Study On The Mechanism Of Protein Kinase C Beta 2 Signaling Pathway In Diabetic Renal Tubular Epithelial Cell Injury Induced By Contrast

Background:Contrast medium-induced nephropathy(CIN)is the third most common cause of acute kidney injury in hospitalized patients.It still has been a big challenge to prevent CIN so far.Diabetes is an independent risk factor for CIN.However,the pathogenesis of contrast-induced nephropathy in diabetic patients is not clear.Objective:This study aims to observe contrast-induced diabetic renal damage,and reveal the mechanisms.Furthermore,observe the Breviscapin’ protection on this damage.Methods:1.In vivo study,8-week male C57/BL6J mice were randomized into 7 experimental groups.The experimental groups were as follows:control group,diabetes group,CIN group,diabetes+CIN group,diabetes+breviscapine group,CIN+breviscapine group,diabetes+CIN+breviscapine group.Hematoxylin and eosin(HE),Masson’s trichrome staining,Periodic Acid-Schiff(PAS),periodic acid-silver methylamine(PASM),Real-time quantitative polymerase chain reaction(RT-qPCR),and Western blotting were used to detect the expression levels of collagen I,CTGF and TGF β1 in kidney tissues.2.In vitro study,renal tubular epithelial cells(HK-2)were divided into normal control group;diatrizoate group(100 mg/ml);AGEs group(50 ug/ml);diatrizoate+AGEs group.CCK-8 and flow cytometry experiments were used to detect cell proliferation and apoptosis,respectively.Autophagy was observed under confocal fluorescence microscope and transmission electron microscope.The expression levels of kidney injury molecule(KIM-1)and neutrophil gelatinase-associated lipocalin(NGAL)in renal tubular epithelial cells were detected by RT-qPCR.Bcl2、Bax、Caspase3、Beclin-1、LC3、P62、p-PKCP2、PKCβ2、p-ERK、ERK、p-JNK、JNK、p-p38 and p38 expression were detected by Western blot.3.siPKCβ2、PKCβ2 inhibitor LY333531 and 3-MA(autophagy inhibitor)were used respectively,when observed apoptosis,autophagy and serial factors expression of renal tubular epithelial cell,to explore the protection PKCβ2/Akt/c-JNK1/p38 in this progress.Results:1.In vivo diabetic mice model,breviscapin reduced proteinuria and serum creatinine levels in diabetic+CIN model mice.Moreover,breviscapin reverses tubular atrophy and fibrosis and glycogen accumulation in the glomerular capillary basement membrane.2.In vivo diabetic mice model,the expression of PKCβ2 phosphorylation in both diabetes group and diabetic+CIN group increased,while breviscapine could down-regulate PKCβ2 phosphorylation.Phosphorylation of Akt,c-JNK,and p38 was significantly increased in the diabetic/diabetic+CIN group compared to control mice.Breviscapine can down-regulate the expression levels of Akt,c-JNK1/2 and p38 phosphorylation in diabetic+CIN mice.It imply that breviscapine could protect renal through PKCβ2/Akt/c-JNK1/2/p38 signal pathway.3.In vitro HK-2 cell experiments,meglumine diatrizoate and AGEs significantly disrupted the morphology of renal tubular epithelial cells and decreased ability of HK-2 cells.The diatrizoate+AGEs group significantly promoted the apoptosis of renal tubular epithelial cells.In the diatrizoate+AGEs group,the apoptosis rate of renal tubular epithelial cells was the most significant.In addition,meglumine diatrizoate and AGEs promote the expression of the apoptosis marker caspase3 in renal tubular epithelial cells.The results of RT-qPCR showed that both meglumine diatrizoate and AGEs promoted the mRNA expression levels of KIM-1 and NGAL.4.In vitro HK-2 cell experiments,the meglumine diatrizoate increased the protein expression level of Beclin-1 in renal tubular epithelial cells and increased the proportion of LC3 Ⅱ/LC3 Ⅰ most significantly under AGEs incubation.However,in the meglumine diatrizoate group,AGEs group and diatrizoate+AGEs group,the expression of p62 was significantly decreased in renal tubular epithelial cells,and the most significant decrease was observed in the diatrizoate+AGEs group.Compared with the blank control group,PKCβ2 and p-PKCβ2 were significantly increased in the diatrizoate+AGEs group.5.Cell flow meter showed that the apoptosis rate of renal tubular epithelial cells was significantly increased in AGEs incubation.Furthermore,siPKCβ2 caused a significant decrease in the apoptotic rate of renal tubular epithelial cells as well as PKCβ2 inhibitor LY333531.Under the AGEs incubation,meglumine diatrizoate caused a significant increase in the expression of proapoptotic factors Caspase3 and Bax in renal tubular epithelial cells,whereas the expression of apoptosis inhibitor Bcl-2 decreased.Western blot results showed that inhibition of PKCβ2 declined the activity of the ERK/JNK/p38 pathway in renal tubular epithelial cells induced by meglumine diatrizoate under AGEs incubation.6.Under AGEs incubation,both PKCβ2 mRNA expressionin and protein expression increased in renal tubular epithelial cells induced by meglumine diatrizoate.The autophagy inhibitor 3-MA inhibited the expression of PKCβ2 and phosphorylated PKCβ2 in renal tubular epithelial cells induced by meglumine diatrizoate.PKCβ2 inhibitor LY333531 promoted autophagy in renal tubular epithelial cells.Conclusions:(1)Breviscapine,significantly improved diabetic renal fibrosis and apoptosis,may attribute to inhibit PKCβ2/Akt/JNK1/2/p38 signal pathway.(2)Meglumine diatrizoate significantly inhibited the proliferation and protective autophagy of renal tubular epithelial cells under AGEs environment,but promoted apoptosis.(3)PKCβ2 might a key signal factor in contrast induced renal injury in diabetes.