SIK2 Protects Against Renal Tubular Injury And The Progression Of Diabetic Kidney Disease Via HSF1/HSPA6 Pathway

BackgroundDiabetic kidney disease（DKD）is one of the most common diabetes complications,whose incidence is nearly 40%.At present,the methods of DKD treatment are mainly to improve lifestyle,and control both blood glucose and blood pressure by medicine.Despite these methods could reduce the urine microalbumin creatine ratio（UACR）to a certain extent,many patients with DKD still progress to end-stage renal disease.Therefore,it is necessary to study the DKD pathological mechanism further,and identify the novel biomarkers that are expected to become therapeutic drug targets for DKD.Salt induced kinase 2（SIK2）,a serine/threonine protein kinase,is a member of the AMP-activated protein kinase（AMPK）family.Existing studies have revealed that SIK2participates in glucose and lipid metabolism.The knockdown of SIK2 downregulates GLUT4expression and reduce glucose uptake in muscle and white adipocytes.What‘s more,SIK2modulates the efficiency of insulin signal transduction by phosphorylating insulin receptor substrate 1（IRS-1）on Ser794 and it also functions as a key gluconeogenic suppressor in the liver when combined with liver kinase B1（LKB1）.It has been identified that SIK2 is related to many metabolic diseases,such as obesity,nonalcoholic fatty liver,and diabetes retinopathy.However,there is no research on the role of SIK2 in DKD.Whether SIK2 affects renal function still needs to be illuminated.This study firstly investigates on the role and molecular mechanism of SIK2 in DKD.Research purposesThe aim of this study is to clarify the role of SIK2 in kidney tubular function and the development of DKD,providing a new idea for the clinical treatment of DKD.Methods1.Construct type 1 diabetes mellitus（T1DM）and type 2 diabetes mellitus animal models（T2DM）.（1）C57BL/6 male mice were divided into control group and T1DM group.Mice in T1DM group were intraperitoneally injected with Streptozocin（STZ,50mg/kg,p H=4.5）,and control mice were intraperitoneally injected with saline at the same dose;Fasting blood glucose（FBG）>13.8m M after 2 weeks was considered as successful modeling of T1DM.（2）The db/db mice with leptin receptor gene deletion(BKS Lepr^-/-)were rasised as the T2DM mouse model,and the control group were the mice with normal leptin receptor gene expression(BKS Lepr^+/+).They were fed routinely.T1DM and T2DM mice showed impaired renal function and developed to DKD mice at 16 weeks after STZ and aged 20weeks respectively.The location of SIK2 in kidney was observed by Immunohistochemical staining（IHC）.The m RNA and protein expression level of SIK2 in the kidney of DKD mice and control group was examined by real-time quantitative PCR（q RT-PCR）and western blot（WB）.2.10-week-old SIK2 KO and WT mice were divided into two groups,non-diabetes nephropathy group（N-DKD）and diabetes nephropathy group（DKD）.KO and WT mice in DKD group were intraperitoneally injected with STZ to induce T1DM,while mice in N-DKD group were intraperitoneally injected with saline at the same dose.Body weight and FBG were monitored monthly.After STZ 16 weeks,samples including urine,serum and kidney tissue were collected.Kidney failure of mice was detected as follows:（1）The concentration of urinary creatinine was determined by Jaffe colorimetry and the concentration of microalbumin in urine was determined by ELISA,then the Urine microalbumin creatine ratio（UACR）of mice was calculated;The excretion of urinary N-acetylglucosaminidase（N-NAG）and blood urea nitrogen（BUN）concentration were measured by colorimetry;（2）The morphology of kidney was observed after HE and Masson staining;（3）The relative m RNA expression of genes involved in fibrosis and inflammation（TGF-1,COL1A1,COL4A1,IL-6,IL-1,TNF–）was detected by q RT-PCR;（4）The expression of AQP1 and NGAL expression was detected by Immunofluorescence staining（IF）,AQP1 was a marker of renal tubular function and NGAL was a marker of renal tubular injury;（5）The level of apoptosis and reactive oxygen species（ROS）in renal tubular epithelial cells were detected by TUNEL staining and Mito Sox staining;（6）The morphology of mitochondria in renal tubular epithelial cells was observed by transmission electron microscopy（TEM）,and the ratio of longitudinal and transverse axes of mitochondria was quantified.3.The small molecule inhibitor ARN-3236 was used to inhibit the SIK2 kinase activity in