Effect Of Necroptosis On The Progression Of Chronic Kidney Disease And Its Possible Mechanism

Part one:Induction and activation of necroptosis in the rat remnant kidney after subtotal nephrectomy (SNX) surgeryObjective:Necroptosis, an alternative mode of programmed cell death, has crucial pathophysiological roles in many diseases, but its effect on chronic kidney disease (CKD) is poorly understood. Therefore, we assessed necroptosis and its pathophysiological effects in a widely used remnant-kidney rat model and detected necroptosis in the remnant kidney of subtotal nephrectomised rats at various time points to conform necroptosis participated in the excessive loss of cells of the remnant kidney during the sustained progression of renal injury.Methods:1. Eight-week-old healthy male Sprague-Dawley (250 g to 300 g in body weight) rats used in this study. SD rats were randomly assigned to the subtotal nephrectomy (SNX) group (n=32) or control group (n=32). SD rats were subjected to subtotal nephrectomy surgery in the SNX group and sham surgery in the control group. Then, rats in the SNX group and the control group were divided respectively into 4 sub-groups by the time points after surgery:0 week、4 weeks、8 weeks、12 weeks (n=8).2. Serum creatinine and blood urea nitrogen concentrations were used to assess renal function, GSI and TIS were evaluated on the same PAS-stained sections, and the ultrastructure of kidney tissues was observed by electron microscopy.3. Expression of RIP 1, RIP3 and LC3B in the rat remnant kidney were assessed by quantitative PCR (qPCR), Western Blot analysis and immunohistochemistry.Results:1. The blood urea nitrogen and serum creatinine levels of subtotal nephrectomised rats increased notably by 4 weeks after SNX surgery (P< 0.05) and attained the further elevation by 8 weeks (P<0.05). By 12 weeks, no change than those of the 8 weeks group was found (P> 0.05). The glomerular sclerosis index (GSI) and tubulointerstitial damage scores (TIS) were dramatically higher by 4 weeks after SNX surgery (P<0.01) and attained the further elevation by 8 weeks and 12 weeks (P<0.01).2. In the rat remnant kidney, necroptotic cells with typical necrotic morphological features were observed at 8 weeks after surgery by TEM. However, obvious necrotic changes were not found at 4,12 weeks.3. The mRNA and protein expression of RIP1 and RIP3 in the rat remnant kidney increased notably by 4 weeks after SNX surgery (P<0.01) and attained the peak elevation by 8 weeks (P<0.01). After that, a sharp decrease of RIP 1 and RIP3 mRNA level was found by 12 weeks (P<0.01). But no decrease of RIP1 and RIP3 protein level was found (P>0.05). Immunohistochemistry results revealed that RIP3 protein was mainly sited in cytoplasmic compartments of renal tubular epithelial cells in the remnant kidney, and scarcely or not scattered in glomeruli.Conclusions:1. Necroptosis, an alternative cell death pathway, participated in the excessive loss of cells in the remnant kidney of subtotal nephrectomised rats.2. The highest peak of necroptosis in the rat remnant kidney occurred at 8 weeks after SNX surgery.Part two:Inhibition of necroptosis by necrostatin-1 can alleviate chronic kidney injury of subtotal nephrectomised rats and its mechanisms.Objective:To further conform necroptosis participated in the excessive loss of cells in the rat remnant kidney, Nec-1 and Z-VAD were administered to rats underwent SNX surgery for exploring the effect of necroptosis on subtotal nephrectomised rats and its mechanisms.Methods:1. SD rats were randomly assigned to the subtotal nephrectomy (SNX) group (n=18) or the control group (n=6). These rats were subjected respectively to subtotal nephrectomy surgery in the SNX group and sham surgery in the control group. Then, rats in the SNX group were divided into 3 sub-groups:the SNX+vehicle group, the SNX+Z-VAD group, the SNX+Nec-1 group (n=6). Rats in the SNX+Z-VAD group and the SNX+Nec-1 group were injected respectively with Z-VAD or Nec-1 for 4 weeks.2. Effect of Z-VAD or Nee-lon renal function and renal pathologic changes was assessed.3. Effect of Z-VAD or Nec-1 on cell death in the rat remnant kidney tissues was observed by TUNEL staining and electron microscopy.4. Effect of Z-VAD or Nec-1 on RIP1、RIP3、MLKL、PGAM5 and Drp1 in the rat remnant kidney was assessed by qPCR, western blot analys is, and the localisation of RIP 1 and RIP3 in remnant kidney cells were observed by immunofluorescence analyses.5. The ROS level in the kidney tissues was detected by a DCFH-DA analysis.Results:1. In the remnant kidney tissue of Z-VAD-treated and vehicle-treated rats, renal cells with the typical morphological features of necroptotic cell death were found by TEM. However, obvious necroptotic cells were not found in the renal tissue of Nec-1-treated and control rats.2. Nec-1 inhibited significantly the elevated mRNA and protein expression of RIP1 and RIP3 in the rat remnant kidney (P<0.05); however, ZrVAD did not reduce elevated RIP1 and RIP3 levels (P>0.05).3. Treatment with Nec-1 or Z-VAD obviously reduced the increased concentration of the urea nitrogen and creatinine, the glomerular damage and tubular and interstitial lesions and TUNEL-poshive cells of subtotal nephrectomised rats (P<0.01).4. The increased expression of PGAM5 and Drp1 mRNA was dramatically reduced with