The Mechanism Of Decreased Glomerular Filtration Rate Induced By Tumor Necrosis Factor-α In Hepatorenal Syndrome

The mechanism of decreased glomerular filtration rate induced by tumor necrosis factor-αin hepatorenal syndromeAimThe reduction in glomerular filtration rate (GFR) is the key pathogenesis of hepatorenal syndrome(HRS). Glomerular mesangial cells(GMCs) are closely associated with glomerular filtration area. Cell contraction is closely related to changes in the intracellular calcium concentration([Ca²⁺]i). The inositol 1,4,5-trisphosphate receptors (IP₃Rs) are the main intracellular Ca²⁺ release channels. Obviously high plasma level of tumor necrosis factor-α(TNFα) is also found in HRS. There is little known about the precise mechanisms of TNFαin decreased GFR. Is there some interaction between the increased TNFαlevels and the contraction of GMCs? Theoretically, the change in IP₃RI expression level has been linked to regulation of [Ca²⁺] i. In order to explore the possible pathogenesis, we examined the effect of TNFαon [Ca²⁺]i and the contraction of GMCs which were stimulated by endothelin(ET). Next we explored the IP₃RI expression levels of GMCs treated by TNFαwith immunohistochemistry, Western blot and real time quantitative PCR. We also observed the effect of TNFαon the activity of IP₃RI promoter. Furthermore, we focused on the effect of TNFαon protein kinase C-α(PKCα)activity and the signal transduction mechanism of PKCαduring IP₃RI overexpression.Materials and methods1,MaterialsGMCs were originally isolated and cultured from glomeruli by sieving kidney cortex of male wistar rats. Cells were characterized as GMCs by positive immunofluorescence staining for smooth muscle-specific actin and negative immunofluorescence staining for kevatin.2,Methods (1)Primarily cultured GMCs were divided into TNFα-treated 0h,2h,4h,8h,24h groups and control group (D) ,TNFα-treated 8h group (T), L-threo-dihydrosphingosine (safingol) -treated 8h group (S),TNFα-saflngol cotreated 8h group (TS). The results were repeated 4 times at differrent passages cells.(2)The role of TNFαon [Ca²⁺]i was identified by the Ca²⁺-sensitive dye Fluo-3/AM with confocal imaging.(3)The effect of TNFαon contraction levels of GMCs was determined by accessing the surface area of cells before and after contraction stimulated by ET.(4)Immunocytochemistry, western blot and real time PCR methods were used to test the effect of TNFαon expression of IP₃R I protein and mRNA in GMCs.(5)PGL₃^- IP₃R₁ promoter recombined plasmid transfection assays were performed to determine the effect of TNFαon the activity of IP₃RI promoter.(6)Immunofluorescence staining, PKCαactivity assays and western blot analysis were used to test the effect of TNFαon the translocation activation of PKCαand expression of PKCα.(7)Real time PCR method was used to test the effect of PKCαinhibitor on upexpression of IP₃RI mRNA caused by TNFα.Results1,Measurement of [Ca²⁺]iWith Ca²⁺-sensitive dye Fluo-3/AM we found that ET caused a rapid increase in [Ca²⁺]i, that was obviously enhanced by TNFa(P＜0.05 ). In experiments we found that 2-APB could totally inhibit ET -induced release of stored Ca²⁺. Pretreatment of GMCs with the IP3Rs inhibitor 2-APB for 20min fully eliminated the difference in changes of [Ca²⁺]i between control groupand TNFα-treated groups.2,Measurement of the GMCs surface areasThe surface areas of GMCs were decreased in control group after ET stimulation. The changes of the surface area in TNFα-treated group were bigger than control group(P＜0.01 ).3,Immunocytochemistry staining of IP₃RI IP₃RI protein was localized to the endoplasmic reticulum cytoplasm region of GMCs and expressed at low levels in control group. The percentages of IP₃RI-staining positive cells were globally increased in TNFα4h-24h groups. The maximal effect was seen at 24h group (P＜0.05).4,Western blot analysis of IP₃RI proteinOur results showed low level expression of IP₃RI protein in control group. The expression of the IP₃RI protein obviously increased in TNFα-treated 4h-24h groups.The maximal effect was seen at 24h group (P＜0.05).5,Real time PCR analysis of IP₃RI mRNAWe found an increase of IP₃RI mRNA expression in TNFα-treated 2h-24h groups compared with control group. The maximal effect was seen at 8h(P＜0.05).6,Measurement of the activity of IP₃RI promoterOurresults showed that the activity of IP₃RI promoter was very weak in control group. TNFαcan remarkably increase the activity of IP₃RI promoter in GMCs and the maximal effect was seen at 8h(P＜0.05).7,Immunofluorescence staining of PKCaIn unstimulated cells, PKCa was diffusely detected in the cytoplasm. TNFαmarkedly induced translocation activation of PKCαfrom cytoplasm to perinucleus and into nucleus.8,PKCαactivity assaysThere was middling activity of PKCαin control group. TNFαcould strikingly increase the activity of PKCαand the maximal effect was seen at 8h(P＜0.05).9,Western blot analysis of PKCαThere was high level expression of PKCαprotein in control group cells. TNFαcould not affect the expression levelof PKCαbecause there was no difference between control group and TNFα-treated groups (P＞0.05).10,Real time PCR analysis of the PKCαsignal transduction roleOur results demonstrated that the expression level of IP₃RI in TS -cotreated group was much lower than that of TNFα-treated cells (P＜0.05). Conclusion1,GMCs treated by TNFαwere more sensitive to ET that stimulate IP₃-mediated release of stored Ca²⁺. ET caused a rapid increase in [Ca²⁺]i , that was obviously enhanced by TNFα.2,IP₃Rs were the target of TNFα. The interaction between TNFαand IP₃Rs probably leaded to important modulatory influence on [Ca²⁺]i changes and GMCs contraction.3,TNFαenhanced the contraction responses of GMCs to ET.4,TNFαhas a potent ability to increase the protein expression of IP₃RI in GMCs. The increased protein expression of IP₃RI may be due to increased synthesis of IP₃RI protein as we found an increase in mRNA levels prior to observing an increase in protein levels. Our finding that TNFαincreased IP₃RI mRNA levels could be explained by an effect on transcription of IP₃R I gene.5,TNFαcould increase the activity of IP₃RI promoter which perhaps was the first target of TNFαand was an origination of the high expression of IP₃RI mRNA and IP₃RI protein.6,GMCs treated by TNFαshowed subcellular localization of PKCαto perinucleus and into nucleus which was the sign of PKCαactivation. The result of PKCαactivity analysis showed that TNFαinduced an increase in PKCαactivity.7,The phenomenon of TNFα-induced-overexpression of IP₃RI mRNA was successfully blocked by safingol, which was the specific inhibitor of PKCα. We concluded that PKCαmay have an important signal transduction role.