| Part one The expression of gasdermin D in diabetes kidney and podocytes induced by high glucoseObjectives:diabetes is a chronic disease threatening human health worldwide,with high incidence rate,high mortality,multiple organ involvement and poor prognosis.Statistics show that it will reach 783million by 2045.During this period,the world population is estimated to increase by 20%,while the number of patients with diabetes is estimated to increase by 46%;Statistical analysis in 2021 shows that 6.7 million adults died of diabetes or its complications,and about 1/3 of diabetes deaths were younger than 60 years old.The chronic complications of diabetes affect the kidney,heart,eyes,blood vessels and peripheral nerves.Diabetes is the most common cause of chronic kidney disease（CKD）and the main cause of end stage renal disease（ESRD）.As one of the most serious microvascular complications in diabetes patients,the incidence rate of DKD is also increasing year by year.Current studies suggest that DKD is a metabolic disease mediated by a variety of inflammatory factors,with complex physiological and pathological mechanisms,and the pathogenesis is unknown.Current studies have confirmed that hyperglycemia,lipid metabolism disorders,oxidative stress,advanced glycation end products and other injury related molecules can be recognized by some specific pattern recognition receptors in cells to induce cell death,leading to renal injury and function decline.NLRP3 and IL-1βwere found during the development of diabetes nephropathy A large amount of activation can induce the pyroptosis of renal innate cells,renal fibrosis,glomerulosclerosis and renal tubular injury.Inhibition or knockout of NLRP3 shows obvious renal protection.However,GSDMD（gasdermin D）is a downstream molecule of NLRP3,and the role of GSDMD in diabetes renal tissue has not been studied.GSDMD is a newly discovered pyroptosis executive protein,which has a specific affinity with membrane phospholipids and acidic lipids.It exists in the cytoplasm in a self-inhibitory state and can be activated by a variety of risk factors,such as ROS,lipid metabolism disorders,advanced glycation products and other metabolic factors,as well as bacterial and viral infections.Since the discovery of GSDMD,the release of IL family has not been solved.The current research shows that GSDMD is necessary for the secretion of interleukin like proteins in cell membrane perforation,and interleukin-1 is closely related to podocytes.The activation of interleukin-1 receptor can limit the susceptibility of glomerulus to injury by stimulating Akt signal cascade.Therefore,it is speculated that the pyroptosis executive protein GSDMD may also be involved in podocyte injury.Methods:1.Expression of GSDMD in renal tissue of patients with diabetes nephropathy:there were two groups:DN group:20 patients with type 2 diabetes nephropathy diagnosed by renal pathology,and 10 patients in control group（Ctr,control group）.Non diabetes patients with clinical manifestations of mild hematuria or proteinuria were diagnosed as mild glomerular disease by renal biopsy,and were not complicated with other glomerular diseases（such as Ig A nephropathy or membranous nephropathy）.Routine paraffin embedding of kidney tissue,4μm,The expression and localization of GSDMD protein in each group were detected by HE,PAS,PASM,Masson staining and immunohistoche-mistry.2.Expression of GSDMD in kidney tissue of DM rats:establishment of animal model and collection of samples:40 healthy male SD rats were randomly selected as control group（Ctr,control group）after 2 weeks of adaptive feeding,and fed with normal diet（energy ratio:fat 12.11%,protein 22.47%,carbohydrate 65.42%）.The other groups were given high-fat diet（energy ratio:fat 45.65%,protein 16.46%,carbohydrate 37.42%）.At the end of the fourth week,15 rats in the diabetes group（DM）and 15 rats in the intervention group were intraperitoneally injected with 1%streptozotocin solution（sigma,USA）,a single dose of 40mg/kg,once a day,for three consecutive days.Rats in the Ctr group were intraperitoneally injected with 0.1 mol/l sterile citric acid sodium citrate buffer.After 72h,fasting blood glucose（FBG）of rats in each group was measured.When FBG>16.7mmol/L,it was determined as diabetes.From the 5th week,the intervention group was given Empagliflozin（15mg/kg/d）by gavage,and the Ctr and DM groups were given the same amount of water for injection by gavage every day for 8 weeks.The animals were killed at the end of the 8th week after successful modeling,fasted for 6-8 hours,and were anesthetized and weighed by intraperitoneal