Mechanisms Of Endoplasmic Reticulum Stress In The Progress Of Diabetic Kidney Injury

BackgroundDiabetic nephropathy is a common and serious complication of diabetes mellitus, which leads to renal failure in up to 30% of individuals with diabetes and thus becomes the most important cause of end-stage renal diseases (ESRD). The pathogenesis of diabetic nephropathy is a chronic and complicated process, and more and more evidences support that apoptosis plays pivotal role in the development of nephropathy.Apoptosis is the process of gene-controlled programmed cell death that occurs under physiology or pathology condition to keep the homeostasis. Cell apoptosis with cell proliferation control the balance of total cell number in each organ. Under diabetic condition, factors inducing the apoptosis increase which result in the intense of apoptosis overwhelming proliferation, then the number of functional cells decreases and finally leads to the organ failure. At present 3 different pathways are considered that mediate the process of cell apoptosis:mitochondrial pathway, death receptor pathway and newly found endoplasmic reticulum (ER) stress associated pathway.ER is the largest organelle in the cell and involved in the metabolism of membrane/secreted protein, glycosaminoglucan, phospholipid, cholesterol, and calcium, especially in the secreted protein maturation, folding and transport. Once the insulting factors destroy the balance of protein production, maturation and degrading, the ER stress is activated. Several complex homeostatic signaling pathways, known as the unfolded protein response (UPR), have evolved to cope with the ER stress. The processes of UPR include:temporary suspension the general protein synthesis, up regulation the expression of ER resident proteins and molecular chaperons, inducing the ER associated degradation (ERAD). Though moderate ER stress could alleviate the damage caused by stress, prolonged or severe stress response leads to apoptosis and removes the damaged cells. But if the intense or the extent of apoptosis is out of control, excess functional cells lose, which result in the premature organ failure.Renal cells, including mesangial cell, podocyte, tubular epithelial cell and et al, have well-developed ER, which is tightly related to the cell physiological function. Mesangial cells are the primary manufacturers of basal membrane and mesangial matrix. They are also involved in the degrading of mesangial immune complex, and secreting the bioactive substances such as renin and angiotensin. Podocytes are the components of the glomerular filtration barrier. Glycoprotein connected to the outer membrane of the cells plays the vital role in the filtration. Besides re-absorbing and degrading the filtrated protein, tubular cells also produce the cytokines such as bradykinin. Above all, ER fulfills multiple protein related functions in the renal cells and ER stress might be easily activated in the renal disease.Under diabetic condition, ER stress insulting factors are prone to increase, including hyperglycemia, oxidative stress, proteinuria, and cellular electrolyte disorder. However, fewer publications are found between ER stress and diabetic kidney injury. So the aim of this research is to investigate the role of ER stress in the diabetic nephropathy, and then provide possible strategies on the kidney protection.Objectives1. To establish the diabetic rat model, and demonstrate the increment of apoptotic renal cells.2. To investigate the activation of ER stress in the diabetic kidney.3. To explore the signaling pathways of ER associated apoptosis in the diabetic kidney injury.Methods1. Establishment of diabetic rat model30 male Wistar rats (7 weeks of age; mean body weight 280±10 g) were used. After one week of acclimatization, of the 30 Wistar rats,20 were given a single intraperitoneal dose (65 mg/kg) of streptozotocin (STZ, dissolved in citrate buffer (pH 4.5)), while the remaining 10 rats were given the same dosage of citrate buffer (pH 4.5). Hyperglycemia was confirmed by measuring the venous circulating plasma concentrations of glucose. Seven days post-STZ injection, blood samples were obtained from the rat tail vein after 12 hours of fasting, and the rats with glucose concentration higher than 300 mg/dl were