Effects Of Lentivirus Mediated PKC-α Interference On Glomerular Filtration Rate In Hepatorenal Syndrome Resulting From Fulminant Hepatic Failure

Aim:Advanced hepatic failure is often accompanied with acute kidney injury（AKI）and has a poor prognosis due to the deterioration of both renal and liver function.Complex mechanisms are likely involved in renal malfunction during severe liver failure,such as alteration in systemic hemodynamics,subsequent renal hypoperfusion,activation of vasoconstrictor systems and reduced activity of vasodilator systems.Ultimately,renal vasoconstriction leads to a pronounced reduction in the glomerular filtration rate.Glomerular mesangial cells（GMCs）regulate glomerular filtration pressure（GFP）by changing glomerular capillary filtration surface area through their contraction.Vascular smooth muscle cells（VSMCs）in preglomerular resistance vessels（interlobular artery and afferent arteriole）mediate constriction or dilation of the vessels（i.e.changes in luminal diameter）,in turn regulating renal vascular resistance,renal blood flow and GFR.Multi-factorial mechanisms are involved in abnormal regulation of renal blood flow and GFR.Plasma levels of angiotensin II,endothelin-1（ET-1）,norepinephrine（NE）and tumor necrosis factor-α（TNF-α）are significantly increased in patients with severe liver injury.Cytosolic Ca²⁺concentration([Ca²⁺]i)in GMCs and VSMCs responds to a couple of external signals,and activates various effectors,including cellular contraction.Theoretically,the changes in IP₃R1 expression levels have been linked to regulation of[Ca²⁺]i.The contraction of mesangial cells involves the interplay of many signal transduction pathways which include both protein kinase C（PKC）and intracellular calcium.TNF-αwas shown to potentiates intracellular Ca²⁺signaling by enhancing of IP₃R1expression through TNFR1/PC-PLC/PKC-αand TNFR2 signalling pathways in HMCs.There is little know about the precise mechanisms of TNF-αin decreased GFR.Small interfering RNAs（siRNAs）directed against the PKC-αgene were designed and transfected into GMCs.Quantitative real-time polymerase chain reaction and Western blot analysis were to examine the effects of TNF-αon PKC-α,IP₃R1 mRNA and protein expression and SP-1 protein expression.Chromatin immunoprecipitation was to examine the effects of TNF-αon IP₃R1 promoter activity.Cell intracellular calcium concentration([Ca²⁺]i)was measured.Lentiviral vectors can deliver and express si RNA（Short interference RNA）in a wide variety of dividing and nondividing cell.In order to detect kidney function and GFR after using RNA interference technology,to interfere expression of PKC-αon rat kidney by constructing recombinant lentivirus that express rat PKC-αsiRNA.Sprague-Dawley rats treated with a combination of D-galactosamine plus lipolgsaccharid（D-GalN/LPS）can be used as a model with FHF and acute renal failure.In order to explore the possible pathogenesis,we examined the the IP₃R1 expression levels of renal with Western blot and Real Time PCR and determined GFR.We focused on the effect of TNF-αon PKC-αsignal transduction mechanism in order to point a new direction of molecular invervention by directly targeting this novel pathway in severe liver failure with multiple-organ.Methods:1.The full-length PKC-αgene sequence was retrieved from Gen Bank（NM₀01105713）and the siRNA design principles were obtained from Invitrogen special siRNA design software.The siRNA target rat PKC-αgene sequence was selected based on the Western Blot and Real Time PCR results.2.Western blot and Real Time PCR methods were used to test the effect of TNF-αon expression of IP₃R1,PKC-αprotein and m RNA in GMCs.3.Western blot and Real Time PCR methods were used to test the effect of si RNA-PKC-αon suppress TNF-αinduced expression