| BackgroundAs one of the most common microvascular complications of diabetes mellitus(DM),diabetic nephropathy(DN)is one of the leading causes of end stage renal disease(ESRD).Increasing evidences demonstrate that tubulointerstitial lesions are independent risk factors for the development and progression of DN.Therefore,it will be illuminating to study the roles that changes of renal tubules,peritubular capillaries(PTC)and interstitial microenvironment played in the development of DN.A1 Adenosine Receptor(A1AR)is widely expressed in kidney.It received extensive attention due to its important role in tubuloglomerular feedback(TGF).Previous studies showed that A1AR also had protective roles independent of TGF,but the exact mechanism reminded unknown.AimsIn this study,we studied the protective role of A1AR,which is independent of TGF,played in DN progression of albuminuria and renal fibrosis,which alleviate the tubular microenvironment(renal tubule,interstitium and PTC)damage in diabetic patients,A1AR knockout(A1AR-/-)DN mice and in vitro cell experiments.1.Established a DN model in A1AR-deficient mice.mRNA gene chip analysis was performed to search differentially expressed genes and find the possible molecular regulatory pathway that A1AR participates in DN progression.2.By DN patients and mice models,to explore the effect of A1AR on renal tubular tight junction destruction,microphage infiltration caused by detachment of pericytes,megalin loss associated albuminuria and tubulointerstitial fibrosis.3.In vitro cell experiment,to demonstrate the protective role of A1AR to renal proximal tubular endothelium in hyperglycemic environment.Thus,we provided potential intervention target for DN treatment.4.To observe the effect and possible mechanism of A1AR in water-salt metabolism-related hypertension regulation in DN.MethodsPart 1:Establishment of DN model and molecule pathway screening.1.DN patients confirmed by renal biopsy were included.Age and sex matched glomerular minimal lesion(GML)were used as control.Clinicopathological feature were analyzed.Urine were collected to extract exosomes,which was identified by morphology,particle size and specific markers TSG101.A1AR,SGLT2 and NCC in urinary exosomes were also tested by western blot.The localization of A1AR in DN patients were confirmed by immunofluorescence staining.The differential expressions of A1AR in DN patients and control were analyzed by immunohistochemistry and semi-quantitative analysis.2.We established type 1 DN mice model by intraperitoneal injecting streptozotocin(120mg/kg)to wildtype and A1AR-/-C57/BL mice,respectively.Blood glucose was measured by strips.Twenty-four-hour urine sample was collected in metabolic cages.Blood pressure were obtained by tail cuff method.Body weight,blood glucose,urine volume,and urinary albumin were recorded.Renal pathologies of wildtype and A1AR knockout mice were by evaluated by Haemotoxylin and Eosine(H&E)staining and transmission electron microscopy.Lesions of glomeruli,tubules,vascular and interstitium were employed to evaluate the DN model.3.mRNA of renal cortical tissue of wild type control group,wild type DN group,A1AR-/-control group,and A1AR-/-DN group was extracted.mRNA chip sequencing was used to screen genes that express differently.KEGG pathway cluster analysis was utilized to find possible molecular pathway that A1AR protect kidney from diabetic damage.Part 2:The protective effect of A1AR on the micro-environmental homeostasis of diabetic nephropathy.1.CD34,the marker of vascular endothelial cell and PDGFRβ,the marker of pericyte,underwent immunofluorescence staining in DN patients with A1AR to specify the relative location.Perivascular capillary and pericyte injuries were evaluated by immunohistochemical staining.Renal cortical A1AR and A2AR expression in different stage were evaluated by western blot.The positional relationship between interstitial vessels and pericytes in DN mice was observed by transmission electron microscopy,to