injection of 10%chloral hydrate.Blood was taken from the abdominal aorta for biochemical indicators;The capsule of bilateral kidneys was removed by laparotomy and weighed.The blood was washed with normal saline.The right kidney was fixed in 10%paraformaldehyde tissue fixative for subsequent HE,PAS,PASM,Masson staining and immuneo-histochemical detection;The left kidney was treated with liquid nitrogen and frozen in a-80℃refrigerator for preparing tissue homogenate to extract kidney tissue protein and RNA.Immunohistochemistry and Western blot were used to detect GSDMD,caspase-1 and nephrin protein in renal cortex.The maximum contour images of 20 glomeruli with blood vessels and/or urine poles were measured,and the glomerular mesangial area and capillary bundle area were measured.3.Culture,grouping and sample collection of mouse podocytes:immortalized mouse podocytes were cultured at 33℃in a 5%CO2incubator.The culture medium contained 10%fetal bovine serum,100u/ml penicillin and100μg/ml streptomycin,10u/mlγ-IFN was cultured in DMEM/F12（5:1）medium.Transfer to 37℃and 5%c CO2incubator for removalγ-IFN was cultured in the above medium,and podocytes gradually differentiated and matured.The solution was changed once every 2 days,and the cells grew to80%-90%for passage.The differentiated and mature podocytes were divided into normal glucose culture group（NG,normal glucose 5.5mmol/l）and high glucose culture group（HG,high glucose 30mmol/l）.The cells were collected at6 hours,12 hours,24 hours and 48 hours to extract total protein and RNA.The expression of GSDMD/GSDMD-N protein at different time points was detected by Western blot.The localization and distribution of GSDMD were detected by cellular immunofluorescence staining.Results:1.The results of biochemical indexes showed that there were significant differences in plasma albumin,triglyceride,BUN and blood UA in DN patients;Age,hemoglobin,Scr and 24-hour urinary protein in the control group were significantly different from those in the control group（p<0.01）;2.Immunohistochemical results showed that the expression of GSDMD was significantly increased in DN group.GSDMD was mainly distributed in renal tubular epithelial cells,glomerular podocytes,endothelial cells and mesangial cells;3.The weight of rats in diabetes group decreased significantly Ctr group increased（P<0.01）;Urinary protein excretion and blood glucose increased（p<0.01）;4.HE,PAS,PASM and Masson staining showed that the glomerular mesangial matrix in DN group was widened,the basement membrane was significantly thickened,K-W nodules,focal tubular atrophy and interstitial fibrosis were visible.In DM group,the glomerular volume of renal tissue increased,the renal vesicle space narrowed,the mesangial area widened,the glomerular fibrosis area increased,some renal tubular epithelial cells became vacuolar degeneration,cells fell off,and the basement membrane was exposed;5.Western blot showed that the expression of GSDMD in renal cortex of diabetes rats was significantly higher than that of normal control group（P<0.01）;The expression of GSDMD density was significantly increased by immunohisto-chemistry（P<0.01）;6.The results of Western blot and QRT PCR showed that the expression of GSDMD induced by high glucose changed with time,and reached the peak at 24hours of HG culture（P<0.01）;The expression of GSDMD-N and the detection of GSDMD m RNA were consistent.Immunofluorescence showed that the fluorescence intensity of GSDMD was significantly higher than that of NG and mainly concentrated in the cytoplasm.Part two Gasdermin D on inflammatory factors and apoptotic proteins in mouse podocytes induced by high glucose via JNK pathwayObjective:The early stage of diabetes nephropathy mainly involves glomerular damage.At present,proteinuria is still the staging standard of DN.Podocytes,as a component of glomerular filtration barrier,play an important role in DN glomerulosclerosis.It is believed that podocyte inflammation and apoptosis are involved in the occurrence and development of diabetes nephropathy.A large number of data showed that compared with non-diabetes mice,the expression of inflammatory molecules and pro-inflammatory cytokines in the circulation of diabetes mice was up-regulated,and the inflammatory factor interleukin（IL）-1βAnd IL-18 promote the progress of DN.High glucose induced podocyte apoptosis and increased cleavage of Caspase-3 and Bax/Bcl-2.GSDMD is the ultimate executor of pyroptosis.It serves as the direct substrate of activated caspase-1 in typical inflammatory signaling pathways and caspase-11/4/5 in non-classical pathways.It