of IP₃R1 protein and mRNA in GMCs.4.Cell intracellular calcium concentration([Ca²⁺]i)was measured to test the effect of TNF-αenhanced[Ca²⁺]i release and siRNA-PKC-αblunt the trend.5.Western blot was used to test the effect of TNF-αon expression of SP-1protein.ChIP assay is performed to examine the effect of TNF-αenhanced SP-1 binding with IP₃R1 promoter.6.Western blot was used to test the effect of si RNA-PKC-αon suppress TNF-αinduced expression of SP-1protein.ChIP assay is performed to examine the effect of siRNA-PKC-αon suppress TNF-αenhanced SP-1 binding with IP₃R1 promoter.7.Western blot was used to test the effect of TNF-αon expression of JNK and p-JNK protein.8.Western blot was used to test the effect of si RNA-PKC-αon suppress TNF-αinduced expression of JNK and p-JNK protein.9.Western blot was used to test the effect of JNK inhibitor,SP600125on suppress TNF-αinduced expression of SP-1protein and IP₃R1 mRNA and protein.10.Lentivirus vectors were commissioned by ShangHai GenePharma Co.,Ltd.11.Rats were injected 1.0×10⁸TU lentivirus 2 weeks before examined the the PKC-αexpression levels of renal with Western blot and Real Time PCR.The infection rate was observed by fluorescence microscopy.12.Infection with LVs in vivo.40 rats were random divided into 4 groups:the blank control group（NS group）,D-GalN/LPS group,infected by negative control letivirus plus D-GalN/LPS treatment group（LV-shRNA-NC group）,infected by PKC-αshRNA lentivirus plus D-GalN/LPS treatment group（LV-shRNA-PKC-αgroup）.Infected by lentivirus after differentiation for 7d,the microosmotic pumps with FITC-inulin were implanted into rats abdominal cavity.On day 7 post-pump implantation saline,GalN/LPS were injected into tail vain.13.Liver and kidney specimens were fixed in 10%formalin and 2.5%glutaraldehyde,the embedded in paraffin for histopathological analysis.Suplus specimens were stored at-80℃until further preparation.14.Serum levels of alanine aminotransferase（ALT）,aspartate transaminase（AST）,Urea nitrogen（BUN）,creatinine（Cr）,potassium ino（K⁺）,sodium ion（Na⁺）,chloride ion（Cl^—）,tumor necrosis factor-α（TNF-α）and endothelin-1（ET-1）were determined using commercial kits.15.Serum were collected to determine FITC-inulin after 12 hours.Fluorescence of FITC was determined using a Thermo Scientific Varioskan Flash with 485-nm excitation and read at 538-nm emission.GFR was evaluated using the equation inulin clearance=inulin infusion rate/steady-state blood inulin concentration.GFR was expressd in microliters per minute（GFR₁）,microliters per minute per kilogram body weight（GFR₂）,microliters per minuter per gram kidney weight（GFR₃）.The model of FHF accompanied with AKI was considered to be successful,according to GFR and other results determined above.16.Western blot and Real Time PCR methods were used to test IP₃R1 protein and mRNA in kidney.17.Isolation of rat Glomruli,measurements of intracellular[Ca²⁺]I in glomeruli.18.Isolation of rat Glomruli,determination of glomerular inulin space.19.Urine samples were selected and the level of proteinuria,albuminuria and urine N-acetyl-β-D-glucosaminidase（NAG）were performed by ELISA.Results:1.SiRNA sequence selectionThe siRNA target rat PKC-αgene was selected based on the Western Blot and Real Time PCR results.2.Effect of TNF-αon expression of IP₃R1 protein and mRNA in GMCsA significant induction of IP₃R1 mRNA occurred by 2 hrs after TNF-αtreatment and reached a maximum at 8 hrs（P<0.01）compared to basal levels,and returned to a basal level at 24 hrs.Compared to the basal level,IP₃R1 protein levels started to increase at 4 hrs after TNF-αtreatment and increased to the highest levels at 8 hrs,and sustained till 24 hrs（P<0.05）.Thus,the increment in mRNA for IP₃R1 preceded the increment in protein.3.Effect of TNF-αon