investigate the impact of A1AR deficiency on microvessles and cell tight junction.Pericytes,lymphatic vessels,and macrophages infiltration were analyzed by immunohistochemical staining and semi-quantitatively analysis of PDGFRβ,podoplanin,F4/80,respectively.2.The positional relationship between A1AR and megalin in diabetic patients were observed by immunofluorescence staining.The relationship between megalin/cubilin loss and proteinuria was analyzed semi-quantitatively by immunohistochemical staining.Microvilli of tubular brush border were evaluated by transmission electron microscopy.3.The positional relationship between A1AR and collagen I in diabetic patients were observed by immunofluorescence staining.H&E and Masson staining were used to measure the level of renal fibrosis and extracellular matrix deposition in DN mice.The impact of A1AR deficiency on Caspase 1/IL-18 pyroptosis pathway and NLRP3/IL-1β inflammasome signal pathway were analyzed by western blot.The fibrogenic factors TGFβ and collagen(Ⅰ/Ⅲ/Ⅳ)expression in tubulointerstitium were employed to evaluate the impact of A1AR deficiency on inflammation and fibrosis,by Immunohistochemical staining and semi-quantitative analysis.Part 3:Protective effect of A1AR on proximal tubular epithelial cell injury in environment with high concentration glucose.1.The human proximal tubular epithelial cell line was cultured in DMEM/F12 medium in vitro.Passage culture was performed when adherent cells had grown about 80%～90%confluency.The morohological characteristics were observed under inverted microscope.Immunofluorescence staining of CK18 and megalin was used to identify whether the cells were renal tubular epithelium and proximal tubular epithelial cells,respectively.2.To evaluate the trans-differentiation from proximal tubule epithelial cell to macrophage,CD68/NLRP3/Caspasel/IL-18 were detected by immunofluorescence staining,after stimulation in environment with high concentration glucose for 24h.To evaluate impact of environment with high concentration glucose on proximal tubular epithelial cells,cell lysate when cultured 72h were collected,and western blot were used to measure the level of megalin,occludin,NLRP3/IL18,TGFβ and Collagen 1.3.A1AR agonist(CCPA)or A1AR inhibitor(DPCPX)in different concentration were co-incubated with cells.CCK-8 and LDH release assays were used to detect cell proliferation and drug toxicity,respectively.Appropriate concentration(0.1umol/L)was selected for subsequent stimulation experiments.Occludin,NLRP3/Caspasel/IL18 and collagen 1 expression were analyzed by western blot to investigate the impact of CPPA or DPCPX on proximal tubular epithelial cells in environment with high concentration glucose.Part 4:The role of A1AR in regulation of abnormal water and salt metabolism associated hypertension in diabetic nephropathy1.To observe the blood pressure changes in DN patients and mice.Immunofluorescence staining was performed to determine the position of water-salt transporter and SGK1 expression in DN patients.Immunohistochemical staining and semi-quantitative analysis were performed to evaluate the expression of sodium and water transporters(SGK1\NCC\NKCC2\SGLT2)in DN patients and mice.2.To evaluate the effect of A1AR deficiency on blood pressure in DN mouse model.Immunohistochemical staining and western blot were performed to evaluate the expression of sodium and water transporters(NCC\NKCC2\SGLT2\rENac).Statistical analysisAll normally distributed data were presented as Mean ± SEM,unless otherwise indicated.Unpaired t-test was used to compare two values between different animals.Multivariable determinations were analyzed One-way Analysis of Variance.P values less than 0.05 were considered to indicate statistically significant differences.All statistical analyses were performed with SPSS software(Chicago,IL).ResultsPart 1:Establishment of DN model and molecule pathway screening.1.This study included 13 patients with DN,with an average 24-hour urine