The Role And Mechanism Of Endoplasmic Reticulum Stress In The Development Of Diabetic Nephropathy

Diabetic nephropathy (DN) is a common and serious complication of diabetes mellitus. As diabetic nephropathy progresses, apoptosis, which causes excessive renal cells loss, is considered as one of vital mechanisms.Endoplasmic reticulum (ER) is a highly dynamic organelle responsible for multiple cellular functions such as the folding of secretory and membrane proteins. However, various conditions can disturb the functions of the ER and result in ER stress (ERS), including ER-Ca2+ depletion, ischemia, hypoxia, exposure to free radicals, elevated protein synthesis, and so on. Several signaling pathways are initiated to cope with ERS, which are designated as the unfolded protein response (UPR). One major pathway of UPR is to increase the expression of ER-localized molecular chaperons, such as glucose-regulated protein 78 (GRP78), which can contribute to repairing unfolded proteins. GRP78 serves as a master modulator for the UPR network by binding to the ER's sensors such as protein kinase R (PKR)-like ER kinase, inositol requiring 1 (IRE1), and activating transcription factor 6 (ATF6), at the same time inhibiting their activation, so it plays a vital role in the recognition of unfolded proteins. Thus the early UPR enhances cell survival by that the adverse effects of ERS are dealt with in a timely and efficient manner. Though moderate ER stress could alleviate the damage caused by stress, prolonged or severe stress response leads to apoptosis and may thus be an important factor in the pathogenesis of many diseases. Although the signaling pathways leading to apoptosis involved in ERS are not yet fully understood, two pathways of ER-associated death(ERAD) have been definited, which characterized by the activation CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP/GADD153) and the caspase12-dependent pathway. Glomerular podocytes are unique, highly specialized, terminally differentiated cells in the renal glomerulus. Exhaustion of podocytes, common to glomerular diseases, plays an important role in the pathogenesis of glomerulosclerosis, and apoptosis is invoved in this process. But whether there is a relationship between ERS and program that initiate the cell death is still unknow.Accumulative evidence has demonstrated that apoptosis initiated by the ERS was involved in the pathogenesis of several renal diseases, and plays a vital role in diabetes. In order to obtain insight into the molecular mechanisms underlying diabetic nephropathy, we investigated the involvement of ER-associated apoptosis in kidney disease of STZ-induced diabetic rats and mouse glomerular podocytes exposed to high glucose, and analysized the interconnection between ERS and traditional mitochondria apoptosis pathway. At the same time, we detected the effect of specific chemical unfolded protein response inducer-thapsigargin on the apoptosis and pathological progress in order to identify potential therapeutic targets, which might explore an appropriate approache to minimize the risks.Methods:1 Assessment of apoptosis rate and the expression of GRP78, GADD153/CHOP, Caspase-12 in the kidney of STZ-induced diabetic rats and mouse glomerular podocytes exposed to high glucoseIn vivoUninephrectomized male Wistar rats were used. The rats of diabetic group received a single intraperitoneal injection of STZ dissolved in 0.1mol/L sodium citrate (pH4.5) at a dose of 65mg/kg body weight, and rats in the control group only received an injection of the same volume of 0.1mol/L sodium citrate. The model of diabetes was considered to be successful when the blood glucose was≥16.7mmol/L and the glucose in urine was +++~++++ after 48 hours of the injection. Normal control group and diabetic group rats were raised for 2 weeks, 4 weeks, 8 weeks and 12 weeks respectively until they were sacrificed. Blood samples were collected through abdominal aorta, and renal cortex was removed, cleaned, and washed immediately saved in 4% formaldehyde and 4% glutaraldehyde for light microscopic, electron microscopic observation, TUNEL staining and immunohistochemical staining. Partial renal cortices were fixed in 70% alcohol for flow cytometry. Partial renal cortices were freezed at -80℃for Western blot.In vitro1)Cell cultureMouse glomerular podocytes were cultured at 33°C in 95% air and 5% CO2 in DMEM-F12 media containing 10% fetal calf serum, 100 U/ml penicillin and 100μg/ml streptomycin and 2-3 drops Mouse Interferonγ, at which point they were trypsinized and reseeded in fresh flasks. Before thermoswitching to 37°C, cells were grown to 70 to 80% confluence. Then change DMEM-F12 media containing 10% fetal calf serum, 100 U/ml penicillin and 