The Role And Regulation Mechanism Of ER(Endoplasmic Reticulum)-derived Transcription Factor CHOP In Proteinuria Inducing Apoptosis Of The Renal Tubular Epithelial Cells

Heavy proteinuria is one of the most common clinic manifestations in chronic kidney disease(CKD). It is not only an important symbol which reflects the degree of the renal glomerular injury, but also causes renal tubulointerstitial injury promoting pathological changes of the kidney. It is an independent risk factor for progression of CKD. Albumin is the major component of the proteinuria in glomerulus diseases. Many studies have shown that overloaded albumin can activate the renal tubular epithelial cells to generate a large amount of inflammatory mediators, cytokines, growth factors and extracellular matrix proteins, mediated apoptosis and transdifferentiation of the renal tubular epithelial cell ,and lead to renal interstitial fibrogenesis. Research shows that excessive apoptosis of renal tubular epithelial cell is one of the important pathophysiological bases of renal tubular atrophy. At present, relatively affirmation mechanisms of proteinuria induceing apoptosis in the renal tubular epithelial cells are Fas-FADD-caspase8 track, PPAR-γ, NF-κB and mitochondrion apoptosis. Apoptosis caused by endoplasmic reticulum stress is a new track discovered in recent years, it activates the downstream apoptosis signal molecules,such as CHOP(CCAAT/enhancer-binding protei-homologous protein,alsocalled GADD153),c-Jun N-terminal kinase(JNK),caspase-12, Bcl-2 kindred and target genes leading to the cell apoptosis through PERK(PKR-like ER kinase),ATF6(activating transcription factor 6) and IRE-1(inositol requiring kinase 1) .More and more research shows that ERS-induced cell apoptosis plays an important role in the development of many diseases, such as malignant tumors, diabetes, nervous system degenerations and cardiovascular diseases.However, we know very little about the role and mechanism of the ER-associated death pathway in chronic kidney disease process . CHOP is the ERS-specific transcription factor, belonging to the C/EBP transcription factor family. CHOP activation caused by ERS is the most important apoptosis pathway. PERK, ATF6 and IRE-1-activation all induce CHOP transcription. Among them, PERK plays a key role in CHOP expression. Research showed that ERS inducer tunicamycin could significantly increase kidney CHOP gene expression resulting in apoptosis of renal tubular epithelial cells and kidney damage, while CHOP-I- mice showed a much lower responsiveness. Both cases indicated that CHOP plays an important role in the kidney damage caused by ERS. Hereby, we speculate: proteinuria can cause apoptosis of tubular epithelial cells by activating CHOP and PERK is a key link in this pathophysiological process.Methods1. Relationship between endoplasmic reticulum stress and apoptosis of renal proximal tubular cells of patients with chronic kidney disease(1) Subjects According to the new definitions of CKD, promulgated by the United States kidneydisease Foundation (NKF),1999, we selected 21 patients with CKD confirmed by clinically diagnosis and renal biopsy, and divided them in to three groups according to their 24h -urinary protein levels. The groups were slight proteinuria group (<1.5 g/24h, 7 cases, of which IgA nephropathy 4 cases, mesangial proliferative glomerulonephritis 3 cases), moderate proteinuria group (1.5 ~ 3.5 g/24h , 7 cases, of which IgA nephropathy 4 cases, mesangial proliferative glomerulonephritis 3 cases), serious proteinuria group (> 3.5 g/24h, 7 cases, of which IgA nephropathy, 3 cases ,mesangial proliferative renal Nephritis ball 4cases). The control group were 7 cases, which derived from the patients with kidney cancer and the kidney samples were taken from the part away from the tumor. The tissues were confirmed to be normal by pathological examination , urine protein <150 mg/24h.(2) Test index and methodsThe plasma and urinary biochemical parameters, including serum albumin, lipoprotein, Scr and urinary protein levels, were analyzed by routine biochemical method. The histologic changes were examined by HE,PAS,PASM and Masson staining. ORP150, GRP78 and CHOP expression in renal tubular epithelial cells were tested by immunohistochemical method. Cell apoptosis was detected by TUNEL.2. Role and regulation mechanisms of ER-derived transcription factor CHOP in the apoptosis of tubular epithelial cells induced by albumin overload(1) The dose- and time-effect correlation between albumin overload and GRP78, CHOP,PERK expression and PERK phosphorylation and apoptosis(a) 0 mg/ml,5 mg/ml,10 mg/ml and 20 mg/ml of HSA stimulated HKCs for 24 hours respectively, measured GRP78, CHOP, PERK expression and PERK phosphorylation by western blot ,and analyzed cell apoptosis by Annexin V-FITC/PI dual-flow cytometry.