The Role Of Autophagy Mediated By MiR-30a-targeting Beclin 1 In Delaying The Progression Of Podocytopathies

Background and Objective:Podocytopathies like minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are common cause of idiopathic nephrotic syndrome. The pathogenesis of MCD and FSGS are podocyte injury. Podocyte dysfunction and loss play a key role in the emergence of glomerulosclerosis and the progression of kidney disease. As terminally differentiated cells, how to maintain podocyte function is particularly important. Autophagy has been shown to be an important mechanism for cells to maintain homeostasis, responded to a variety of cell injury and stress. In the previous study we have found that microRNA30a also plays an important role in the pathogenesis of podocyte injury. In recent years, it is confirmed that Beclin 1, a key autophagy-promoting gene, is a potential target for miR-30a. miR-30a could negatively regulate Beclin 1 expression resulting in suppressed autophagy acivity in cancer cells. This study is established to identify the role of miR-30a in the regulation of Beclin 1 mediated autophagy in podocyte, and the role of autophagy in the podocyte injury.Methodology:Part One:30 patients with biopsy proven MCD and 30 patients with biopsy proven FSGS having urine protein≥3.5g/24h were enrolled in the study. The study also included 21 patients with biopsy proven MCD who were received repeat renal biopsy.12 patients of them were remaining MCD and 9 patients progressed to FSGS at repeat biopsy time. The podocyte and autophagysome number of kidney tissue were observed by electron microscopy. The podocyte containing autophagysome occupying the total number of podocytes is definited as podocyte autophaysome positive rate. Autophagy was measured by formation of LC3-punctum by Immunofluorescence and LC3 or Beclin 1 expression by Immunohistochemical.Part Two:(1) Three types of cultured human podocyte (untreated, miR-NC, miR-30a overexpression podocyte) were treated with PAN. After 6hr/12hr of PAN (50ug/ml) treatment, the expression of miR-30a in podocyte was measured by RT-PCR, Beclin 1 and LC3-Ⅱ bands were measured by western blot. The number of LC3-punctum was measured by Immunofluorescence. Moreover, podocyte were pretreated with autophagy inhibitor (3-MA, chloroquine, Beclin 1 siRNA translated) and autophagy stimulator (Rapamycin) followed by treated with PAN. The apoptotic cells were measured by Flow cytometry. (2) Wistar rats were used to generate a PAN-induced podocyte injury model. PAN (4mg/100g) was diluted in 0.9% NaCl (10mg/ml) and was injected into the rats through the left jugular vein with a single dose of 4mg/100g body weight at day 0. PAN model Rats were pretreated with 3-MA, miR-30a overexpression plasmid and rapamycin. The vehicle-treated group received 0.9% NaCl in the same approach. Renal tissues were processed using the standard method for histological and immunofluorescence microscopy studies. Urine samples were collected at day 1-7,10,14 and 20. Blood samples were collected at day 4,7 and 20. Autophagy was measured by detecting LC3, Beclin 1 and P62 in methods of western blot, immunofluorescence and immunohistochemisty.Results:Part One:The present of autophagy somes in podocytes was demonsrated by electron microscopy and immunofluorescence. Podocyte autophaysome positive rate in the patients with FSGS was lower than that in patients with MCD (26%±0.19 vs 37%±0.16, P<0.05). There was significantly decreased of podocyte autophaysome positive rate in patients with FSGS at repeat biopsy as compared with first biopsy (22%±0.11 vs 45%±0.10, P<0.05). However, no change of podocyte autophaysome positive rate was observed in patients with MCD remaining (39%±0.08 vs 39%±0.10, P>0.05).Part Two:(1) In this study, PAN could decreased the expression of CD2AP, podocin and synaptopodin in podocyte, increased the apoptotic rate of podocyte, and downregulated the expression of miR-30a in podocyte over time. PAN also could induce activating autophagy in podocyte displayed as LC3-punctum by Immunofluorescence and LC3-Ⅱ and Beclin 1 band by western blot. (2) As compared with miR-NC group, overexpression of miR-30a in podocytes inhibited PAN induced the increase of autophagysome, Beclin 1 and LC3-Ⅱ/LC3-Ⅰ, however, increased PAN induced podocyte apoptosis (PAN 20ug/ml:14.8±0.85% vs 9.6±1.54%; PAN 35ug/ml:15.4±1.41% vs 10.95±0.75%, P<0.05), decreased the expression of synaptopodin, CD2AP and podocin in podocytes. (3) Autophagy inhibitor (3-MA and Chloroquine) pretreated podocyte or Beclin 1-siRNA translated podocyte significantly increased the PAN-induced podocyte apoptosis, decreased the expression of synaptopodin, CD2AP and podocin in podocytes (P<0.05). (4) 3-MA and miR-30a overexpression plasmid pretreated rats had earlier and more proteinuria, more entensive footprocess effacement and decreasing podocyte marker as compared with control rats. They also had attenuated upregulation of LC3-Ⅱ, Beclin 1 and downregulation of P62 in glomeruli. Rapamycin pretreated rats had decreased peak proteinuria, less footprocess effacement and increased of LC3-Ⅱ/LC3-Ⅰ as compared with PAN control group, however, there was no difference of the expression of podocin or synaptopodin between these two groups.Conclusions:(1) Podocyte autophagy positive rate decreased in patients with MCD progressing to FSGS and was less in patients with FSGS than that with MCD. It suggested that podocyte autophage play a key role in podocyte injury and the progress of podocytepathies.(2) miR-30a had the role of negative regulation Beclin 1 expression in podocyte and could reduce Beclin 1-mediated autophagy activity. It could enhance PAN-induced podocyte injury.(3) Inhibiting autophagy activity could promote the PAN-induced rats having earlier, more protienturia and more entensive podocyte injury. While activating autophagy may ameliorate the PAN-induced podocyte injury. It suggested that podocyte autophage play a key role in maintaining its function and preventing podocyte from injury.