Protective effects of Klotho protein on acute kidney injury in septic mice and its mechanism / 中华危重病急救医学

ABSTRACT

Objective? To? investigate? the? protective? effects? of? Klotho? protein,? a? kind? of? single-pass?transmembrane?protein,?on?acute?kidney?injury?(AKI)?in?septic?mice?and?its?mechanism.? Methods? Sixty?SPF?healthy?male?C57BL/6?mice?(6-8?weeks)?were?randomly?divided?into?sham?operation?group?(Sham?group),?sepsis?model?group?(CLP?group)?and?Klotho?protein?injection?group?(CLP+KL?group),?with?20?in?each?group.?The?septic?AKI?mice?model?was?established?by?cecal?ligation?and?puncture?(CLP);?Sham?group?had?the?same?procedure?except?that?the?cecal?was?not?ligated.?The?CLP+KL?group?was?received?Klotho?protein?(0.02?mg/kg)?by?intraperitoneal?consecutive?injection?for?4?days?after?operation;?Sham?group?and?CLP?group?were?injected?with?the?same?amount?of?saline.?Blood?samples?were?obtained?at?24?hours?after?operation,?the?levels?of?serum?creatinine?(SCr)?and?urea?nitrogen?(BUN)?were?measured?by?sarcosine?oxidase?and?urease?method.?The?mice?were?sacrificed?under?anesthesia?at?5?days?after?operation?to?harvest?renal?tissues,?and?the?pathological?damage?of?the?kidney?was?evaluated?by?hematoxylin-eosin?(HE)?staining.?The?ultrastructure?of?mitochondria?in?mouse?renal?tubular?epithelial?cells?was?observed?under?transmission?electron?microscope.?The?levels?of?reduced? glutathione?hormone?(GSH),?malondialdehyde?(MDA)?and?nitric?oxide?synthase?(NOS)?in?mitochondrion?were?determined?by?micro-enzyme?method,?thiobarbituric?acid?method,?colorimetry?method,?respectively.?The?protein?expressions?of?Klotho,?Bcl-2?and?cytochrome?C?(Cyt?C)?were?detected?by?Western?Blot.? Results? The?pathological?structure?of?the?kidneys?in?the?Sham?group?was?clear?and?intact.?Compared?with?the?Sham?group,?the?renal?tissue?edema?of?the?mice?in?the?CLP?group?was?significant,?and?the?transparent?tube?type?was?observed?in?the?small?lumen,?and?the?interstitial?inflammatory?cells?infiltrated;?the?levels?of?SCr?and?BUN?were?significantly?increased?[SCr?(μmol/L)?182.60±6.97?vs.?47.20±5.37,?BUN?(mmol/L)?53.70±5.12?vs.?18.70±2.62,?both?P?<?0.01];?the?mitochondria?were?swollen?and?deformed,?the?sputum?structure?was?destroyed,?the?matrix?density?was?decreased,?the?outer?membrane?was?lost,?and?the?levels?of?MDA,?GSH?and?NOS?were?significantly?increased?[MDA?(μmol/g)?1.172±0.046?vs.?0.746±0.094,?GSH?(μmol/g)?5.765±0.059?vs.?4.223±0.072,?NOS?(kU/g)?0.91±0.05?vs.?0.68±0.03,?all?P?<?0.01];?the?protein?expressions?of?Klotho?and?Bcl-2??in?renal?tissue?were?decreased,?and?the?protein?expression?of?Cyt?C?was?increased?(Klotho/β-actin?0.188±0.020?vs.?0.538±0.024,?Bcl-2/β-actin?0.311±0.010?vs.?0.391±0.015,?Cyt?C/β-actin?0.226±0.010?vs.?0.135±0.006,?all??P?<?0.01).?Comparing?with?the?CLP?group,?the?glomerular?and?tubular?tissue?epithelial?edema?and?the?small?lumen?in?the?CLP+KL?group?were?reduced;?the?levels?of?SCr?and?BUN?were?significantly?decreased?[SCr?(μmol/L)?85.70±7.23?vs.?182.60±6.97,?BUN?(mmol/L)?35.30±3.50?vs.?53.70±5.12,?both?P?<?0.01];?the?mitochondrial?structure?was?relatively?intact;?the?levels?of?MDA,?GSH?and?NOS?were?significantly?decreased?[MDA?(μmol/g)?0.958±0.072?vs.?1.172±0.046,?GSH?(μmol/g)?4.756±0.107?vs.?5.765±0.059,?NOS?(kU/g)?0.79±0.02?vs.?0.91±0.05,?all?P?<?0.01];?the?protein?expressions?of?Klotho,?Bcl-2?were?significantly?increased,?but?the?protein?expression?of?Cyt?C?was?significantly?decreased?(Klotho/β-actin?0.336±0.011?vs.?0.188±0.020,?Bcl-2/β-actin?0.474±0.017?vs.?0.311±0.010,?Cyt?C/β-actin??0.168±0.006?vs.?0.226±0.010,?all?P?<?0.01).? Conclusion? Klotho?protein?has?significant?protective?effects?on?AKI?in?septic?mice,?and?its?mechanism?is?related?to?maintaining?mitochondrial?structural?integrity?and?oxidative?stress?response.