Role Of IQGAP1in Angiotensin Ⅱ-Induced Podocyte Apoptosis

Objective:Activation of the renin-angiotensin-aldosterone system (RAAS) promotes the progression of CKD, and inhibition of the RAAS retards proteinuria and progression to end-stage renal disease. Angiotensin II (AngⅡ), as a major effector of the RAAS, is the principal molecule mediating its function. AngⅡ has diverse nonhemodynamic effects on renal tissue, and elevated levels can lead to oxidative stress, expansion of the extracellular matrix, production of inflammatory cytokines, disruption of the cytoskeleton, cell cycle abnormalities, and apoptosis. Podocytes are terminally differentiated epithelial cells that are important components of the glomerular filtration barrier and their injury or depletion plays a pivotal role in the onset of proteinuria and progression of glomerular diseases. Our previous studies demonstrated that AngⅡ could induce podocyte apoptosis, but the precise underlying mechanism is unclear. A critical molecule associated with apoptosis in numerous types of cell is the scaffolding protein IQ domain GTPase-activating protein1(IQGAP1), which is ubiquitously expressed. Here, we explored the role of IQGAP1in AngⅡ-induced podocyte apoptosis and its molecular mechanism.Methods:Part1:Thirty-six male Wistar rats were randomly assigned to receive either saline or AngⅡ by osmotic mini-pump, or be used as normal control. The systolic blood pressure and proteinuria were measured at day7,14,21,28. After the animals were sacrificed at day14,28respectively, the kidneys were collected. Renal pathological changes, glomerular podocyte ultrastructure changes and podocyte apoptosis were observed. The glomerular expression of IQGAP1was assessed by immunohistoche-mistry, immunofluorescence, real-time PCR and Western blotting. The activation of caspase-3was determined by Western blotting. The correlation test between IQGAP1expression and caspase-3activation is conducted using partial correlation analysis.Part2:Conditionally immortalized mouse podocytes were cultured in vitro. Differentiated mouse podocytes were serum-free for12hours prior to treatment.(1) Podocytes were exposed to AngⅡ at10-8M for variable incubation time and at different concentrations for6h.(2) Podocyte apoptosis was assessed by flow cytometry and Hoechst-33258staining.(3) Expression of IQGAP1was analyzed by immunofluoresc-ence, real-time PCR and Western blotting.(4) IQGAP1siRNA was introduced to investigate the role of IQGAP1in AngⅡ-induced podocyte apoptosis.Part3:Conditionally immortalized mouse podocytes were cultured in vitro. Differentiated mouse podocytes were serum-free for12hours prior to treatment.(1) Mitogen-activated protein kinases (MAPK) pathway inhibitors were introduced to investigate the effects of MAPK signalings on the expression of IQGAP1and AngⅡ-induced podocyte apoptosis.(2) IQGAP1siRNA was introduced to investigate the role of IQGAP1in MAPK phosphorylation.(3) Co-immunoprecipitation was used to evaluate the interaction between IQGAP1and ERK1/2.Results:Part1:(1) AngⅡ-infused rats developed significant hypertension and marked proteinuria. Mild glomerular mesangial cell proliferation and mesangial matrix increase were also observed in AngⅡ-infused rats.(2) The transmission electron microscope analysis revealed diffuse foot process fusion or effacement, accompanied by chromatic agglutination, karyopyknosis, and nuclear fragmentation in podocytes of AngⅡ-infused rats on days14and28respectively.(3) The number of apoptotic podocytes in AngⅡ-infused rats was significantly more than that in normal control which was detected by TUNEL assay(P<0.05).(4) The expression of IQGAP1in glomeruli distributed linearly along the capillary loops, and colocalized with nephrin. AngⅡ infusion up-regulated glomerular expression of IQGAP1, which had an significantly positive correlation with activation of caspase-3.Part2:(1) AngⅡ promoted podocyte apoptosis in a dose-and time-dependent manner.(2) IQGAP1was located in celluar membrane and cytoplasm of cultured podocytes. Exposure to AngⅡ stimulated IQGAP1expression in a dose-and time-dependent manner.(3) Knockdown of IQGAP1with siRNA obviously prevented AngⅡ-induced apoptosis of podocytes.Part3:(1) Pretreatment with SB202190, SP600125, or U0126dramatically prevented AngⅡ-promoted podocyte apoptosis respectively. But the protein level of IQGAP1was not altered.(2) Knockdown of IQGAP1with siRNA obviously reduced AngⅡ-induced phosphorylation of ERK1/2, but not that of P38and JNK. This was accompanied by a reduced interaction between ERK1/2and IQGAP1. Conclusion:(1) AngⅡ promotes podocyte apoptosis in vivo and in vitro.(2) IQGAP1was located in celluar membrane and cytoplasm of podocyte.(3) AngⅡ elevated the IQGAP1expression in podocytes, and IQGAP1contributes to AngⅡ-induced apoptosis of podocytes by interacting with the ERK1/2signaling protein.