is the key molecule linking pyroptosis and apoptosis.JNK is an important member of mitogen activated protein kinase family,which has a wide tissue distribution.The activation of JNK regulatory pathway can trigger a variety of pathophysiological processes.Inhibition of JNK pathway plays an important protective role in high glucose stress cell injury through its anti-inflammatory,anti-proliferation and anti-oxidation.In the previous study,we found that the expression of GSDMD was up-regulated in podocytes of mice with diabetes nephropathy and high glucose environment,which was positively correlated with the pathological damage of diabetes kidney tissue.It is speculated that GSDMD may be involved in the pathogenesis of diabetes nephropathy.However,the exact mechanism is unknown.Whether it affects inflammatory factors and apoptosis related proteins through JNK signaling pathway needs further study.Methods:1.The optimal transfection conditions were screened.Lipofectamine 3000transfection reagent was mixed with GSDMD si RNA in different proportions and amounts to transfect podocytes.The expression of GSDMD was detected by Western blot and q RT-PCR.2.The effects of GSDMD si RNA on GSDMD,GSDMD-N and synaptopo-din protein of mouse podocytes under high glucose environment were detected by Western blot.The mice were divided into normal glucose group（NG,glucose5.5mmol/l）,high glucose group（HG,glucose 30mmol/L）,HG+NC（high glucose+si RNA negative control）,high glucose+GSDMDsi RNA plasmid transfection group(high glucose（30mm）+GSDMD si RNA（20μM）.After 24hours of stimulation,the cells were collected.The expression of GSDMD,GSDMD-N and synaptopodin was detected by Western blot and q RT-PCR.The expression of synaptopodin in GSDMD si RNA podocytes was detected by laser confocal microscope.3.To detect the effect of GSDMD knockout on high glucose induced inflammatory factors in podocytes,and divide into three groups:normal glucose group（NG,glucose 5.5mmol/l）,high glucose group（HG,glucose 30mmol/l）,high glucose+NC（HG+NC,high glucose+si RNA negative control）,high glucose+HG+GSDMD si RNA plasmid transfection group（high glucose（30mm）+GSDMD si RNA（20μM））.Cells were collected 24 hours after stimulation and expression of IL-6、IL-1βand TNF-a was detected by Western blot.4.The effect of knockout of GSDMD on apoptosis related proteins of podocytes induced by high glucose was divided into normal glucose culture group（NG,glucose 5.5mmol/l）,high glucose culture group（HG,glucose30mmol/l）,high glucose+empty body group（HG+NC,high glucose+si RNA negative control）,high glucose+GSDMD si RNA plasmid transfection group（high glucose（30mm）+GSDMD si RNA（20μM））.After 24 hours of stimulation,the cells were collected and the expressions of cleaved-caspase-3and Bax/Bcl-2 were detected by Western blot.5.The effect of high glucose on JNK signal pathway activity.Grouping:normal glucose group（NG,glucose 5.5mmol/l）,high glucose group（HG,glucose 30mmol/l）,normal glucose culture group+JNK inhibitor（NG+SP:normal glucose+SP600125）;High glucose culture group+JNK inhibitor（HG+SP:high glucose+SP600125）.The cells were collected 24 hours after stimulation.The expression of JNK and p-JNK was detected by Western blot,and the expression of JNK specific inhibitor SP600125 was detected by immunofluorescence.6.The effect of JNK specific inhibition on the activation of inflammatory factors in podocytes under high glucose environment.Groups:normal glucose group（NG,glucose 5.5mmol/l）,high glucose group（HG,glucose 30mmol/l）,normal glucose culture group+JNK inhibitor（NG+SP:normal glucose+SP600125）;High glucose group+JNK inhibitor（HG+SP:high glucose+SP600125）.After 24 hours of stimulation,the cells were collected and the expression IL-6、IL-1βand TNF-a was detected by Western blot.7.The effect of JNK specific inhibition on apoptosis related proteins of podocytes in high glucose environment.Groups:normal glucose group（NG,glucose 5.5mmol/l）,high glucose group（HG,glucose 30mmol/l）,normal glucose group+JNK inhibitor（NG+SP:normal glucose+SP600125）;High glucose culture group+JNK inhibitor（HG+SP:high glucose+SP600125）.After24 hours of culture,the cells were collected and the expressions of cleaved Caspase3 and Bax/Bcl-2 were detected by Western blot.8.The effect of GSDMD si RNA gene knockout on the production of mitochondrial ROS induced by HG.Groups:normal concentration glucose culture group（NG,glucose 5.5mmol/l）,high glucose+GSDMD si RNA plasmid transfection group(high glucose（30mm）+GSDMD si RNA（20μM）.The level of mt ROS was detected with Mito SOX red fluorescent probe.9.Western blot was used to detect the effect