| Part One:Abnormal cholesterol metabolism in the kidneys of type 2 diabetes rats Background and Aims:Diabetic Nephropathy (DN) is one of the main chronic complications of Diabetic Mellitus (DM), and is the leading cause of end-stage renal disease (ESRD) as well. It seriously affects the patients’health and their quality of life. Evidence has suggested that lipids accumulation in the kidneys of type 2 diabetic animal models and DN patients. However, the mechanisms were still unclear. This study aimed to investigate the potential mechanism of the lipids deposition in the kidenys of type 2 diabetic rats and to explore whether the renal lipids accumulation have a pathogenetic function in the development and progression of DN.Methods:After one week adaptation, part of the SD rats were given a normal diet (Normal control, NC), and another part of the rats were given a high fat/sucrose diet. Four weeks later, the latter rats were intraperitoneally injected with low-dose streptozocin (STZ,30mg/kg). After 72 hours, only rats with non-fasting blood glucose exceeding 16.7 mmol/L were considered diabetic and selected for additional studies. Then, the diabetic rats were divided into DM group and DM+AT (intragastric administration of Atorvastatin,10 mg/kg/d) group.24-hour urine of rats was collected in individual metabolic cages to measure urine protein and urinary neutrophil gelatinase-associated lipocalin (u-NGAL). At the end of the 8th week, the rats were fast food overnight, and then sacrificed. The blood was collected to separate the serum used for testing fasting blood glucose (FBG), creatinine (Cr), blood urea nitrogen (BUN), total triglyceride (TG), total cholesterol (TC), and low density lipoprotein (LDL). Part of the kidney was fixed in 4% paraformaldehyde, then dehydrated and embedded in OCT for Oil Red O staining. Another part of the kidney was fixed in 10% neutral formalin and embedded in paraffin for immunohistochemical staining, hematoxylin-eosin (HE) staining, periodic acid Schiff (PAS) staining, and periodic acid-silver metheramine (PASM) staining. The left renal tissue was immediately stored at-80℃ for quantitative RT-PCR and Western-blot.Results:1. The weight of the diabetic rats was lower than that of the normal rats (P<0.05), but the ratio of kidney weight to body weight (KW/WT) of the diabetic rats was higher (P<0.05). Compared with the NC group, the blood glucose of diabetic rats was significantly increased, and the levels of serum Cr, BUN, TG, TC, and LDL were also markedly higher in the diabetic rats (P<0.05). AT treatment for 8 weeks had no effect on the weight, KW/WT, and the lever of serum glucose of diabetic rats (P>0.05). However, the levels of serum TC and LDL were markedly reduced by AT treatment (P<0.05), though the change of BUN and TG were not obvious (P>0.05).2. The 24-hour urine protein of the DM group was significantly increased, and it continued to elevate with the progress of diabetes (P<0.05). At the end of 8th week, the u-NGAL was also elevated in the DM rats (P<0.05). Compared with the DM group, the 24-hour urine protein and u-NGAL were improved in the DM+AT group (P<0.05).3. HE staining showed many vacuolar cells in the kidneys of DM rats, while no vacuolar cells was found in normal rats. Oil Red O staining showed that lipid droplets accumulation in the kidneys of the diabetic rats, especially in the renal tubules. However, no lipid was found in the kidneys of NC group. AT treatment reduced the lipid droplets in the kidneys of the diabetic rats. PAS staining showed mesangial expansion in the renal glomeruli, and PASM staining showed the basement membrane thickness in the glomeruli and tubules of diabetic rats, comparied with NC group. However, AT alleviated the mesangial expansion in the renal glomeruli and the basement membrane thickness both in the glomeruli and tubules.4. The mRNA and protein expression of HMG-CoAR, LDLr, SREBP-2 and SCAP were significantly upregulated in type 2 diabetic rats compared with the normal rats (P<0.05). AT treatment upregulated the mRNA and protein expression