protein of 5.7±3.8g and an average serum creatinine of 129.6±61.2umol/L.The typical renal pathological manifestations of DN were diffuse thickening of the glomerular and tubule basement membrane,formation of K-W nodule,thickening of vascular wall with hyaline degeneration and focal inflammatory cell infiltration and fibrosis in tubulointerstitium.The extracted exosomes were typical double membrane structure,with average diameter of around 100nm.TSG101,the marker protein of exosome was detected by western blot,so were the renal tubular markers SGLT2,A1AR and NCC.Immunofluorescence staining suggested that A1AR mainly expressed on proximal tubular epithelial cells.According to immunohistochemical staining,compared to GML group,the expression of A1AR in renal tubules was significantly increased about 1.38 times in patients.2.Three days after injection of streptozotocin,the blood glucose of the wild-type and A1AR-/-mice was significantly increased,which was greater than 16.7 mmol/L.suggesting successful establishment of DM model in mice.The mice were observed at 4 and 16 weeks,respectively.The blood glucose was consistently higher than 16.7 mmol/L.Meanwhile,mice showed significant polyphagia,polydipsia and polyuria,with significant increased kidney size,lower body weight,higher 24h urinary albuminuria(129.4±12.2 vs 16.7±2.5 μg/d,P<0.001).Compared with wide type(WT)group,AlAR-/-(KO)group had more severe albuminuria(183.8±9.7 vs 129.4± 12.2 pg/d,P<0.001),suggesting successful establishment of DN model in mice.Electron microscopy showed glomerular and tubular hypertrophy,significant thickening of the basement membrane and vascular wall,and significant tubulointerstitial fibrosis.3.mRNA chip sequencing results of renal cortical tissue of WT control group,WT DN group,KO control group and KO-DN group,combined with subsequent KEGG pathway cluster analysis,suggesting that the main molecular mechanism that A1AR involved in DN damage include cytokine pathway,protein endocytosis pathway and extracellular matrix deposition.Part 2:The protective effect of A1AR on the micro-environmental homeostasis of diabetic nephropathy.1.Immunofluorescence staining suggested that PDGFRβ,a marker of pericytes,mainly expressed around CD34,a,marker of microvascular endothelial cells.A1AR was widely expressed in proximal tubular cells,vascular endothelial cells and interstitial inflammatory cells.The western blot showed that expression of A1AR is elevated in WT-DN group compared to WT-control group(about 1.38 times).In both diabetic patients and mice,separation of pericytes and vascular endothelium could be observed.It could also be observed expression of PDGFRβ increased as well as decreased occluding(0.4±0.06 比 0.7 ± 0.04 比 1.0±0.03,P=009)and CD34 expression(0.3±0.03 vs 0.6±0.08 vs 1.0±0.05,P<0.001),with tubulointerstitial inflammatory cell infiltration,which implied the impairment of tubular microenvironment in DN.Above lesions in the kidney of KO-DN mice are heavier than those in the WT-DN group.2.Immunofluorescence staining demonstrated that A1AR and megalin were co-expressed on proximal tubular epithelial cells.Transmission electron microscopy showed that with the increasing age,the microvilli structure of the proximal tubule brush was decreased.In DN patients and DN,the megalin expression were decreased,and were negatively correlated to proteinuria(r=-0.862,P=0.002 and r=-0.927,P<0.001)).With increase of age,the losses were gradually aggravated.Compared with WT-DN group,mice in KO-DN group had more severe megalin-cubulin losses,and proteinuria.3.Conventional pathological staining and transmission electron microscope found that WT-DN mice show significant fibrosis,with a large amount of extracellular matrix deposition.Immunochemical staining of TGFβ1 and Collagen(Ⅰ,Ⅲ,Ⅳ)further confirmed this result.Western blot showed that the expression of Caspasel/IL18 was increased.In KO-DN mice,the expressions of Caspasel/IL18 and inflammasome NLRP3/IL-1 p were further