of GSDMD si RNA on the antioxidant stress protein SOD2 of MPCs in high glucose environment.The groups were divided into normal glucose group（NG,glucose 5.5mmol/l）,high glucose group（HG,glucose 30mmol/l）,high glucose+empty body group（HG+NG,high glucose+si RNA negative control）,high glucose+GSDMD si RNA plasmid transfection group(high glucose（30mm）+GSDMD si RNA（20μM）.After 24 hours of culture,the cells were collected and the expression of SOD2 was detected by Western blot.10.Effects of GSDMD si RNA gene knockout and NAC on the expression levels of JNK and p-JNK in mouse podocytes induced by HG,normal glucose culture group（NG,glucose 5.5mmol/l）,high glucose culture group（HG,glucose 30mmol/l）,high glucose+GSDMD si RNA plasmid transfection group（high glucose（30mm）+GSDMD si RNA（20μM））high glucose+NAC group:（high glucose（30mm）+NAC（15μM））after 24 hours of culture,the cells were collected and the expression of JNK and p-JNK were detected by Western blot.Results:1.Lipofectamine 3000 3.75ul+pcmv3 GSDMD20um+p3000 5ul transfected podocytes obtained the best expression effect of GSDMD.2.Compared with HG group,GSDMD si RNA reduced the expression of GSDMD and GSDMD-N protein in podocytes under high glucose environment（P<0.05）.GSDMDsi RNA improved the decrease of synaptopodin expression induced by high glucose（P<0.05）.3.In HG group,the expression of IL-1β、IL-6 and TNF-a was also high.the expression of these proteins was significantly reduced by transfection of GSDMD si RNA,4.Compared with NG group,the expression of cleaved-caspase-3 and Bax/Bcl-2 in HG group was significantly increased（P<0.05）,GSDMD knockout decreased the expression of cleaved-caspase-3 and Bax/Bcl-2 in podocytes under high glucose environment（P<0.05）.5.P-JNK/JNK increased in high glucose environment,which was statistically different from that in NG group（P<0.05）.At the same time,the expression of JNK and p-JNK decreased after the application of JNK specific inhibitor SP600125（P<0.05）.The results of laser confocal microscopy showed that the expression of synaptopodin in high glucose group decreased significantly.After the application of JNK specific inhibitor SP600125,the expression of synaptopodin increased significantly.6.The results of western blot showed that the expression of TNF-a、IL-6and IL-1βwas significantly lower than that of HG（P<0.05）.7.Compared with the control group,the expression of cleaved Caspase3and Bax/Bcl-2 protein in MPCs increased significantly after 24h stimulation in high glucose environment（P<0.05）.After application of SP600125,the expression of cleaved Caspase3 and Bax/Bcl-2 protein decreased significantly（P<0.05）.8.Knockout of GSDMD can significantly improve the enhancement of mt ROS production induced by HGKnockout of GSDMD significantly improved the HG induced decrease of SOD2 expression（P<0.05）.The expression level of p-JNK protein in HG+GSDMD si RNA and HG+NAC group was significantly lower than that in HG group.There was no significant difference in JNK levels between HG+GSDMD si RNA group and HG+NAC group.Our study shows that GSDMD is a key regulator of mt ROS production,and GSDMD knockout inhibits HG induced JNK activation through ROS.Part three Effects of Empagliflozin on gasdermin D related inflammatory factors in renal tissue and podocytes of diabetes the intervention of apoptosisObjective:Diabetes nephropathy（DN）is the main cause of end-stage renal disease,and increases the risk of cardiovascular events and death.Although a variety of drugs and surgical methods have been used to treat diabetes,the effect is not satisfactory.At present,regulating blood glucose,blood lipid and blood pressure,enhancing antioxidation and inhibiting Ras activation are not enough to prevent the progress of DN.Podocyte loss is closely related to pyroptosis.Pyroptosis is a kind of programmed cell death.When the inflammatory body of nucleotide binding oligomerization domain like receptor protein 3（NLRP3）is activated,it will further activate caspase-1.Then caspase-1 will cut GSDMD and release its N-terminal domain,so as to penetrate the cell membrane and form membrane pores.Membrane rupture,cell osmotic pressure,DNA cleavage,release of cell contents and inflammatory mediators lead to a strong inflammatory response.Sodium glucose cotransporter 2 inhibitor is a new oral hypoglycemic drug,which acts on proximal convoluted tubules and regulates90%glucose reabsorption in glomerular filtration.It was approved to treat adult type 2 diabetes in 2014.Empagliflozin is a selective sodium glucose cotransport-er 2 inhibitor.Clinical studies have shown that SGLT2 can reduce blood glucose,blood