| Objective:In renal ischemia-related diseases,ischemic reperfusion injury（IRI）has always been an important risk factor for the progression of acute and chronic kidney diseases.stanniocalcin（STC）is a glycoprotein hormone discovered early in teleost fishes.Stanniocalcin is divided into STC1 and STC2 according to different protein sequence homology.Among them,STC1 can effectively interfere with the production of superoxide in the body tissue and the progression of inflammatory response,and has a certain protective effect on the kidney.However,the specific mechanism of STC1 in the progression of renal IRI,the therapeutic potential of STC1 on kidney injury,and the role of STC1 in the oxidative stress-mediated apoptosis of renal tubular cells are still unclear.Therefore,exploring the role of STC1 in IRI and the specific molecular pathway mechanism will provide a new therapeutic target for the clinical treatment of acute and chronic kidney injury caused by IRI,and have important theoretical and practical significance for reducing the prevalence of end-stage renal disease and improving the prognosis of patients.Methods:1.Animal Experiment:（1）Establishment of STC1 Over-Expression（OE）SD rats and STC1 Knock-Out（KO）SD rats kidney models:Thirty adult male Sprague-Dawley rats weighing 250-300g（Animal Center Laboratory）were placed in a controlled light condition（12h light and 12h dark）at room temperature of 22±2℃and humidity of 60%.STC1 OE and STC1 KO rat models were established by retrograde injection of STC1 overexpression and knockout adeno-associated virus（AAV）into the left ureter,respectively.After three weeks,AAV was stably expressed,the subsequent surgery was performed.（2）Establishment of renal IRI model in SD rats:After one week of intraperitoneal anesthesia,the right kidney of SD rats was removed and the left renal pedicle was clamped for 45 minutes with non-traumatic arterial clamp.The effect of clamping was judged by observing the color change and shape of the left kidney surface.After clamping the left renal pedicle,the surface of the rat kidney quickly turned dark purple,and the outline size slightly shrank,which confirmed that the unilateral left renal artery obstruction model was successfully established by using non-traumatic vascular clamp.（3）Based on the grouping of STC1 OE AAV and renal STC1 KO AAV,SD rats were divided into 4 groups:sham operation group,renal IRI group,STC1 OE combined with renal IRI operation group,and STC1 KO combined with renal IRI operation group,with 6rats in each group.The right unilateral kidney was resected one week earlier and the left kidney was occluded for 45 minutes.Then the left kidney was harvested after 24 hours of reperfusion.After gross observation of the Kidney,the same part of the kidney was made into paraffin sections,and part of the kidney tissue was homogenized and centrifuged to obtain the supernatant for the expression level of reperfusion injury and histological study,including tissue damage,oxidative stress and inflammation-related products,including kidney injury molecule 1（Kidney injury molecule 1）.KIM-1),tumor necrosis factor（TNFα）,interleukin（IL）-6,reactive oxygen species（ROS）,myeloperoxidase（MPO）,lactate dehydrogenase（LDH）,Malondialdehyde（MDA）,In addition,1m L left ventricular blood samples were collected and centrifuged for biochemical parameters measurement,including blood urea nitrogen（BUN）and serum creatinine（SCR）.（4）High-throughput sequencing and bioinformatics analysis were performed on the above in vivo experimental data,and immunohistochemistry,H&E,PAS,TUNEL and other detection methods were used to explore the specific signaling mechanism of STC1in the progression of renal IRI.2.Cell Experiments:（1）Construction of in vitro model of NRK-52E Hypoxia/Reoxygenation（H/R）cells:NRK-52E cells were cultured in vitro,and H/R model of simulated cells in hypoxia environment was constructed by cobaltous chloride（Co Cl2）,a classical chemical reagent.1)NRK-52E cells were randomly divided into 6 groups:control group and H/R 6-hour induction group treated with 200μM,400μM,600μM,800μM