kept in diabetic group. All rats were killed at the 16th week after saline or STZ injection.2. Blood glucose test and 24-hour proteinuria examinationBlood glucose was detected per 4 weeks. At the end of the 1st and 16th week,24-hour urine was collected with the metabolic cage and the levels of proteinuria were measured.3. Renal histopathological stainingKidneys were transversally sliced and fixed in 4% neutral formalin for over 24 hours, and then embedded in paraffin, cut into 4-μm thick sections for staining with H&E or PAS methods. Then slides were observed under the microscope to determine if the diabetic kidney injury occurred or not.4. TUNEL stainingTUNEL staining was used to detect the expression of apoptotic cells in the diabetic kidney.5. Immunohistochemical stainingImmunohistochemical staining was used to detect the protein expression of GRP78, CHOP, Caspase-12 and p-JNK. The intensity and extent of the positive area were calculated and compared. 6. Western blot analysisTotal proteins of the renal samples were extracted and conducted 10—14% SDS-PAGE. Then the protein expression levels of GRP78, CHOP, Caspase-12, Total-JNK and p-JNK were measured.7. Realtime RT-PCR analysisRealtime RT-PCR was used to detect the mRNA levels of GRP78, CHOP, Caspase-12, and JNK.Results1. General features of the experimental ratsIn the whole experiment,3 diabetic rats died and might be caused by diabetic ketoacidosis, infection or other diabetic complications. Finally 27 rats survived and 10 from control group and 17 from diabetic group. Compared to the control, diabetic rats seemed to be inactive, and with symptoms of lower body weights, polyuria and polyphagia, which are normally associated with diabetic state.2. The diagnosis of diabetic nephropathyThe diagnosis of diabetic nephropathy was determined by blood glucose monitoring,24-hour proteinuria detection and renal pathology. Compared to the control, concentrations of glucose blood in diabetic rats were always higher than 16.7 mmol/L (P<0.05). At the end of 16th week, the level of 24-hour proteinuria increased obviously in diabetic rats (P<0.05). And the examination of renal pathology showed glomerular hypertrophy, glomerular basement membrane thickness, glomerulosclerosis, renal tubular atrophy and interstitial fibrosis in diabetic kidney.3. Enhanced apoptotic state in diabetic kidneyThe results of TUNEL assay showed that apoptosis was observed in both the cortex and medulla of the diabetic kidney. Estimation of renal apoptosis revealed a nearly fivefold increase in TUNEL-positive nuclei in diabetic kidneys compared to the normal (P<0.05).4. Up-regulation of ER chaperone GRP78 in kidneys of diabetesGRP78, one important molecular chaperone localized in ER, has been used extensively as an indicator for the induction of ER stress. Immunohistochemistry study showed that GRP78 was abundantly expressed in the renal tissue from diabetic rats (P<0.05). In contrast, normal rats exhibited modest or weak immunoreactivity for this molecule. The result of western blot analysis confirmed the finding. Furthermore, real-time PCR analysis showed the same trend of grp78 in mRNA level (P <0.05). All these findings suggested the activation of ER stress in the kidneys of diabetic rats.5. Involvement of the ER-associated apoptosis pathwaysCHOP, JNK and caspase-12 are the key molecules in the three branches of ER-associated apoptosis pathways. Firstly, western blots were used to check the involvement of these pathways in diabetic kidneys. The densitometric analysis of bands for CHOP, caspase-12, and p-JNK revealed a significant increase in relative protein content in renal tissue from diabetic rats in comparison with those from normal rats(P<0.05). This means CHOP was induced, and caspase-12 and JNK pathway were activated in the diabetic kidney. Changes in RNA expression of jnk, chop, and caspase-12 were quantified by RT-PCR in control and diabetic rat kidneys. Expressions of chop, and caspase-12 were significantly increased in the diabetic kidneys (P<0.05), paralleled with their enhanced protein expression. However, the result of jnk did not show any differences between the diabetes and control (P>0.05). At last, the results of immunostaining provided more useful information. In diabetic kidney, Caspase-12 positive