The Role Of Podocyte-secreted Microvesicles On Renal Tubular Cells

Focal segmental glomerulosclerosis (FSGS) is a common glomerular disease, whichis named by the morphological characteristics of renal pathology, with the complexity ofthe pathogenesis and limited nature of our knowledge. It is clinically manifested asmassive proteinuria and podocyte injury, but tubulointerstitial injury is also a prominentfeature of FSGS. Possible mechanisms include massive proteinuria, hypoxia ofperitubular capillaries, the activation and release of cytokines. However, thesemechanisms could not fully explain the phenomena observed in the clinical. Therefore, itis important to clarify the mechanisms of glomerular diseases leading to tubulointerstitialinjury.Besides soluble factors, cell-derived microvesicles (MVs) were described as a newmechanism of cell-to-cell communication. MVs are released by various cell types,including glomerular podocytes, tubular epithelial cells and stem cells, etc. MVs mayinteract with target cells by surface-expressed ligands, transfer surface receptors, deliverproteins, mRNA, and bioactive lipids. It was showed that MVs derived from embryonicstem cells could reprogram hematopoietic progenitors by an mRNA-dependentmechanism. And microvesicles derived from MSCs could activate a proliferativeprogram in surviving tubular cells after injury via a horizontal transfer of mRNA.Another study showed that microvesicles derived from endothelial progenitor cellsprotected the kidney from ischemia–reperfusion injury by microRNA-dependentreprogramming of resident renal cells.In the present study, we hypothesized whether injured or activated podocytes-derivedMVs could induce tubular cells injury, revealing a new tubular injury mechanism in theglomerular diseases.Part oneThe identification of podocytes-derived microvesiclesObjective:To purify MVs from human podocytes and urine and then observe its biologicalcharacteristics. Metholodlogy:MVs were purified from culture supernatant of human podocyte cell line and urineusing a three-step purification protocol and observed by electron microscopy. Theexpression of MV specific membrane marker-CD63was dectected by immuno-electronmicroscopy.Results:1. Transmission and scanning electron microscopy showed the spheroid morphologyand their size of purified MVs.2. Western blot and immuno-electron microscopy confirmed the expression of MVs’specific marker CD63.Conclusion:Bothe the normal and damaged podocyte could secret MVs.Part twoThe Role of Podocyte-secreted Microvesicles on Renal Tubular Cells InjuryObjective:To observe the effect of podocyte-secreted microvesicles on renal tubular cellsinjury.Methodology:MVs were isolated from normal and LPS stimulated podocyte and labeled withPKH26, and then incubated with HK-2cells6h in different concentrations (80ug/ml,150ug/ml).β-catenin and Megalin expression in HK-2cells were analyzed byimmunofluorescence. β-catenin, E-cadherin and Megalin mRNA expression weredetected by RT-PCR.β-catenin and E-cadherin protein expression were detected byWestern blot.Result:1. PKH26(granular red fluorescent) could be observed in cytoplasm of HK-2cells by fluorescence microscopy.2．Compared with normal podocyte secreted MVs, β-catenin expression was decreased byWestern blot analysis, with incontinuous staining on HK-2cells incubated with LPSstimulated-podocyte secreted MVs at given concentrations, especially150ug/ml.Immunofluorescence of Megalin expression showed down-regulation. RT-PCR resultsshowed that the mRNA levels of β-catenin and megalin were decreased.Conclusion:1. Podocyte secreted MVs could enter HK-2cells.2. Damaged podocyte secreted MVs induced HK-2cells injury.