The Effect Of Protein Kinase C-alpha On The Renal Artery Vascular Pressure In Rats With Liver Failure

Objective:The main pathogenesis of hepatorenal syndrome?HRS?is the decrease of glomerular filtration rate?GFR?,which is characterized by no pathological and morphological changes in kidney,but dysfunction.The contraction of glomerular mesangial cells?GMCs?can cause the decrease of glomerular filtration area and the contraction of glomerular artery smooth muscle cells?VSMC?,which may lead to a decrease in renal blood flow.So as to explore the signal transduction role of PKC-alpha and IP₃RI in the process of renal artery contraction induced by hepatic necrosis,and to examine the expression level of PKC-alpha,IP₃R1 and renal artery pressure after ET-1 stimulation.SD rats treated with D-galactosamine combined with lipopolgsaccharide can be used as a HRS model resulting from fulminant hepatc failure?FHF?,and the packaged lentiviral vectors that knocked out PKC-alpha and IP₃R1 were injected into the corresponding groups in advance.The renal arterial pressure was detected and the serum biochemical indexes were detected.The expression of PKC-alpha,IP₃R1 protein and mRNA in renal tissue was detected.For the purpose of providing a theoretical basis for the study of the mechanism of GFR decline in HRS.Methods:1.Construction of HRS model,administration before modeling 2 weeks,each gene silencing group rats were injected into the tail vein with pre-packaged lentiviral vector respectively.After 2 weeks of feeding,all rats were weighed and divided into different groups.In group NS,saline solution,D/L group,LV-NC group,LV-PKC-alpha group and LV-IP₃R1 group were given saline solution with D-GalN?400mg/kg?/LPS?32?g/kg?,and the volume was 2ml/kg.2.Renal artery pressure detection:after anesthesia open the 12h Modeling Rats belly,isolate superior mesenteric artery and the right renal artery and left renal vein with a cotton swab,ligation of distal renal artery hemostasis,clipping the proximal renal artery,cut out a small mouth on blood vessels with ophthalmic scissors.Insert the trocar into right renal artery,use hemostatic clamp to fix it,tee pipe connected with the pressure transducer and biological signal acquisition and processing system.3.Detect serum alanine aminotransferase?ALT?,serum aspartate aminotransferase?AST?,creatinine?Cr?,urea nitrogen?BUN?,potassium ion?K⁺?,sodium ion?Na⁺?and chloride ion?Cl^-?.4.The liver and kidney tissues were stained with conventional HE.The necrosis of liver cells and the change of renal tissue structure were observed under ordinary light microscope.5.Western blot and Real-time PCR were used to detect the expression of PKC-alpha and IP₃R1 protein and mRNA in kidney tissues of each group.Results:1.Vascular pressure of renal arteryAfter renal artery pressure is stable,D/L group and LV-NC group compared with NS group slightly increased,there is a significant difference?P=0.021<0.05?;there is no significant difference between LV-PKC-alpha group,LV-IP₃R1 group and NS group;LV-PKC-alpha group compared with D/L group and LV-NC group significantly decreased,the difference is significant,?P=0.004<0.05?;LV-IP₃R1 group compared with D/L group,LV-NC group decreased significantly which is similar with LV-PKC-alpha group,the difference is significant?P=0.023<0.05?.2.Biochemical indexes of serum liver and kidney functionD-L group and LV-NC group ALT,AST,BUN,Cr increased significantly after the administration of 12h,compared with the NS group there are significant differences?P<0.05?,The levels of Cr and BUN in group LV-PKC-alpha are significantly lower than those in group D-GalN/LPS and LV-NC,there is a significant difference?P<0.05?,but compared with the NS group,no significant difference?P>0.05?.There was no obvious change in K⁺,Na⁺,Cl^-concentration.3.Liver and kidney histological examination resultsBeing observe with naked eye,the result show that 12h after sdministration of D-GalN/LPS,the liver is purple black,obviously congestion and swelling,diffuse bleeding point,the texture becomes brittle,the section has a dark red congestion overflow.Under the light microscope,the hepatocytes were necrotic,the anhepatic cells were regenerated,the fibrous septum collapsed,and a large number of red blood cells were infiltrated.Under the light microscope,the glomerular and renal tubule structure of the rats in the necrotic group were not changed compared with the normal group.4.Expression of PKC-alpha,IP₃R1 protein and mRNA after gene silencingD/L group and LV-NC group 12 hours PKC-alpha,IP₃R1 protein and mRNA was in high expression,the two groups compared with NS group the difference is significant?P<0.05?;and the expression of PKC-alpha protein and mRNA decreased significantly in LV-PKC-alpha group,compared with the D/L group and LV-NC group is significant?P<0.05?,but compared with the NS group difference is not significant;the expression of IP₃R1 protein and mRNA decreased significantly is similar with PKC-alpha,compared with the D/L group and LV-NC group is significant?P<0.05?,no significant difference compared with NS group.Conclusion:1.Silencing of PKC-?and IP3R1 has a protective effect which can reduce the renal damage and renal artery pressure increase in rats with acute liver failure.2.LV-shRNA-PKC-?treatment in advance can obviously inhibit the expression of PKC-alpha protein and mRNA in the kidney of rats with acute liver failure.3.LV-shRNA-IP₃R1 treatment in advance can obviously inhibit the expression of IP₃R1 protein and mRNA in the kidney of rats with acute liver failure.