of HMG-CoAR, LDLr, and SREBP-2 (P<0.05), but the mRNA and protein expression of SCAP were unchanged by AT treatment (P>0.05).Conclusions:1. There was lipids deposition in the kidneys of type 2 diabetic rats. Reduction of renal lipid deposition by AT can improve renal morphology and function in diabetic rat models.2. The disorder of the SCAP-SREBP-2-HMG-CoAR/LDLr pathway may cause renal cholesterol accumulation in high fat/sucrose-fed and STZ-induced rat models, resulting in diabetic renal injury.Part Two:Advanced glycation end products mediated renal lipid deposition in diabetic ratsBackground and Aims:The etiological factors and mechanisms of DN are complicated. One of the important pathogenesis of DN is chronic hyperglycemia induced accelerated formation of advanced glycation end products (AGEs). AGEs were regarded as the cause of foam cells formation in atherosclerosis and fatty liver. In recent years, studies have confirmed there is lipid accumulation in the kidney of type 2 diabetic animal models and patients with DN. However, whether AGEs are related to the renal lipid deposition is still unknown. Therefore, the purpose of our research is to explore whether AGEs could cause foam cells formation in the kidney of diabetes, thus injure renal morphology and function.Methods:In vivo study:Diabetic rats were divided into DM group and DM+AG (intragastric administration of Aminoguanidine Hydrochloride,100 mg/kg/d) group.24-hour urine of rats was collected in individual metabolic cages to measure urine protein and u-NGAL. At the end of the 8th week, the rats were fast food overnight, and then sacrificed. The blood was collected to separate the serum used for testing carboxy-methyl-lysine (CML). Part of the kidney was fixed in 4% paraformaldehyde, then dehydrated and embedded in OCT for Oil Red O staining. Another part of the kidney was fixed in 10% neutral formalin and embedded in paraffin for immunohistochemical staining, periodic acid Schiff (PAS) staining, periodic acid-silver metheramine (PASM) staining, and immunohistochemical staining. The left renal tissue was immediately stored at-80℃ for quantitative RT-PCR and Western-blot.In vitro study:According to the different treatments, human renal proximal tubule cells (HK-2) were divided into 6 groups:Ctr group (treated with experimental medium), CML group (treated with medium containing 50 μg/mL CML), CML+anti-RAGE group (treated with medium containing 50 μg/mL CML and 10 μg/mL anti-RAGE), LDL group (treated with medium containing 200μg/mL LDL), CML+LDL group (treated with medium containing 50 μg/mL CML and 200μ.g/mL LDL), and CML+LDL+anti-RAGE group (treated with medium containing 50 μg/mL CML,200μg/mL LDL and 10 μg/mL anti-RAGE). After 24 hours treatment, the effects of cholesterol accumulation were examined by Oil Red O staining and a quantitative intracellular cholesterol assay. The gene transcription and protein translation of HMG-CoAR, LDLr, SREBP-2 and SCAP were analyzed by quantitative RT-PCR and western blot respectively. The translocation of SCAP from the endoplasmic reticulum (ER) to the Golgi was detected by confocal microscopy.Results:In vivo study:1. There was an increase of serum CML in the diabetic rats, and AG markedly reduced the serum CML (P<0.05). Immumohistochemical staining showed significant deposition of CML in the diabetic renal tubules, glomerulus, mesangium, basement membrane and interstitium. However, AG could alleviate the CML deposition in the kidneys of diabetic rats.2.24-hour urine protein of the diabetic group was significantly increased, and it continued to elevate with the progress of diabetes, however,4-week and 8-week treatment of AG improved this alteration (P<0.05). AG treated for 8 weeks also lowered the level of u-NGAL (P<0.05).3. Oil Red O staining showed that AG reduced the lipid droplets in the kidneys of the diabetic rats, and PAS and PASM staining showed AG alleviated the mesangial expansion in the renal glomeruli and the basement membrane thickness both in the glomeruli and tubules.4. Furthermore, AG also downregulated