expression of PKC-αprotein and mRNA in GMCsQuantitative analysis demonstrated that PKC-αmRNA started to increase at 4hrs and reached higher levels at 8 hrs after TNF-atreatment（P<0.05）,which were sustained till 24 hrs.Expression levels of PKC-αprotein expression started to increase at 4 hrs,and reached the highest at 8 hrs（P<0.05）,returned to nearly a basal level by24 hrs.4.Effect of silencing PKC-αon inhibited TNF-αinduced IP₃R1 over-expressionCompared with TNF-αtreatment alone（mRNA 2.53±0.46,P<0.05,protein2.23±0.80,P<0.05）,silencing PKC-αcould inhibited TNF-αinduced expression of IP₃R1 at both mRNA（1.09±0.50,P<0.05）and protein（1.13±0.94,P<0.05）.5.Effect of silencing PKC-αon decrease TNF-αinduced[Ca²⁺]releaseWe found that ET-1 caused a markedly increase in[Ca²⁺]i of GMCs,that was obviously enhanced by TNF-α（P<0.05）.In experiments we found that silencing PKC-αcould totally inhibit ET-1 induced release of stored Ca²⁺.Transfected cells fully eliminated the difference in changes of[Ca²⁺]i between control group and TNF-αtreated group.6.Effect of TNF-αon expression of SP-1 protein and binding with IP₃R1 promoterThe previous finding suggested SP-1 is a necessary binding site of TNF-αinduced regulation of IP₃R1 expression.The expression of SP-1 protein obviously increased in TNF-αtreated 2h²4h groups.The maximal effect was seen at 8h group（2.50±1.06,P<0.05）.Furthermore,the Ch IP assay demonstrated that the TNF-αremarkably enhanced the SP-1 binding with IP₃R1 promoter in GMCs and the maximal effect was seen at 8h.7.Effect of silencing PKC-αon suppressed SP-1 protein expression and binding with IP₃R1 promoterWestern blot analysis showed that the TNF-αinduced increasing of SP-1 protein（2.38±0.69,P<0.05）expression was significantly reduced following transfection（0.81±0.36,P>0.05）.And the transfection also blunted the rise of SP-1 binding with IP₃R1 promoter.8.Effect of silencing PKC-αon JNK/P-JNK expressionSignificant induction of JNK and p-JNK protein occurred by 2hrs after TNF-αtreatment and reached a maximum level at 8hrs（P<0.01 and P<0.05,respectively）compared to the basal levels.JNK and p-JNK protein levels were returned at 24hrs.Compared to the siRNA-NC group,JNK and p-JNK protein expression in siRNA-NC+TNF-αgroup was significantly increased（P<0.05）.Silencing PKC-αsuppressed TNF-α-induced expression of JNK and the p-JNK protein in GMCs（P<0.05）.9.Involvement JNK activation in TNF-α-induced IP₃R1expressionThe addition of SP600125 was sufficient to cause almost completely abrogated TNF-α-stimulated increase in SP-1 protein level（P<0.01）.SP600125 suppressed TNF-α-stimulated IP₃R1expression at both mRNA and protein levels（P<0.01）.10.Rat kidney infected by lentivirus-RNA-interference systemAfter 2 weeks infection,there were no fluorescents in NS group,but the expression of green fluorescence in both of NC group and PKC-αgroup,which could be observed by fluorescence microscopy.Based on the Western Blot and Real Time PCR results,the infection rate was about 50%.11.Histological evaluationLiver and kidney specimens were fixed in 10%formalin and embedded in paraffin for histopathological analysis.Tissue was cut into 4-μm thich sections and were stained with hematoxylin and eoin,then analyzed under a light microscope.Liver tissues stained with hematoxylin-eosin presented histologically extensive hepatic necrosis and severe hemorrhage in part of lobules.Extensive necrosis is accompanied with fracture of hepatocellular cord,loss of lobular structure,sinusoid expansion and Kupffer cell proliferation.Damaged hepatocytes appear to have their nuclei dissolved with nuclear debris or pycnosis.None of the GalN/LPS-exposed rats had obvious renal morphologic alterations in histological examination.12.Serum biochemical analysis and cell factor examinationSerum