pressure and diuresis,and inhibit RAAS activation.Studies have confirmed that empagliflozin reduces oxidative stress and cell death in pancreas and kidney of type 2 diabetic mice.It is unclear whether empagliflozin plays a role through podocyte inflammatory factors and apoptosis.Methods:1.The treatment of SD rat culture and tissue samples and the extraction of renal cortical protein are the same as those in part I and part II.2.To determine the safe and effective concentration of empagliflozin,podocytes were divided into normal control group,empagliflozin 100nm,10nm,1nm,0.1nm and 0.01nm groups.After the cells were subcultured and adhered to the wall,the original medium was discarded and replaced with empagliflozin DMEM/F12 medium with corresponding concentration.After 48h of culture,the viability of podocytes in each group was calculated.3.MTS results showed that 0.01,0.1 and 1nm empagliflozin could maintain stable podocyte activity when cultured with NG for 48h,but empagliflozin inhibited podocyte activity when it was greater than 10nm.When cultured in HG for 48h,empagliflozin at concentrations of 0.01,0.1 and 1nm could protect podocytes from the decrease of cell activity induced by high glucose,and empagliflozin aggravated the cell damage when it was greater than 10nm.In this part of the experiment,empagliflozin concentration of 0.1nm was used for subsequent research.Results:1.Empagliflozin reduces the level of inflammatory factors in renal cortex of diabetes rats.Compared with Ctr group,the expression of nephrin in DM group was significantly down regulated（P<0.01）;DM+Em group reversed the down-regulated expression of nephrin in DM group（P<0.01）.It was significantly higher of Caspase-1,cleave-caspase-1,GSDMD,cleave-IL-1βin DM group than that in Ctr group（P<0.01）.2.Empagliflozin reduces pathological damage in diabetes rats.The mean glomerular size（expressed as mean glomerular diameter）in DM group was larger than that in normal group and empagliflozin group;PASM staining showed that the glycogen deposition in DM group was significantly higher than that in normal group（P<0.01）.After empagliflozin treatment,the glycogen deposition area decreased（P<0.01）.Masson and PASM staining showed that DM group had significant pathological changes compared with normal group,including mesangial matrix expansion and fiber area increase（P<0.01）.3.The molecular mechanism of empagliflozin protective effect on podocytes.The results of laser confocal microscopy showed that the accumulation of GSDMD decreased significantly after empagliflozin co-culture.The expression trend of Caspase-1 was the same as that of GSDMD,and the brightness was stronger than that of GSDMD.Western blot showed that high glucose significantly increased the expression levels of GSDMD and caspase-1in cytoplasm（P<0.01）.In empagliflozin group,the expression of GSDMD and caspase-1 was decreased（P<0.05）.4.Empagliflozin inhibits HG induced podocyte apoptosis.The results of western blot showed that the expression of nephrin in podocytes decreased,and the expression of apoptosis related proteins Bax and caspase-3 increased.Empagliflozin intervention could increase the expression level of nephrin by35%（P<0.01）,and significantly reduce the expression level of cleaved-caspase-3（P<0.01）and the ratio of Bax/Bcl-2（P<0.01）;Flow cytometry analysis showed that the apoptosis rate of HG group was 2.45 times higher than that of NG group（P<0.01）,while that of empagliflozin intervention group was 1.56times higher than that of NG group（P<0.01）.5.Effect of empagliflozin on biological parameters of diabetes rats.Compared with Ctr group,the weight of rats in DM and DM+Em groups decreased（P<0.01）,and the ratio of kidney weight（mg）to body weight（g）in DM and DM+Em groups was higher than that in Ctr group（P<0.01）;Urinary protein excretion in DM group and DM+Em group was significantly higher than that in Ctr group（P<0.01）;FBG levels in DM group and DM+Em group were higher than those in Ctr group（P<0.01）.Conclusions:1.The expression of GSDMD in patients with diabetes nephropathy,renal tissue of diabetes rats and podocytes of mice under high glucose environment is increased,and JNK signal pathway may be involved in this process.2.Knockout of GSDMD and inhibition of JNK signaling pathway can significantly reduce the activation of inflammatory factors,podocyte apoptosis and oxidative stress induced by high glucose.3.Empagliflozin can protect the kidney by reducing the activation of inflammatory factors and apoptosis in renal cortex and podocytes of diabetes. |
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