and 1000μM Co Cl2,respectively.CCK8 and flow cytometry were used to detect the apoptosis rate of cells treated with H/R of different concentrations of Co Cl2.Mrna levels of hypoxia inducible factor（Hif-1α）and Kim-1 were detected by Real-Time s Reverse Transcription-PCR,and the morphological changes of cells in each group were observed by light microscopy.2)NRK-52E cells were randomly divided into 5 groups:control group and Co Cl2-induced simulated H/R for 6 hours,12 hours,24 hours and 48 hours,respectively.The expression levels of anti-apoptotic protein Bcl-2,pro-apoptotic proteins Bax,Hif-1αand KIM-1 of the Bcl-2 protein family were detected by Western blot.（2）STC1 overexpression and STC1 gene knockout cells were constructed:STC1 OE model in NRK-52E cells was constructed by adenovirus transfection,and STC1 KO model in NRK-52E cells was constructed by lentivirus transfection.NRK-52E cells were randomly divided into 4 groups:control group,Co Cl2-induced H/R model group,STC1OE and Co Cl2-induced H/R treatment group,STC1 KO and Co Cl2-induced H/R treatment group.Western blot was used to detect the expression of anti-apoptotic protein Bcl-2,pro-apoptotic protein Bax,Hif-1α,and KIM-1 in the four groups of models,and four signaling pathway proteins were detected.These include Adenosine 5’-monophosphate（AMP）-activated protein kinase（AMPK）,Recombinant Hepatocyte Nuclear Factor 4 Alpha（HNF4α）,The expression of Glucose-6-phosphatase（G6PC）and phosphorylated adenylate-activated protein kinase（P-AMPK）were detected.Results:1.Results of animal experiments:（1）Compared with the sham-operated group,the expression of STC1 increased in STC1-overexpressing AAV group,while decreased in STC1-knockout AAV group.（2）Compared with the sham-operated SD rats,the degree of renal injury,local oxidative stress and inflammation-related products increased in the IRI group;（3）Compared with the IRI group,the levels of renal tissue damage products and inflammatory response were significantly decreased in the STC1 OE+IRI group.（4）High-throughput sequencing analysis showed that STC1 played a protective role in SD rats with renal IRI through AMPK/HNF4α/G6PC pathway protein pathway.2.Results of cell experiments:（1）Under the light microscope,the cells in the normal group were spindle-shaped with intact cell membrane and grew well.After H/R treatment with 600μM,800μM and1000μM Co Cl2,the cells in the STC1 OE and KO groups became round,shrunken and appeared cell debris,while the cells in the STC1 OE and KO groups showed no obvious change.Most of the cells in the STC1 OE and Co Cl2-induced H/R combined treatment group showed normal cell morphology,while a large number of cells in the STC1 KO and Co Cl2-induced H/R combined treatment group were significantly round,shrinking and necrotic,and a large number of cell debris floating.（2）Compared with the control group,the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 was increased,and the expression levels of Hif-1αand KIM-1 were significantly increased after reoxygenation（within 48h）.（3）Compared with the control group,the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 was decreased in the STC1 OE group,and the expression of Hif-1αand KIM-1 was inhibited,while the expression of apoptosis-related genes was increased in the STC1 KO group.（4）Compared with the control group,the expression level of P-AMPK was increased and the expression levels of HNF4αand G6PC were decreased in STC1 OE group,and the expression level of P-AMPK was decreased and the expression levels of HNF4αand G6PC were decreased in STC1 KO group.Conclusion:1.When the reoxygenation time was within 48 hours,with the extension of reoxygenation（reperfusion）time,the apoptosis rate of NRK-52E cells increased gradually and reached the maximum at 24 hours.2.STC1 OE effectively reduced the apoptosis rate of NRK-52E cells induced by H/R induced by Co Cl2,while STC1 KO increased the degree of apoptosis.3.STC1 may play a protective role in alleviating renal IRI injury through AMPK/HNF4α/G6PC pathway proteins. |
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