area only appeared in the glomerulus; p-JNK was mainly expressed in the glomerulus and interstitial area, especially the small vessels; CHOP could be observed in the distal convoluted tubule area, and focal expression in the glomerulus and proximal tubular area. The differences might be caused by the variance of involved cells and insulting factors.Conclusion1. The STZ induced diabetic rats showed classic diabetic nephropathy performances and enhanced apoptotic state.2. The expression of ER chaperone GRP78 was up-regulated in diabetic kidney, which directly demonstrated the activation of ER stress.3. CHOP, JNK and Caspase-12, three branches of ER associated apoptosis, were all stimulated in diabetic kidney. However, the activated modes or the cells involved may be different. BackgroundGlomerulosclerosis, which is characterized by the expansion of mesangium, is the most important pathological change in the early stage of diabetic nephropathy. The expanded mesangium is comprised of a large number of mesangial cells and increased amount of matrix. However, in the later course of the disease, there is an abundance of matrix and a limited number of mesangial cells. And apoptosis is assumed to play the vital role in the mesangial cell loss.Evidences show that intra-renal levels of angiotensinⅡ(ANGII) are elevated in the development of diabetic nephropathy. ANG II plays an important role in the function maintenance of mesangial cells in both normal and diseased states. Mesangial cells are specialized pericytes located among the glomerular capillaries within a renal corpuscle of a kidney. They are not only providing structural support for and regulate blood flow of the glomerular capillaries by their contractile activity, but also are the major contributors to the extracellular matrix which contains fibronectin, type IV collagen, perlecan, and laminin. All the functions mentioned above are tightly related to the ANGII regulation. However, ANGII could insult kinds of cells and induce the apoptosis or necrosis.Ultrastructural analysis results show that endoplasmic reticulum (ER) in mesangial cell is well-developed, and in an active state. The ER is the site of biosynthesis for steroids, cholesterol and other lipids. It is also the sub-cellular entrance for a number of secretory and structural proteins and provides a unique environment for appropriate protein folding and assembly to produce functional, mature proteins. What's more, protein glycosylation is complicated in the ER. As is known, extracellular matrix is mainly comprised by glycoprotein and proteoglycan. And under the diabetic state, matrix is overloaded in the glomerular mesangium. Homeostasis in the ER is maintained by a coordinated adaptive program, so-called the unfolded protein response (UPR). A number of microenvironmental, developmental and pathophysiological insults as well as a wide range of chemical substances cause accumulation of unfolded proteins in the ER and activate ER stress. In response to accumulation of unfolded proteins, cells adapt themselves to the stress conditions via UPR:attenuation of general translation, induction of ER chaperones and activation of ER-associated degradation (ERAD) to eliminate immature proteins. If the stress is beyond the capacity of the adaptive machinery, cells undergo apoptosis Accumulated evidences showed that ANGII could induce the apoptosis of many cells, and the mechanisms might involve the ERS response. However, whether ANGII inducing apoptosis of mesangial cells is mediated by ERS is still unknown. On the one hand, ANGII has been demonstrated to stimulate the generations of reactive oxygen species in mesangial cells, and the relationship between oxidative stress and ERS is undeniable; On the other hand, ANGII promotes the protein synthesis in ER and might result in the unfolded protein accumulation,thus activate the ERS. Therefore the aim of this study is to investigate the role of ERS in the ANGII-induced apoptosis of mesangial cell.Objectives1. To observe the hallmark of ERS activation in ANGII-induced apoptosis of mesangial cell.2. To explore the ERS associated apoptotic pathways in ANGII-induced apoptosis of mesangial cell.Methods1. Study design1) The established rat renal mesangial cell line HBZY-1 was incubated with ANGII at different concentration (0,1,10,100 nmol/L) and duration (0,12,24,48 hours) to select the best treatment condition. After harvesting, apoptotic cells were labeled with Annexin V/PI kit, and the percentage of apoptosis was calculated with flow cytometer. 