Nec-1 treatment (P<0.05); however, no decrease was found in Z-VAD-treated subtotal nephrectomised rats (P>0.05). The western blot analysis showed Nec-1 inhibited significantly the elevated expression of MLKL and Drp1 in the remnant kidney (P<0.05), however, didn’t inhibit PGAM5 protein levels (P>0.05); Z-VAD did not reduce elevated MLKL and PGAM5 protein expression (P>0.05), but Z-VAD reduced elevated Drp1 protein expression in the remnant kidney (P<0.05).Conclusions:1. Necroptosis in the remnant kidney of subtotal nephrectomised rats is mediated by RIP 1 and RIP3.2. Necroptotic cell death mediated by RIP 1 and RIP3 in the rat remnant kidney might be driven by the MLKL、Drp1、ROS signalling molecules.Part three:Necroptosis mediated by RDP1 and RIP3 in the rat remnant kidney might be induced by Ang 2Objective:To further explore what could trigger necroptosis mediated by RIP1 and RIP3 in the rat remnant kidney, valsartan was administered to rats underwent SNX surgery for confirming the necroptotic cell death in the rat remnant kidney might be induced by Ang 2.Methods:1. SD rats were randomly assigned to the subtotal nephrectomy (SNX) group (n=12) or control group (n=6). These rats were subjected respectively to subtotal nephrectomy surgery in the SNX group and sham surgery in the control group. Then, rats in the SNX group were divided into two sub-groups: the SNX+vehicle group, the SNX+valsartan group. Rats in the SNX+ valsartan group were administrated valsartan for 4 weeks.2. Effect of valsartan on Ang 2, RIP 1/3, MLKL、PGAM5、Drp1 in the rat remnant kidney were assessed by qPCR and Western Blot analysis.3. The localisation of Ang 2 in remnant kidney cells was assessed by immunofluorescence analyses.4. The ROS level in the kidney tissues was detected by a DCFH-DA analysis.Results:1. The level of Ang 2 mRNA and protein in the remnant kidney tissue was higher than that of the control group (P<0.01). Ang 2 was mainly expressed in the glomerular and tubular cell cytoplasm in the rat remnant kidney under confocal microscope.2. The increased expression of RIP 1and Drp1 mRNA as dramatically reduced with valsartan treatment (P< 0.05); however, no significent decrease of RIP3 was found (P>0.05). The western blot analysis showed valsartan reduced significantly the elevated expression of RIP1、RIP3、 MLKL and Drp1 protein in the remnant kidney (P<0.05).Conclusions:Necroptotic cell death mediated by RIP1 and RIP3 in the rat remnant kidney might be induced by Ang 2.Part four:Necroptosis, apoptosis and autophagy in the rat remnant kidney influence each otherObjective:Programmed cell death includes three kinds of main forms:apoptosis autophagy and necroptosis. To explore the modes of renal cell death during the progression of CKD, Z-VAD or Nec-1or 3-MA were administered to rats underwent SNX surgery for exploring which forms were involved in the loss of renal cells in the rat remnant kidney, which forms were main modes of renal cell death and what was the relationship between them.Methods:1. SD rats were randomly assigned to the subtotal nephrectomy (SNX) group (n=24) or control group (n=6). These rats were subjected respectively to subtotal nephrectomy surgery in the SNX group and sham surgery in the control group. Then, rats in the SNX group were divided into 4 sub-groups: the SNX+vehicle group, the SNX+Z-VAD group, the SNX+Nec-1 group, the SNX+3-MA group. Rats in the SNX+Z-VAD group, the SNX+nec-1 group and the SNX+3-MA group were administrated respectively with Z-VAD or Nec-1 or 3-MA for 4 weeks.2. Effect of Z-VAD or Nec-1 or 3-MA on caspase-3、LC3B and RIP3 mRNA in the rat remnant kidney were assessed by qPCR.3. Effect of Z-VAD or Nec-1 or 3-MA on caspase-3、LC3B and RDP3 protein in the rat remnant kidney were assessed by western blot analysis.Results:1. Expression of caspase-3、LC3B、RI3mRNA in the remnant kindey of subtotal nephrectomised rats were significantly higher than those of the control group (P<0.01).The increased expression of caspase-3 mRNA was dramatically reduced with Z-VAD treatment (P< 0.01); however, no decrease of LC3B. RIP3 mRNA was found (P>0.05). Nec-1and 3-MA inhib ited significantly the elevated expression of LC3B、RIP3 mRNA in the remnant kidney (P<0.01), however, Nec-1 and 3-MA couldn’t block the expression of caspase-3 mRNA(P>0.05).2. The western blot analysis showed Z-VAD inhibited significantly the elevated expression of caspase-3 protein in the remnant kidney (P<0.05), however, Z-VAD couldn’t block the expression of LC3B、RIP3 protein (P >0.05), on the contrary, Z-VAD prompted the expression of LC3B protein (P< 0.05). The increased expression of RIP3 and LC3B protein was dramatically reduced with Nec-1 treatment (P<0.05); however, no decrease of caspase-3 protein was found (P>0.05). The increased expression of LC3B、RIP3 protein was dramatically reduced with 3-MA treatment (P< 0.01); however, no decrease of caspase-3 protein was found (P>0.05).Conclusions:1. Apoptosis、autophagy and necroptosis were involved in the loss of renal cells during the chronic pathologic advance of remnant kidney. And apoptosis, autophagy and necroptosis are not always mutually exclusive and may occur simultaneously in the same remnant kidney cells and influence one another.2. Autophagy may further reinforce necroptotic cell death, as a downstream molecular event of necroptosis.