100μg/ml streptomycin. At both temperatures, cells were fed with fresh medium 3 times per week. Cells were then plated onto the flasks and grown either at the permissive temperature of 33°C to promote cell propagation as a cobblestone phenotype (undifferentiated) or at the nonpermissive temperature of 37°C allow the cells to differentiate.2) Determination of GRP78, Caspase-12 and GADD153/CHOP protein in mouse podocytesDifferentiated mouse podocytes were divided into three groups: NG (media containing 1g/L glucose); HG group(media containing 4.5g/L glucose); HG +AngⅡ(media containing 4.5g/L glucose and 10-8mol/L AngⅡ). The cells exposed to stimulus were harvested at 12, 24, 48, 72h respectively. TUNEL staining and flow cytometry were used to examine apoptosis of podocytes. The expression of GRP78, Caspase-12 and GADD153/CHOP in cells were assessed by immunochemistry, western blot and RT-PCR. The cells ultrastructure changes at 72 hours were observed by electron microscope.2 Influence of preconditioning with endoplasmic reticulum stress on the kidney of STZ-induced diabetic rats and mouse glomerular podocytes exposed to high glucoseIn vivoMale Wistar rats were randomly divided into 3 groups: normal control group (N), diabetic group (DM) and tunicamycin treated diabetic group (ATM). ATM group were divided into ATM1 group (tunicamycin was administered intraperitoneally to rats at a dose of 0.2mg/kg body weight at 1week prior to injection of STZ) and ATM2 group (tunicamycin was administered intraperitoneally to rats at a dose of 0.2mg/kg body weight at 1week after induction of diabete). The animals were fed for 4 weeks and 12 weeks. Blood and urine samples were collected, the pathological change were estimated by light microscope and electron microscope, TUNEL assay and flow cytometry were used to evaluate apoptosis of kidney tissue, GRP78, GADDl53 / CHOP and Caspase-12 protein levels were evaluated by immunohistochemical staining and western blot.In vitroDifferentiated mouse podocytes were divided into three groups: NG (media containing 1g/L glucose); HG group(media containing 4.5g/L glucose) and CTM (media containing 4.5g / L glucose and 0.1μg/ml thapsigargin). The cells exposed to stimulus were harvested at 24 and 72h respectively. The cells ultrastructure changes at 72 hours were observed by electron microscope. GRP78, GADDl53/CHOP and Caspase-12 protein and mRNA levels were evaluated by immunohistochemical staining, western blot and RT-PCR.3 Influence of preconditioning with endoplasmic reticulum stress on mitochondrial dysfunction of mouse glomerular podocytes exposed to high glucoseDifferentiated mouse podocytes were divided into three groups: NG (media containing 1g/L glucose); HG group(media containing 4.5g/L glucose) and CTM (media containing 4.5g / L glucose and 0.1μg/ml thapsigargin). The cells exposed to stimulus were harvested at 72h respectively. Mitochondrial tranmembrane pitentials (ΔΨm) was detected by flow cytometry. The Caspase-3 protein was examined by immunochemistry and western blot, and the Cytochrome C protein was evaluated by western blot.Results:1 Apoptosis induced by endoplasmic reticulum stress involved in the kidney of STZ-induced diabetic rats and mouse glomerular podocytes exposed to high glucoseIn vivo①The diabetic rats showed slightly glomeruli hypertrophy, mesangium matrix increase, partial tubular epithalial vacuolar degeneration estimated by light microscope.②Ultrastructure changes, including thickened glomerular basement membrane, the extensively effacement of foot process in podocytes and vacuolar degeneration in proximal tubule epithelial cells, the rough endoplasmic reticulum in proximal tubule epithelial cells and podocytes showed particles integration and degranulation, partial mitochondrial crista disappeared in diabetic group detected by electron microscope.③Assessment of apoptosis by TUNEL assay and flow cytometry: Apoptosis was observed in both the cortex and medulla of the diabetic kidney, and renal apoptosis rate increased compared to the control group.④Immunohistochemistry study showed that slight staining with anti-GRP78 antibody was observed in control group, located mainly in cytoplasm of the distal tubule and collecting duck. In contrast, diabetic group produced widespread strong staining with anti-GRP78 antibody in the kidney, especially in cytoplasm of the proximal tubule and glomerulus. Control group rats exhibited weak immunoreactivity for Caspase-12, and it was abundantly expressed in the renal tissue from diabetic rats. GADDl53/CHOP was mainly distributed in cytoplasm of nephric tubule in control group, and in diabetic group, the expression level of it was increased, detected in nucleus of proximal tubule and partial glomerulus, especially in the cortico-medullary boundary area.