elevated(1.52±0.6 vs 1.24±0.3 vs 1.0 ±0.2,P=0.017;1.51±0.3vs 1.3±0.2 vs1.0±0.2,P=0.018),with more severe tubulointersitial fibrosis,compare to WT-DN mice.Part 3:Protective effect of A1AR on proximal tubular epithelial cell injury in environment with high concentration glucose.1.Under the inverted microscope,single cell was polygonal or elliptical.When they were grown about 80%～90%confluency,they were cobblestone-like or paving stone-like.Immunofluorescence staining showed cells were both CK18 and megalin positive,suggesting that the cultured cells were proximal renal tubular epithelial cells.2.After stimulated by high concentration of glucose for 24h,CD68,marked for macrophage,as well as NLRP3/Caspasel/IL-18 could be detected by immunofluorescence staining.After stimulated by high concentration of glucose for 72h,compared with the control group and isotonic group,the expression of Megalin(0.46±0.03 vs 1.1±0.05,P=0.008)and Occludin(0.6±0.02 vs 1.0 ± 0.03,P=0.023)was significantly decreased,while the expression of NLRP3/IL18 was significantly increased,accompanied by increased expression of pro-fibrotic factor TGFβ and collagen Collagen 1.These results suggested that high concentration glucose impair proximal tubular epithelial cells,accompanied by the occurrence of transdifferentiation.3.According to the results of cell proliferation and toxicity test,1μmol/L of CCPA(A1AR agonist)and 0.1μmol/L of DPCPX(A1AR inhibitor)were separately incubated with proximal tubular epithelial cells for 24 h.Western blot confirmed that CCPA could significantly inhibit the upregulation of NLRP3(0.4±0.03 vs 1.0±0.06,P=0.032)、Caspasel(0.6±0.02vs 1.0±0.01,P=0.007)、IL-18(0.6±0.02 vs 1.0±0.01,P=0.007)and Collagen 1(0.6±0.02 vs 1.0±0.02,P=0.024)induced by high concentration glucose.On the contrary,DPCPX could aggregate above upregulation.Part 4:The role of A1AR in regulation of abnormal water and salt metabolism associated hypertension in diabetic nephropathy1.Both DN patients and mice included in this study were complicated with hypertension,Immunofluorescence showed that NCC,NKCC2,SGLT2 and rENac,were all expressed at the brush border of renal tubules in DN patients.Immunohistochemical staining and semi-quantitative analysis showed that compared with the GML group,the expression of NKCC2(1.6±0.3 vs 1.0±0.2,P<0.001)and SGLT2(1.3±0.3 vs 1.0±0.2,P=0.042)significantly increased in DN patients,along with the upregulation of SGK1 expression(1.32±0.2 vs 1.0±0.3,P=0.041).While there was no significant difference in NCC expression.2.Higher increased blood pressure was observed in A1AR-/--DN mice than WT-DN(127±9.4 vs 118±6.0 vs 106±8.5 mmHg,P<0.001),Immunohistochemical staining and western blot analysis results showed that A1AR deletion aggravated upregulation of NCC(38±4.5 vs 20 ± 2.0 vs 10 ± 0.6%,P<0.001),NKCC2(36±5.2vs20±2.3vs 16±0.8%,P<0.001)and rENac(1.32±0.1 vs0.8±0.3vs 1.0±0.1,P=0.02).There was no significant difference in SGK1 expression between A1AR-/--DN and WT-DN mice.ConclusionIn our studies1.A1AR played a protective role in the diabetic injury of microenvironment hemostasis,which consists of proximal tubules,peritubular capillaries,pericytes and interstitium.2.A1AR deficiency aggregated tubular megalin loss related proteinuria by activating macrophage filtration related inflammation(Caspase 1/IL-18).The detachment of pericytes from microvessels could induce tubulointerstitial fibrosis,which was also aggravated by A1AR deficiency.3.A1AR agonists could improve inhibit inflammatory response to maintain homeostasis,thus alleviate the previous mentioned pathophysiological processes.In contrast,A1 AR antagonists further aggravate this injury.4.Elevated blood pressure was associated with abnormal expression of renal water and salt transporters both in DN patients and mice,A1AR has a protective effect on the regulation of water-sodium balance and blood pressure via TGF regulation,and nothing to do with SGK1. |
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