(b) 20 mg/ml HSA to stimulated HKCs for 12 hours,24 hours, and 36 hours ,use 0mg/ml HSA to stimulate HKCs for 0 hour and 24 hours for the control group, measured GRP78, CHOP, PERK expression and PERK phosphorylation by western blot ,and analyzed cell apoptosis by Annexin V-FITC/PI dual-flow cytometry.(2) Effect of CHOP gene silencing on overloaded albumin induced apoptosis of tubular epithelial cells(a) Inhibitory effect of CHOP siRNA on HKC CHOP mRNA expression with albumin overloaded was detected by RT-PCR.(b) Effect of CHOP siRNA on HKC CHOP,PERK expression and PERK phosphorylation induced by albumin was detected by western blot.(c) Effect of CHOP siRNA on HKC apoptosis induced by albumin was detected by Annexin V-FITC/PI dual-flow cytometry.(3) Effect of PERK gene silencing on overloaded albumin induced CHOP expression and apoptosis of tubular epithelial cells(a) Inhibitory effect of PERK siRNA on HKC CHOP mRNA and PERK mRNA expression with albumin overloaded was detected by RT-PCR.(b) Effect of PERK siRNA on HKC CHOP,PERK expression and PERK phosphorylation induced by albumin was detected by western blot.(c) Effect of PERK siRNA on HKC apoptosis induced by albumin was detected by Annexin V-FITC/PI dual-flow cytometry. Results1. Relationship between endoplasmic reticulum stress and apoptosis of renal proximal tubular cells of patients with chronic kidney disease In control group, the expression of ORP150, GRP78 and CHOP were very low while the expression of ORP150 and GRP78 in CKD groups significant increased (P <0.01); expression of CHOP in nucleus was strong positive (P <0.01); apoptosis also increased significantly (P <0.01); The expression of ORP150,GRP78 and CHOP and cell apoptosia were positively correlated with the 24-hour proteinuria level (r = 0.695, r = 0.760, r = 0.753, r = 0.835, P <0.01). Significantly positive correlation was observed between CHOP expression and apoptosis (r = 0.765, P <0.01).2. Role and regulation mechanisms of ER-derived transcription factor CHOP to the albumin overload induced apoptosis of tubular epithelial cells(1) The dose- and time-effect correlation between albumin overload and GRP78, CHOP,PERK expression ,PERK phosphorylation and apoptosis(a) The expression of GRP78, CHOP, PERK ,PERK phosphorylation and apoptosis were up-regulated by HSA with dose-dependent (P <0.01).(b) Stimulating HKC cells with20 mg/ml HSA, GRP78, CHOP ,PERK expression, PERK phosphorylation and cell apoptosis were up-regulated and the effects were time-dependent (P <0.01).(2) Effect of CHOP gene silencing on albumin overload induced apoptosis of tubular epithelial cellsCHOP siRNA significantly inhibited albumin-induced HKC CHOP mRNA as well as protein expression (P <0.01), while had no effect on PERK expression . CHOP siRNA significantly up-regulated PERK Phosphorylation (P <0.01), and inhibited albumin-induced HKC apoptosis (P <0.01).(3) PERK siRNA significantly inhibited albumin-induced HKC cells CHOP mRNA, PERK mRNA expression, and their protein expression (P <0.01), significantly inhibited HSA-induced PERK phosphorylation (P <0.01), and the albumin-induced HKC apoptosis were also significantly inhibited (P <0.01). Conclusions1. Endoplasmic reticulum excessive stress may be involved in the CKD patients with renal tubular epithelial cells damage. CHOP up-regulation and the expression of nuclear transfer may have closely relationship with proteinuria induced epithelial cells apoptosis.2. HSA can increase the expression of GRP78,CHOP,PERK and PERK phosphorylation, and the effect is dose- and time-dependent, which is same as the change of apoptosis, suggesting that PERK-CHOP way may be have closely relationship with albumin overload induced HKCs apoptosis.3. ER-derived transcription factor CHOP expression may be the key link of the process of albumin overload induced apoptosis in the epithelial cells.4. Albumin overload can cause ER-associated apoptosis of epithelial cells by activating PERK and up-regulating CHOP expression, this may be the main mechanism of proteinuria-caused injury.