The Mechanisms Of Endoplasmic Reticulum Stress Triggering SET7/9Expression And Its Role In The Pathogenesis Of Diabetic Nephropathy

Background and objectiveDiabetic nephropathy (DN) is one of the most serious microvascular complications ofdiabetes, as well as a major cause of end stage renal disease (ESRD). So far themechanisms involved in the pathogenesis of DN are still unknown and the effectivetreatment methods are lacking. It is widely known that DN is a glomerular inflammationactivation disease. Thus, exploring the activation mechanism of inflammation in DN is veryimportant to suppress the development of DN. Recent research showed that endoplasmicreticulum stress (ERS) play a key role in the pathogenesis of diabetes and diabeticcomplications relevant vessels. Our previous studies have confirmed that ERS is involvedin the pathogenesis of DN via regulation of oxidative stress and inflammation activation.Attenuation of ERS by4-PBA could effectively reduce ERS strength, inhibit renaloxidative stress and inflammation activation in DN rats, ultimately prevent the developmentand progression of DN. Epigenetics is also involved in the pathogenesis of DN, which is thejunction of genetic factors and environment, but it is not clear whether ER stress andepigenetic modifications have a direct molecular link. On these grounds, our present studyutilize db/db mice, an animal model representative of type2diabete, observe variationalregularity of ERS and ERS-related transcription factors in the kidneys of db/db mice, aswell as the alternation of epigenetics, inflammation activation, renal pathology andbiochemistry indices via regulating ERS with betaine. Moreover, this study utilizes themethod of siRNA knockdown to further clarify the mechanisms of ERS triggering SET7/9expression and investigate the effect of ERS-SET7/9-MCP-1pathway on the pathogenesisof diabetic nephropathy.Subjects and methods1. To confirm the relationship between ERS and SET7/9utilizing DN mice models1.1. Male4-week-old C57BL/KsJ db/db mice (initial weight,20-26g), an animal model representative of type2diabetes, were randomly divided into diabetic nephropathymodel group (DN group, n=18) and betaine treatment group (DN+B group, n=18).Age-matched18nondiabetic db/m mice (initial weight,14-16g) were defined as normalcontrols group (NC group). Betaine was administered intragastrically once a day for12weeks to the DN+B group at a dose of800mg/kg·day. The DN group was administered thesame amount of physiological saline.1.2. At the end of4,8and12weeks,6mice were randomly drawn from each group.Blood and24-hour urine samples were collected and the kidneys were harvested after theirbody weight (BW) was measured. Renal pathological changes of mices were observedusing special staining of periodic acid-schiff (PAS). A glomerulosclerosis index (GSI) wasused to evaluate the glomerulosclerosis score.1.3. Blood Glu (BG), serum creatinine (Scr), and blood urea nitrogen (BUN) weredetermined using an automatic biochemistry analyzer. Furthermore, the urinary protein andurinary MCP-1concentration with a quantitative sandwich enzyme-linked immunosorbentassay (ELISA).24hUPER was calculated with the following formula: UPER (mg/24h)=24h total volume of urine (ml)×urinary protein concentration (mg/ml).1.4. The mRNA expressions of GRP78, SET7/9and MCP-1in the kidneys of all micewere measured by real-time fluorescence PCR (qRT-PCR).1.5. The protein expressions of GRP78, ATF6α, SET7/9, XBP1s and H3K4me2in thekidneys of all mice were determined by western blot.1.6. The H3K4me1and SET7/9recruitment at MCP-1promoters and the XBP1srecruitment at SET7/9promoters in the kidneys of all mice were determined by chromatinimmunoprecipitation assay.2. To further clarify the mechanisms of ER stress triggering SET7/9expression duringDN formation using siRNA knockdown.2.1. Male4-week-old C57BL/KsJ db/db mice (initial weight,20-26g) were randomlydivided into physiological saline group, control siRNA group, SET7/9siRNA group andXBP1s siRNA group (n=6) receiving physiological saline (1ml), control siRNA, SET7/9siRNA and XBP1s siRNA (50ug in1ml PBS) once every10days by tail vein injection.After2months, the kidneys were harvested.2.2. Renal pathological changes of mices were observed using PAS. GSI was used in order to evaluate the glomerulosclerosis score.2.3. The protein expressions of SET7/9and XBP1s in the kidneys of all mice weredetermined by western blot.2.4. The H3K4me1and SET7/9recruitment at MCP-1promoters in the kidneys of allmice were determined by CHIP assay.2.5. The mRNA expressions of SET7/9, XBP1s and MCP-1in the kidneys of all micewere measured by qRT-PCR.Results1. To confirm the relationship between ERS and SET7/9utilizing DN mice models1.1Dynamic changes of renal pathology in db/db mice and the effect of betaineGlomerular and tubular lesions were not detected in NC group at any age. At the endof4weeks, the glomerular lesions were not obvious in DN group. At the end of8weeks,glomerular basement membrane thickening, increased mesangial matrix began to emerge.At the end of12weeks, it revealed typical renal pathological