STZ-induced DKD mice.10-week-old C57BL/6 male mice were intraperitoneally injected with STZ to induce T1DM.At week 12 of STZ,mice were divided into two groups after matching body weight and FBG:control group（STZ-Ctl）and SIK2 activity inhibition group（STZ-ARN）.In STZ-ARN group,ARN-3236 was orally administered at a concentration of60mg/kg daily.After treatment with ARN-3236 for 21 days,body weight and FBG were measured,urine,serum and kidney tissue were collected.The renal injury level of mice was evaluated comprehensively by detecting renal function indicators,morphological changes,renal tubular epithelial cells apoptosis percentage and fibrosis percentage.The specific method is the same as the second point in the method.4.Overexpressing SIK2 by transfecting plasmid in HK2 cell line.Exploring the downstream pathway and genes regulated by SIK2 by RNA sequencing.Silencing/overexpressing SIK2 by si RNA/plasmid in HK2 cells.the differentially genes which were screened by RNA sequencing were verified by q RT-PCR and WB.The apoptotic cells ratio of HK2 cells after silencing or overexpressing SIK2 was detected by flow cytometry.The regulation of SIK2 on HSF1 transcriptional activity was detected by luciferase reporter gene assay.The binding of SIK2 to p300 and the effect of SIK2 on HSF1phosphorylation and acetylation level were detected by co immunoprecipitation（Co-IP）and WB.5.Both SIK2-WT and SIK2-KO male mice were divided into two groups,the control group（WT-C,KO-C）and the overexpression HSF1 group（WT-HSF1,KO-HSF1）.In order to induce DKD,both WT and KO mice were intraperitoneally injected by STZ.After STZ 12weeks,mice in the overexpression HSF1 group were transduced with adeno-associated recombinant virus（AAV9-pCDH16-HSF1）via tail vein injection,while mice in the control group were injected with control vehicle（AAV9-pCDH16）.Body weight and FBG were measured,urine,serum and kidney tissue were collected 4 weeks after AAV infection.The HSF1 expression in the kidney and renal injury level of mice were compared in the 4 groups.The specific method is the same as the second point in the method.6.To evaluate the roles of SIK2 in tubular injury,STZ-induced diabetic and db/db mice were transduced with AAV9-pCDH16-SIK2 or control vehicle AAV9-pCDH16 via tail vein injection.Body weight and FBG were measured,urine,serum and kidney tissue were collected 4 weeks after AAV infection.The SIK2 expression in the kidney and renal injury level were compared between control group and overexpressing-SIK2 group mice.The specific method is the same as the second point in the method.Results1.SIK2 was mainly expressed in renal tubular epithelial cells,but not in glomeruli.The expression of SIK2 was significantly decreased in the renal tubules of two DKD mouse models（STZ and db/db）compared with the control group.2.Under N-DKD condition,there was no difference in body weight,FBG,or renal morphology and function between WT and KO mice.However,under DKD condition,KO-STZ mice displayed a decrease in body weight and an increase in UACR,BUN and-NAG compared with WT-STZ group（P<0.05）.HE and Masson staining showed tubular atrophy and an expansion of the tubulointerstitial compartment in kidneys of diabetic WT mice,which were aggravated in diabetic KO mice（P<0.05）.Furthermore,the staining of kidney sections with AQP1（a marker of renal tubules）and NGAL（a marker of tubular injury）demonstrated graver tubular injury in KO mice compared with WT mice under diabetic condition（P<0.05）.The relative m RNA expression of genes involved in the signal pathway of fibrosis and inflammation was higher in kidney of KO-STZ mice（P<0.05）.TUNEL staining showed that an obvious increase in renal tubular epithelial apoptosis KO-STZ mice（P<0.05）.What‘s more,TEM images showed that the deformation of mitochondria was mainly swollen in WT-STZ mice.In KO-STZ mice,the deformation was aggravated,reflected by angulation of mitochondria and disintegration of cristae.KO-STZ mice also generated more ROS than WT-STZ mice.3.STZ-induced diabetic mice was administrated with ARN-3236,in order to detect the effects of SIK2 kinase deficiency on tubular function.The level of phosphorylation of SIK2 at Thr175,which was the key kinase activity site of SIK2,was obviously reduced in kidney of STZ-mice after ARN-3236 treatment.Similar to the results which were observed in KO mice,ARN-3236 treatment had no significant effect on FBG and body weight,but aggravated