2) Cultured cells were treated with ANGII at the selected concentration and duration. Then Immunofluorescence staining, western blot and realtime PCR were used to measure the expression of GRP78, CHOP, Caspase-12 and p-JNK on protein and mRNA levels.3) According to result of 2nd step, to synthesize the siRNA against ERS apoptotic molecules. Treated cells with siRNA before ANGII incubation, and then detected the apoptotic percentage with flow cytometer.2. Cell cultureThe established rat renal mesangial cell line HBZY-1, which was purchased from Wuhan cell center, was cultured in low-glucose DMEM. Cells in generation 10-20 were used in the treatment.3. Immunofluorescence stainingCells were grown on glass slides in the presence of selected concentration of ANGII for the proper duration. Then the cells were fixed with 4% paraformaldehyde for 15 minutes at 4℃and permeabilized in 0.1% Triton X-100 for 10 minutes at 4℃. The cells were then blocked in 10% rabbit blocking serum at 37℃for 30 minutes followed by incubation with GRP78 goat polyclonal IgG (1:50) overnight at 4℃. After washes with PBS three times, cells were incubated with FITC-conjugated rabbit anti-goat IgG (1:50) at 37℃for 1 hour. The results were evaluated under fluorescing microscope.4. Annexin V/PI kit to measure the percentage of apoptosis When the stimulation was over, cells were digested with 0.25% trypsin and collected, then resuspended in binding buffer with annexin V-FITC and PI. After staining, cells were immediately analyzed with flow cytometer.5. Western blot The total protein was extracted from the harvested cells. Western blot was used to detect the protein levels of GRP78, CHOP, Caspase-12 and p-JNK.6. Realtime RT-PCR analysis The total RNA was extracted from the harvested cells with Trizol. Realtime RT-PCR was used to detect the mRNA levels of GRP78, CHOP, Caspase-12 and JNK.Results1. ANGII induced apoptosis of mesangial cells in a dose-and time-dependent mannerTreated mesangial cells with ANGII of different concentration (0,1,10,100 nmol/L) for 24 hours and measured the percentage of apoptosis, the results showed that the apoptotic ratio was increased gradually, were 1.37%±0.41%, 3.72%±1.23%,9.27%±3.53%,17.74%±5.19%respectively (P <0.05) Exposed cells with 100nmol/L ANGII for different duration (0, 12,24,48 hours) and the results of apoptosis tests are 1.53%±0.49%,5.72%±1.97%,19.14%±6.53%,24.74%±9.19% for each time point (P<0.05). Thus ANGII induced apoptosis of mesangial cells in a dose-and time-dependent manner. And the best ANG concentration and duration selected were 100nmol/L and 24 hours.2. Up-regulation of ER chaperone GRP78Immunofluorescence staining, Western blot and Realtime PCR, three methods were used to detect the expression of GRP78 after the ANGII treatment in the mesangial cell. And the results showed that GRP78 was obviously upregulated on both mRNA and protein levels compared to the control group (P<0.05).3. The expressions of CHOP and Caspase-12 were elevated in ANGIIinduced mesangial cell apoptosis Three key molecules of ERS associated apoptosis, CHOP, Caspase-12 and JNK, were examined with western blot and real time PCR after cell exposed to ANGII at selected concentration and duration. Results showed that the expressions of CHOP and Caspase-12 were increased dramatically compared to the control (P<0.05), but there was no change for JNK expression (P>0.05).4. CHOP and Caspase-12 siRNA decreased the apoptosis induced by ANGII in mesangial cell Cells were transfected with CHOP and Caspase-12 siRNA 24 hours before ANGII treatment. And the results of apoptosis percentage showed that CHOP and Caspase-12 siRNA could decrease the apoptosis induced by ANGII in mesangial cell. The apoptotic percentages for control siRNA group, group CHOP siRNA and group Caspase-12 siRNA are 31.74%±8.57%, 20.61%±6.33%,18.94%±5.49%(P<0.05)。Conclusion1. ANGII could activate the ERS in renal mesangial cells.2. ANGII-induced ERS associated apoptosis of mesangial cell might be mediated by CHOP and Caspase-12 pathways.