Inhibiting PKCβ2 protects HK-2 cells against meglumine diatrizoate and AGEs-induced apoptosis and autophagy

Abstract

Background: Contrast induced diabetic nephropathy (CIN) is an important cause of hospital-acquired acute renal failure. Our aim was to observe the effect of protein kinase C beta 2 (PKC beta 2) knockdown on human proximal tubular epithelial cells (HK-2 cells) against meglumine diatrizoate and advanced glycation end products (AGEs)-induced apoptosis and autophagy. Methods: Cell viability was detected using cell counting kit-8 (CCK-8) assay in HK-2 cells after disposal with meglumine diatrizoate and AGEs with or without PKC beta 2 siRNA/inhibitor LY333531. Flow cytometry and western blot were used to test cell apoptosis and the related protein levels in meglumine diatrizoate and AGEs co-treated HK-2 cells with or without PKC beta 2 siRNA/inhibitor LY333531. Autophagy related proteins were detected using western blot. Immunofluorescence staining was used to examine the autophagyspecific protein light chain 3 (LC3), and autophagosome and autolysosome formation was observed under a transmission electron microscopy. Results: CCK-8 assay results showed that meglumine diatrizoate inhibited AGEs-induced HK-2 cell viability. Furthermore, meglumine diatrizoate promoted cell apoptosis and the expression level of caspase3 in AGEs-induced HK-2. Western blot results showed that meglumine diatrizoate elevated the expression levels of PKC beta 2 and p-PKC beta 2 in AGEs-induced HK-2 cells, and up-regulated the expression level of Beclin-1 and the ratio of LC3 II/LC3 I, and down-regulated the expression level of p62 in AGEs-induced HK-2 cells. We found that PKC beta 2 knockdown alleviated meglumine diatrizoate and AGEs-induced HK-2 cell apoptosis and autophagy. Intriguingly, PKC beta 2 inhibitor LY333531 reversed 3-methyladenine (3-MA)-induced autophagy inhibition in meglumine diatrizoate and AGEs-induced HK-2 cells. Conclusions: Our findings reveal that inhibiting PKC beta 2 protects HK- 2 cells against meglumine diatrizoate and AGEs-induced apoptosis and autophagy, which provide a novel therapeutic insight for CIN in diabetic patients.