The Protective Effect Of Necrostatin-1 On Sepsis Associated Acute Kidney Injury

BACKGROUNDAcute kidney injury(AKI)is a common clinical syndrome that is characterized by the rapid loss of kidney function and abrupt kidney damage.It is associated with high incidence about 2-12%in in-patients,and the incidence even reach up to 30%in patients admitted in ICU.AKI patients have higher in-hospital mortality,long-term mortality and incidence of end-stage kidney disease(ESRD).The causes of AKI are various,including ischemia reperfusion injury,sepsis,renal-toxic drugs,burn,rhabdomyolysis and so on.Sepsis associated AKI(SA-AKI)is an important cause.Sepsis,a systemic inflammatory response syndrome due to severe infection,could result in multiple organ dysfunction and leads to death ultimately.The incidence of sepsis increased year by year and was about 5‰ in the past decade.Patients with sepsis in ICU is nearly 30%and have high mortality about 18-26%.SA-AKI is the most common complication of sepsis.Sepsis patients with SA-AKI have worse prognosis than those without SA-AKI.SA-AKI is the leading cause of community-acquired AKI and secondary cause of hospital-acquired AKI.Currently incidence of SA-AKI is still increasing and mortality staying at a high level.So it is a challenge to prevent and cure SA-AKI.Mechanism of S A-AKI is different from AKI of other causes.There are paucity of renal tubular epithelial cell death compared with ischemia reperfusion AKI.Mechanisms of SA-AKI include mitochondrial impairment,oxidative stress,inflammatory response,microcirculation disturbance and so on.Necroptosis is an important type of regulated necrosis and is involved in various pathological conditions.Receptor-interacting protein kinase(RIP)-1 receives signals from various death stimuli,and subsequently recruits RIP-3 via RIP homotypic interaction motif domain-mediated interactions and promotes RIP-3 phosphorylation.Phosphorylated RIP-3 mediates the phosphorylation of mixed-lineage kinase domain-like(MLKL),ultimately leading to rupture of the plasma membrane.Previous studies have identified necroptosis in acute tubular necrosis(ATN)induced by various stimuli.Necrostatin-1(Nec-1)a small molecular about 259.3KD is a kind of alkaloid.It could specifically inhibited RIP-1 kinase activity and prevent phosphorylation of RIP-3,eventually reduce necroptosis.In I/R or cisplatin induced AKI,Nec-1 could protect renal tubular cell through inhibiting necroptosis.However ATN is not the major mechanism of SA-AKI.Could Nec-1 also alleviate kidney injury caused by sepsis?In our pre-research,we found the level of RIP-3 phosphorylation increased.So we hypothesized that RIP-1→RIP-3 signaling pathway also play an injury role in sepsis AKI and necrostatin-1 could alleviate AKI through inhibiting RIP-1 phosphorylating RIP-3.OBJECTIVEThe purpose of this study was to investigate whether Nec-1 could alleviate SA-AKI and the probable mechanism of Nec-1 protective effect on SA-AKI.METHODS(1)Establishment of SA-AKI mice model We administered 10 mg/kg LPS i.p.to 8-week-old adult C57BL/6J mice and observed 18 hours.(2)Experimental animal groupingAll the experimental mice were randomly assigned to 3 groups as described below.(3)Detect content in animal experiment1.Blood samples are acquired from Inferior vena cava and kidneys were cut.2.Determination of renal function:Serum BUN and creatinine measurements were obtained using the i-STAT Portable Clinical Analyzer and CHEM8+ cartridges.3.HE and PAS staining was used in kidney histopathology.4.PGC1-α detection by immunohistochemistry.5.Western blot were performed for p-RIP-3、p-MLKL、p-DRIP-1、LC3、p62.6.Mitochondrion and autophagosome/autolysosome in renal tubular epithelial cells were observed by electron microscope.2.SA-AKI tubular epithelial cell injury in vitro(1)Cell culture.The HK-2 human kidney proximal tubular cell line was maintained in a mixture of Ham’s F12 and Dulbecco’s modified Eagle’s medium supplemented with 10%fetal bovine serum(FBS)and 1%penicillin/streptomycin in a humidified atmosphere of 5%C02 and 95%air at 37℃.The cells were seeded at an appropriate cell density for different assays and allowed to grow to 80%confluence.Cell synchronization was routinely performed by incubating cells in serum-free medium for 24 h prior to each experiment.Finally,the cells were exposed to different experimental conditions.