Abstract 12279: CD40, a Significant Mediator of Hypertensive Renal Injury, Activates Pro-Apoptotic P53 Signaling in Human Renal Proximal Tubule Cells

Abstract

Background: We have shown clinical evidence that circulating levels of the proinflammatory receptor CD40 contribute to the progression of hypertensive renal disease and that the ligand for CD40 (sCD40L) is significantly elevated in these patients. Experimentally, we developed a CD40 knockout (CD40KO) rat model on the Dahl-S background that is prone to hypertensive renal injury, and demonstrated significantly reduced renal inflammation and proteinuria following high salt diet in the CD40KO vs Dahl-S controls. On this background we used HK-2 cells and created a stable CD40 knockout cell line (HK-2CD40KO) and performed the following experiments. Hypothesis: Renal proximal tubule-specific CD40 signaling significantly contributes to the development of renal injury. Methods: HK2 cells and HK2-CD40KO cells were stimulated with 100 ng/ml of soluble CD40 ligand (sCD40L) for 24hrs. RNA from duplicate samples was isolated for RNA sequencing followed by bioinformatic pathway analysis (R program). Additionally, one-week high salt diet (2% NaCl) was administered to seven-week old Dahl S wild-type and Dahl S CD40KO male rats (n=8 per group) and kidney cell apoptosis was evaluated by TUNEL staining. Results: Bioinformatic analysis identified 1231 differentially expressed genes when comparing sCD40L-stimulated HK2 vs HK2-CD40KO cells. KEGG pathway analysis demonstrated p53 signaling as one of the most significantly altered pathways. The pro-apoptotic gene PMAIP1 was found to be among the most highly upregulated in sCD40L treated HK2 cells vs HK2 control (2.6-fold change), which was confirmed by qPCR and Western blot. Furthermore, CD40KO rats demonstrated significantly reduced renal cell death (3-fold decrease vs. S rat, p<0.01) compared to wild-type Dahl S rats following high salt diet. Conclusion: RNA sequence analysis demonstrates that disruption of CD40 in renal proximal tubule epithelial cells significantly alters p53 signaling pathways and the pro-apoptotic gene PMAIP1 is upregulated upon sCD40L treatment. Disruption of CD40 significantly attenuates kidney cell death following high salt diet induced renal injury. Our data indicates that PMAIP1 contributes to CD40-mediated renal injury.