PKA/CREB Signaling Prevented Against Podocyte Apoptosis Via Upregulation Of Mitochondrial OXPHOS Complexes

Background: Glomerular vascular epithelial cells(podocytes) are terminal differentiated cells with plenty of foot processes. Slit diaphragm formed between adjacent podocyte foot processes is the key molecule barrier for plasma protein filtration. The decrease in the number of podocytes, foot process fusion or reduced slit diaphragm protein expression was associated with pathological proteinuria. Therefore, podocyte apoptosis or necrosis is the important events for the progression of glomerular disease. Our previous studies showed that activation of metabolic glutamate receptor(m Glu R) 1/5 prevented puromycin aminonucleoside(PAN)-induced apoptosis via cyclic adenosine monophosphate(c AMP)/protein kinase A(PKA) signaling. Classic PKA signaling performs its biological function via activation of downstream transcription factor c AMP response element binding protein(CREB), which binds c AMP response element and regulates target gene transcription. The role of CREB in c AMP/PKA-induced protection is still unknown. We showed that PKA signaling prevented PAN-induced mitochondria fission in podocytes, suggesting that mitochondira function may be important. Actually, the main function of mitochondria is the production of energy for tissue cells by oxidative phosphorylation. We have no idea for the role of oxidative phosphorylation dysfunction in podocytes.Objective: To explore the role of transcription factor CREB in the PKA-induced protection in podocytes, as well as the effects on mitochondrial oxidative phosphorylation and ATP production.Methods: Conditionally immortalized differentiated murine podocytes between 15 and 25 generation were used in the present experiments. Cell toxicity was examined by using a cell count kit-8. Annexin V/PI staining and flow cytometry were uesd to detect cell apoptosis. Immunofluorescence staining and confocal laser scanning microscopy were used to estimate the localization and expression of p-CREB. RNA interference(RNAi) was used to inhibit the CREB expression. We also used Agilent expression profile chip to screen the m RNA expression in differential podocytes treated with or without PKA agonist in the presence or absence ADR. The message RNAs of respiratory chain complexes subunits encoded by mitochondrial genes were detected by using real-time PCR. Luciferase chemiluminescence was used to detect the production of ATP in podocytes. Western blot was used to detect protein expression.Results: We found that Adriamycin(ADR) induced podocyte apoptosis. Pretreatment with p CPT-c AMP(PKA specific agonist) prevented podocytes against ADR-induced increase of cleaved caspase-3 and the loss of podocytes. Treatment with p CPT-c AMP 5 min and 15 min resulted the increase of CREB phosphorylation in total cell Cell lysis solution(increased by 105.64±38.21% and 98.61±41.03% respectively, compared with control group p<0.05) and in nuclear lysis(increased by 114.52±11.28% and 118.84±14.44% respectively, compared with control group p<0.05). Confocal immunofluorescence showed treatment with p CPT-c AMP mainly upregulate phosphorylated CREB(p-CREB) expression in nucli. RNA interference decreased CREB protein expression to 25.1±2.44%. Pretreatment with p CPT-c AMP was unable to prevent ADR-induced increase of cleaved caspase 3 expression in CREB RNAi treated podocytes. Data of Agilent expression profile chip studies showed that ADR prodominatly decreased the m RNA expression of respiratory chain complex I subunits encoded by mitochondrial genes in podocytes, which was prevented by pretreatment with p CPT-c AMP. Real-time PCR verified these findings. Immunoblot experiments showed that activation of PKA prevented ADR-induced decrease of mitochondrial respiratory chain complexes I subunits ND1/3/4 protein expression. Inhibiting CREB expression by using RNAi prevented p CPT-c AMP-induced ND3, but not ND1/4 protein overproduction in podocytes. CREB RNAi can also block p CPT-c AMP-induced increase of ATP and the expression of Peroxisome proliferator-activated receptor gamma coactivator-1 alpha(PGC-1 a).Conclusion: PKA signaling pathway might prevent podocyte injury in a CREB dependent manner. Upregulation of mitochondrial respiratory chain protein at least played a partial role in the protection of PKA/CREB signaling.