tubular damage and interstitial fibrosis in diabetic mice.Compared to control group,inhibition of SIK2 activity increased UACR,BUN and-NAG in diabetic mice（P<0.05）.IF staining showed that AQP1 expression decreased,while NGAL expression increased in STZ-ARN group.The relative m RNA expression of genes related with fibrosis and inflammation in kidney was up-regulated by ARN-3236（P<0.05）.Moreover,ARN-3236increased the apoptosis of tubular epithelial cells.Mitochondria deformation and ROS generation were exacerbated in STZ-ARN mice（P<0.05）.4.To elucidate the cellular molecular mechanisms,RNA sequencing of HK2 cells transfected SIK2 overexpression plasmid or control vectors was performed.GO and KEGG analysis showed that SIK2 affected the biological process of cellular response to stress and protein processing in ER.Subsequently,the proteins involved in ER stress,such as p-e IF2and CHOP,were obviously downregulated in HK2 cells overexpressing SIK2,when treated with thapsigargin（Tg）.In contrast,knockdown of SIK2 increased the expression of p-e IF2and CHOP（P<0.05）.In addition,compared with relative control group,overexpression SIK2decreased apoptosis by 58.6%in HK2 cells but knockdown of SIK2 increased by 61.4%of apoptosis（P<0.05）.Moreover,treatment HK2 cells with tauroursodeoxycholic acid（TUDCA）attenuated the increased protein levels of p-e IF2and CHOP,and the cellular apoptosis induced by knockdown of SIK2.5.According to RNA-sequencing data,overexpression of SIK2 could up-regulate the expression of HSPA6,which is a member of the heat shock 70 protein（HSP70）family（P<0.05）.Knockdown of HSF1,a prerequisite for the transactivation of HSP70,attenuated the upregulation of HSPA6 and blunted the inhibition of ER stress and apoptosis mediated by overexpressing SIK2 in HK2 cells.In a dual-luciferase reporter gene assay,SIK2 distinctly promoted the HSF1-mediated transcriptional activation of HSPA6 in a dose-dependent manner（P<0.05）,but the promotion was abated by truncated mutation of the kinase domain.Besides,we found that SIK2 interacted with p300 via co-IP assay.Overexpressing SIK2promoted the phosphorylation of p300（Ser89）and knockdown of SIK2 repressed the phosphorylation of p300（P<0.05）.6.The expression of HSF1 and HSPA6 was upregulated in mice transfected with AAV9-pCDH16-HSF1.Compared with diabetic WT mice,diabetic KO mice had no difference in FBG levels,whether HSF1 was overexpressed or not.However,the decreased body weight,and the aggravated albuminuria,interstitial fibrosis,tubular injury and epithelial apoptosis observed in diabetic KO mice were attenuated by the tubular-specific overexpression of HSF1.Meanwhile,in diabetic mice,overexpressing HSF1 abated the upregulation of genes involved in tubular injury and interstitial fibrosis induced by knockout of SIK2.7.The expression of SIK2 was upregulated in STZ and db/db mice transfected with AAV9-pCDH16-SIK2.Overexpressing SIK2 did not affect the FBG levels.However,restoration of SIK2 significantly reduced the albuminuria,BUN and-NAG,improved tubular injury and reduced interstitial fibrosis（P<0.05）.Diabetic mice overexpressing SIK2showed decreased tubular epithelial apoptosis and ROS production,but an obvious improvement in mitochondrial morphology and function.Moreover,the protein levels of HSPA6 were notably increased,and ATF4,CHOP and cleaved Caspase 3 were decreased in kidney of diabetic mice,when SIK2 was specifically overexpressed.Taken together,these results indicate that targeting SIK2 might be a potential therapeutic strategy for tubular injury in DKD.ConclusionsOur study found that SIK2 was mainly expressed in renal tubular epithelial cells,and the expression of SIK2 was significantly decreased in the renal tubules of DKD murine models.SIK2 deletion aggravated the process of renal injury,while overexpression of SIK2 in renal tubules reduced renal tubular injury and improved renal function in DKD mice.SIK2 protects against tubular epithelial apoptosis via HSF1/HSPA6（member of HSP70）.Specifically,SIK2enhanced the transcriptional activity of HSF1 by inhibiting p300 HAT activity,and consequently up-regulated the transcriptional expression of HSPA6.Up-regulation of HSPA6induced by HSF1 could attenuate ER stress and ER stress-induced apoptosis in renal tubules.Overall,these findings indicate that SIK2 protects against renal tubular injury and the progression of DKD,make a compelling case for targeting SIK2 for clinical therapy in DKD.