severe in Trpc6 wt/wt and Trpc6 del/del rats. TRPC6 inactivation did not protect against the substantial declines in renal function (increases in blood urea nitrogen, serum creatinine and kidney:body weight ratio) in NTS-treated animals, and increases in a urine maker of proximal tubule pathology (ß2-macroglobulin) were actually more severe in Trpc6 del/del animals. By contrast, glomerular pathology, blindly scored from histology, and from renal cortical expression of podocin suggested a partial but significant protective effect of TRPC6 inactivation within the glomerular compartment, at least during the autologous phase of the NTS model.
ABSTRACT
Objective:To explore the protective effect of rapamycin and the role of autophagy on podocyte injury induced by pu-romycin amino nucleoside.Methods:The mice podocytes cell 5(MPC5)were randomly divided into four groups:Control group,PAN group(50 μg/ml PAN);rapamycin group(RAP group,100 ng/ml,200 ng/ml,300 ng/ml rapamycin)and PAN+rapamycin group (PAN+RAP group,treated with rapamycin before PAN).The apoptosis of podocytes were detected by Annexin V/PI.Transmission electronic microscopy was used to observe the formation of autophagosome.The expression of autophagic markers,LC3,p62,and 4EBP1,P70S6K,mTOR were detected by Western blot.Results: The podocyte apoptotic rate was significantly decreased when the podocytes were treated with rapamycin compared with PAN group.Further,more autophagosomes appeared in cytoplasm of podocytes in RAP and PAN+RAP group compared with PAN group.And the expression of LC3Ⅱ was significantly increased in rapamycin treated podocytes,whereas the expression of p62 was decreased markedly.Phosphorylation of mTOR,4EBP1 and P70S6K was decreased rapamycin treated groups.Conclusion: Podocyte autophagy was inhibited by PAN, and the apoptosis of podocytes was promoted.Rapamycin protect puromycin amino nucleoside induced podocyte injury by upregulating autophagy and it may be concerned with mTOR 4EBP1 and P70S6K signaling pathway.