⑤Western blot study showed that in diabetic group, GRP78 protein was increased since 2 weeks, reached the peak at 4 weeks and then decreased. Caspase-12 protein gradually increased with time. GADDl53/CHOP protein reached the peak at 8 weeks and then decreased slightly.In vitro①The podocytes exposed to high glucose showed villus and foot process increased, and a lot of vacuolus in cytoplasm. There is no change in mitochondrium and endoplasmic reticulum. Similar manifestation was observed in HG+AngⅡgroup.②Assessment of apoptosis by TUNEL assay and flow cytometry: Apoptosis rate increased in HG compared to the control group. Exposed to HG+AngⅡpromote this lesion in a time-dependent manner. The result of TUNEL assay confirmed the findings.③The GRP78, GADDl53/CHOP, Caspase-12 protein levels was increased in HG compared to NG, and enhanced in HG+AngⅡgroup evaluated by immunohistochemistry.④Western blot study showed that in HG group GRP78 protein was increased at 12 h and 24 h and then decreased, approximately reached to the original level at 72h. Caspase-12 protein gradually increased with time. GADDl53/CHOP protein reached the peak at 48 hours and then decreased. The expression of those proteins were strengthened in podocyte exposed to HG+AngⅡin the same tendency.⑤The results of RT-PCR analysis are consistent with Western blot results in HG and HG+AngⅡgroup.2 Effect of preconditioning with endoplasmic reticulum stress on the apoptosis and expressions of GADDl53/CHOP, Caspase-1, GRP78 in STZ-induced diabetic rats and mouse glomerular podocytes exposed to high glucoseIn vivo①The diabetic rats showed slightly glomeruli hypertrophy, mesangium matrix increase, partial tubular epithalial vacuolar degeneration, treatment with tunicamycin could ameliorate the severity by light microscope.②Compared with diabetic group, the ultrastructure manifestations was dramatic improved in ATM group by electron microscope.③TUNEL assay and flow cytometry results show that apoptosis was significantly lower in the ATM group than that in the diabetic group.④Immunohistochemistrically treatment with suitable density of tunicamycin significantly enhanced GRP78 protein expression and decrease the expression of GADDl53 / CHOP, Caspase-12 protein at 12 week.⑤Western blot study showed that ATM group significantly enhanced GRP78 protein expression and at the same time decreased the expressions of Caspase-12, GADDl53/CHOP protein at 12 week.In vitro①Compared to HG group, the severity of ultrastructure changes were ameliorated in the podocytes exposed to suitable density of tunicamycin by electron microscope.②Assessment of apoptosis by TUNEL assay and flow cytometry: the amount of apoptosis cells were cut down in CTM2 group, but dramatically increased in CTM3 group by flow cytometry. The result of TUNEL assay was consistent with the finding of FCM.③The expression of GRP78 was enhanced and GADDl53/CHOP, Caspase-12 protein levels were decreased in CTM group by immunohistochemistry.④Western blot study showed that GRP78 expression was significantly enhanced and Caspase-12, GADDl53 / CHOP protein expression were decreased in treatment group with suitable density of tunicamycin.3 Effect of preconditioning with endoplasmic reticulum stress on mitochondrial dysfunction of mouse glomerular podocytes exposed to high glucose①Change of mitochondrial tranmembrane pitentials (ΔΨm): Compared with N group,ΔΨm significantly decreased in HG, however it was amelioratd by treatment with suitable density of tunicamycin.②Slight staining with anti-Caspase-3 antibody was observed in NG. Compared with NG, the Caspase-3 protein levels was upregulated in HG, and decreased in CTM group by immunohistochemistry.③Western blot study showed that the degree of Cytochrome c and Caspase-3 protein were clearly upregulated in HG group, and those protein were decreased in CTM group . Conclusions:①As diabetic nephropathy progresses, the endoplasmic reticulum stress was actived, and apoptosis induced by ERS occurred in diabetic kidney, which may contribute to the development of diabetic nephropathy.②Tunicamycin with suitable density can ameliorate the pathological changes in diabetic rats and glomerular podocytes exposed to high glucose, and effectively improved cells tolerance and survival, dramatically decreased apoptosis rate. In contrast, high concentrations of TM might lead to severe stress and eventually evoke apoptosis greatly.③Tunicamycin with suitable density attenuated pathways of ER-associated death, meanwhile participated in the modulation of m itochondrial function in some degree, which might provide an evidence of a considerable new therapeutic target in diabetic nephropathy.