(3)LPS administration to simulate SA-AKI tubular epithelial cell injury HK-2 cells were treated with LPS 10μg/ml for 18 hours.(4)Cells were divided into four groups as follows:(4)Detect content in cells experiments① ANEXIN V/propidium iodide were analyzed by flow cytometry.② Western blot analysis were performed to detect p-RIP-3、p-MLKL、p-DRIP-1、LC3、p62.③ Mitotracker detection by immunofluorescence were performed to analyze mitochondria impairment.3.Statistical analysisAll values are expressed as the mean ± standard deviation.Multiplecomparisons among the groups were conducted by one-way ANOVA followed by a least significant difference multiple comparison test.All statistical analyses wereperformed using SPSS software(version 21.0;IBM SPSS,Armonk,NY,USA),with values of P<0.05 considered to indicate statistically significant differences.RESULTS1.Protective effect of Nec-1 on renal function of sepsis mice Mice in LPS group has worse renal function which was detected by BUN(LPS group 32.54±5.46mmol/L vs.control 13.87±4.83mmmol/L,p<0.001)or Cr(LPS group 30.08±4.18μmol/L vs.control 9.80±1.84μmol/L,p<0.001).While pretreatment with Nec-1 could reverse BUN(LPS+Nec-1 group 14.15±4.14mmol/L vs.LPS group 32.54±5.46mmol/L,p<0.001)or Cr(LPS+Nec-1 group 11.50±1.67μmol/L vs.LPS group 30.08±4.18μmol/L,p<0.001).This result indicated Nec-1 protect kidneys from sepsis injury.2.Inhibition effect of Nec-1 on p-RIP-3 and p-MLKL in SA-AKIThe phosphorylation of RIP-3(p-RIP-3)and MLKL(p-MLKL)were assessed by western blotting.Level of p-RIP-3 and p-MLKL is elevated in LPS treatment mice and Nec-1 could reverse p-RIP-3 and p-MLKL level.The similar effect of Nec-1 inhibiting p-RIP-3 and p-MLKL was also observed in HK-2 cells administrated by LPS.3.The effect of Nec-1 on renal tubular epithelial necrosis in SA-AKIThere were no significant difference of renal tubular epithelial cell necrosis.That means Nec-1 protect kidney function not through inhibition of necroptosis.The HK-2 cell death manner and quantity was also analyzed by Anexin V/PI.Similarly,no difference of necrosis was detected between 3 different intervention cell groups.4.The effect of Nec-1 on renal tubular epithelial autophagy in SA-AKIWestern blot analysis showed LC3-II which is a marker of autophagosome and p62 which is an eliminated substrate of autophagy are both increased.While QT-PCR showed neither LC3-II nor p62 mRNA are not increased.Beclin,a protein participating in autophagy production,was not changed.Massive autophagosomes and paucity of autolysosome were observed by electron microscope.These results indicated sepsis do not induce autophagosome production but impair autophagosome elimination and autolysosome.When mice was pretreated with Nec-1,LC3-Ⅱ and p62 decreased.A large amount of autophagosomes were observed by electron microscope in Nec-1 pretreatment group.From these results,it was concluded that Nec-1 promote autophagosome elimination and autolysosome generation.In vitro,LPS induced increases of LC3-Ⅱ and p62 of HK-2 cells.While Nec-1 pre-treatment inhibited level of LC3-Ⅱ and p62.5.Protective effect of Nec-1 on mitochondrion of renal tubular epithelial cell in SA-AKI.Electron microscope showed the amount of mitochondria is reduced and mitochondrion swell in LPS treated mice tubular cells.Nec-1 could increase the amount of mitochondria and relieve swollen mitochondria.In the cell model,LPS induced mitochondria reduction and fragment which detected by mitotracker and Nec-1 could alleviate mitochondrial injury.High level of p-DRP1,which mediating mitochondrial fission,was detected by western blot in LPS treated mice.The elevated p-DRP1 could be reduced by Nec-1.Expression of PGC1-α,a key regulator of mitochondria function and biogenesis,was decrease in tubular cells of LPS treated mice.RT-PCR also show mRNA of PGCl-a is decreased.Nec-1 pretreated mice showed better PGC1-α transcription and expression of PGC1-a.CONCLUSIONSThis research confirmed the protective effect of Nec-1 on SA-AKI.The Nec-1 protective effect is not through inhibition of necroptosis,but may be through other two ways:1.Nec-1 promotes autophagosome elimination and autolysosome generation.2.Nec-1 reduces the mitochondrial impairment induced by sepsis.