ALT,AST,BUN,Cr TNF-αand ET-1 were elevated at 12h in the D-GalN/LPS and LV-shRNA-NC group than those in the control group（P<0.05）.The degree of ALT,AST,TNF-αand ET-1 in LV-shRNA-PKC-αgroup was not significant compare with D-GalN/LPS and NC group（P>0.05）.BUN,Cr in PKC-αgroup were markedly decreased than those in D-GalN/LPS and LV-shRNA-NC group（P<0.05）.13.GFR of NS,D-GalN/LPS,LV-shRNA-NC,LV-shRNA-PKC-αgroupGFR was estimated frome plasma inulin levels on day 7 and the known pump infusion rate.GFR was significantly decreased in D-GalN/LPS and LV-sh RNA-NC group than those in control group.GFR in LV-sh RNA-PKC-αgroup was increased compared with D-GalN/LPS treatment group（P<0.05）.14.Expression of IP₃R1 protein and mRNA with treatment of LV-sh RNA-PKC-αA high level of expression of IP₃R1 protein was showed in D-GalN/LPS and LV-shRNA-NC group and there is no difference between two group（P>0.05）.But the level was significant decreased in LV-shRNA-PKC-αgroup（P<0.05）.Our results demonstrated that the expression level of IP₃R1 mRNA in LV-shRNA-PKC-αgroup was much lower than that of D-Gal N/LPS and LV-shRNA-NC group（P<0.05）.15.Effect of LV-shRNA-PKC-αtreatment on glomerular[Ca²⁺]iThe basal[Ca²⁺]i was calculated according to the equation;and sustained[Ca²⁺]i was calculated as the average value of 0.1s exposure at 20.0s intervals during ET-1exposure for 10 min.Since cell numbers of each glomerulus could be different,which might affect the value of[Ca²⁺]i,we used the ratio of sustained over basal[Ca²⁺]i to represent the effects of LV-shRNA-PKC-α.GalN/LPS exposure increased[Ca²⁺]i robustly compared to N.S.controls.The pre-administration of LV-shRNA-PKC-αreduced the[Ca²⁺]i level markedly.16.Effect of LV-shRNA-PKC-αtreatment on GISThe changes in GIS reflect the glomerular size,and indirectly indicate the vascular dilation/constriction status.The ratio of GIS2（treated by ET-1）over GIS1（control）shown that GalN/LPS exposure markedly decreased GIS ratio compared to N.S.controls.GIS ratio in LV-shRNA-PKC-αgroup was improved compared to that in the GalN/LPS and LV-shRNA-NC group.Conclusion:1.TNF-αhas a potent ability to increase the protein and mRNA expression of IP₃R1 and in GMCs,an increase in mRNA levels prior to observing an increase in protein levels.2.TNF-αhas a potent ability to increase the protein expression of PKC-αin GMCs.3.Silencing PKC-αcould inhibited TNF-αinduced expression of IP₃R1 at both mRNA and protein.4.Silencing PKC-αcould blunt ET-1 caused rapid increase in[Ca²⁺]i,that was obviously enhanced by TNF-α.5.TNF-αcould increase the expression of SP-1,and enhanced SP-1 binding with IP₃R1 promoter which perhaps was an origination of the high expression of IP₃R1mRNA and IP₃R1 protein.6.Silencing PKC-αcould inhibited TNF-αinduced expression of SP-1 at protein and binding with IP₃R1 promoter.7.TNF-αcould increase the expression of JNK and p-JNK protein.Silencing PKC-αcould inhibited TNF-αinduced expression of JNK and p-JNK protein.8.SP600125 could abrogated TNF-αinduced SP-1 protein level and IP₃R1expression.9.Sprague-Dawley rats model which was induced by D-galactosamine plus lipopolysaccharide can develop functional renal failure typical for fulminant hepatic failure.10.In LV-shRNA-PKC-αgroup the elevated BUN,Cr was markedly decreased and GFR obviously improved,but TNF-αand ET-1 obviously increased.11.GFR in LV-shRNA-PKC-αgroup was improved compared with D-GalN/LPS group and LV-shRNA-NC group.12.The expression of IP₃R1 protein and mRNA was significantly elevated in FHF accompanied with AKI.In LV-shRNA-PKC-αgroup the over-expression of IP₃R1 protein and was successfully blocked.13.The phenomenon of increasing[Ca²⁺]i was successfully blockd by pre-treatment of LV-shRNA-PKC-α.14.In LV-shRNA-PKC-αgroup GIS obviously increased.