changes in DN group,including mesangial cells proliferation, mesangial matrix expansion and glomerularbasement membrane thick. The glomerular lesions were obviously attenuated in DN+Bgroup than in DN group. At the end of12weeks, the GSI was significantly higher in DNgroup (0.56±0.13) than in NC group (0.05±0.02). The GSI was significantly decreased inDN+B group (0.24±0.08) than in DN group (P<0.05). These results indicated that DN indb/db mice developed progressively with time and betaine could markedly prevent theprogression of DN.1.2Dynamic changes of BW, BG, Scr, BUN, UPER and urinary MCP-1levels in db/dbmice and the effect of betaineThe BW, BG, BUN, UPER and urinary MCP-1levels were significantly higher in DNgroup than in NC group in a time-dependent manner, which became significantly lowerafter betaine treatment (P<0.05). But there was no obviously difference in Scr betweengroups. These results indicated that DN in db/db mice developed progressively with timeand betaine could markedly prevent the progression of DN.1.3Dynamic changes of renal GRP78, ATF6α and XBP1s in db/db mice and the effectof betaineRenal GRP78mRNA expression in DN group was significantly higher than in NC group, which became significantly lower after betaine treatment (P<0.05). Renal GRP78,ATF6α and XBP1s protein expression were also increased in DN group than in NC group,which significantly decreased after betaine treatment (P<0.05).This indicated that ER stresswas activated in the kidneys of db/db mice, and the renal ER stress strength was increasedwith time, which significantly decreased after betaine treatment.1.4Dynamic changes of renal MCP-1in db/db mice and the effect of betaineRenal MCP-1mRNA expression in DN group was significantly higher than in NCgroup, which became significantly lower after betaine treatment (P<0.05). This indicatedthat inflammation was obviously activated in the kidneys of db/db mice and the attenuationof ER stress strength using betaine could prevent this increase.1.5Dynamic changes of renal SET7/9in db/db mice and the effect of betaineRenal SET7/9mRNA expression in DN group was significantly higher than in NCgroup, which became significantly lower after betaine treatment (P<0.05). Renal SET7/9protein expression were also increased in DN group than in NC group, which significantlydecreased from8weeks after betaine treatment (P<0.05).This indicated that SET7/9expression in the kidneys of db/db mice were significantly increased and the attenuation ofER stress strength using betaine could inhibit this increase.1.6Dynamic changes of renal H3K4me2in the kidneys of db/db mice and the effect ofbetaineRenal H3K4me2protein expression were increased in DN group than in NC group,which significantly decreased from8weeks after betaine treatment (P<0.05).This indicatedthat H3K4me2expression in the kidneys of db/db mice were significantly increased and theattenuation of ER stress strength using betaine could inhibit this increase.1.7The SET7/9recruitment and H3K4me1levels at MCP-1promoters in the kidneysof db/db mice and the effect of betaineRenal SET7/9recruitment and H3K4me1levels at MCP-1promoters were increased inDN group than in NC group, which significantly decreased from8weeks after betainetreatment (P<0.05). This indicated that SET7/9-mediated H3K4me1may be involved in theinduction of MCP-1in the kidneys of db/db mice and regulating ER stress with betaine mayinhibit SET7/9-mediated H3K4me1expression.1.8The XBP1s recruitment at SET7/9promoters in the kidneys of db/db mice and the effect of betaineRenal XBP1s recruitment at SET7/9promoters were increased in DN group than inNC group, which significantly decreased from8weeks after betaine treatment (P<0.05).This indicated that XBP1s may play a key role in the induction of SET7/9.2. To further clarify the mechanisms of ER stress triggering SET7/9expression duringDN formation using siRNA knockdown.2.1The effect of SET7/9or XBP1siRNA on renal pathology in db/db miceThe glomerular basement membrane was thicker, the number of mesangial cells wasgreater, and more mesangial matrix was accumulated in control siRNA group than in NCgroup, which significantly alleviated after SET7/9or XBP1siRNA. These results indicatedthat SET7/9or XBP1s knockdown markedly attenuated glomerular lesions.2.2The effect of SET7/9siRNA on renal SET7/9, H3K4me1, MCP-1in db/db miceCompared with saline-and control siRNA group, renal SET7/9, H3K4me1, MCP-1in SET7/9siRNA group were significantly decreased. These results indicated thatSET7/9-mediated H3K4me1play a key role in the induction of MCP-1．2.3The effect of XBP1siRNA on renal XBP1s, SET7/9, SET7/9recruitment,H3K4me1, MCP-1in db/db miceCompared with saline-and control siRNA group, renal XBP1s, SET7/9, SET7/9recruitment, H3K4me1, MCP-1in XBP1siRNA group were significantly decreased. Theseresults indicated that XBP1s was responsible for SET7/9-H3K4me1induced MCP-1expression.Conclusions1. ERS-related transcription factor XBP1s is involved in the pathogenesis of DN viaregulating SET7/9.2. Regulation of ERS-related XBP1s and SET7/9could effectively prevent thedevelopment and progression of DN.