the expression of LDLr, HMG-CoAR, SREBP-2 and SCAP at both mRNA and protein levels (P<0.05).In vitro study:1. Compared with Ctr, the CE value of CML group was markedly increased (36.66±11.55 μmol/L vs.110.00±21.79 μmol/L, P<0.05), but the CE value of CML+anti-RAGE group was lower than that of CML group (110.00±21.79 μmol/L vs.61.67±18.93 μmol/L, P<0.05). The CE value of CML+LDL group was elevated significantly when compared with LDL group (111.67±18.93 μmol/L vs.150.00±10.00 μmol/L, P<0.05), however, the CE value of CML+anti-RAGE+LDL group was lower than that of CML+LDL group (150.00±10.00 μmol/L vs.83.33±15.28 μmol/L, P<0.05). Oil Red O staining showed there was an increase of lipid droplets in HK-2 cells in the presence of native LDL or CML, and more significant lipid droplets accumulation in LDL plus CML-treated cells compared with cells treated with LDL alone. Anti-RAGE reduced the lipid droplets accumulation in the CML-treated cells in the absence or presence of a high concentration of LDL.2. Compared with Ctr, LDL downregulated the mRNA and protein expression of HMG-CoAR, LDLr, SREBP-2 and SCAP (P<0.05). However, CML upregulated both the mRNA and protein expression of HMG-CoAR, LDLr, SREBP-2 and SCAP in the absence or presence of a high concentration of LDL (P<0.05), and this could be inhibited by anti-RAGE (P<0.05).3. Increased SCAP accumulation in the Golgi in CML group compared with Ctr. However, compared with CML group, SCAP accumulation was reduced by anti-RAGE. Though native LDL loading reduced SCAP accumulation in Golgi, CML could enhance the localization of SCAP to the Golgi even in the presence of native LDL loading. Compared with CML+LDL group, SCAP accumulation in the Golgi was reduced in CML+anti-RAGE+LDL group.Conclusions:1. CML disrupts the cholesterol feedback regulation in the renal tubular epithelial cells, resulting in lipid deposition in the kidneys. The ectopic deposition of lipids may cause renal structure and function damage in DN.2. Inhibitiing AGEs (CML) synthesis and blocking CML-RAGE pathway might be potential renoprotective roles in the treatment of DN.Part Three:Advanced glycation end products mediated renal tubular lipid deposition though endoplasmic reticulum stressBackground and Aims:Our previous studies have discovered AGEs could cause lipid accumulation in the renal tubules, but the mechanisms responsible for this phenomenon are still unknown. Some studies have shown that ERS plays an important role in intracellular lipid accumulation. However, the mechanisms were still unclear. The present study was to explore whether the lipid deposition in renal tubule cells is caused by AGEs-triggered ERS.Methods:According to the different treatments, HK-2 cells were divided into 4 groups:Ctr group (treated with experimental medium), CML group (treated with medium containing 50 μg/mL CML), CML+4-PBA group (treated with medium containing 50μg/mL CML and 5mmol/L 4-PBA), and 4-PBA group (treated with medium containing 5mmol/L 4-PBA). After 24 hours treatment, cell viability was determined by CCK-8 dye and cell apoptosis was determined by Annexin V-FITC Kit. The effects of cholesterol accumulation were examined by Oil Red O staining and a quantitative intracellular cholesterol assay. The gene transcription and protein translation of HMG-CoAR, LDLr, SREBP-2, SCAP, glucose regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were analyzed by quantitative RT-PCR and western blot respectively.Results:1. Compared with Ctr, redueced cell viability and increased cell apoptosis were found in CML group after CML treatment for 24h (P<0.05). However, the cell viability and cell apoptosis were alleviated in CML+4-PBA group compared with CML group (P<0.05), and 4-PBA alone did not affect the cell viability and cell apoptosis of HK-2 cells (P>0.05).2. Compared with Ctr, the CE value of CML group was markedly increased (53.33±11.55 μmol/L vs.116.67±32.15 μmol/L, P<0.05), and the CE value of 4-PBA group did not change obviously (53.33±11.55 μmol/L vs.53.33±5.77 μmol/L, P>0.05). However, the CE value of CML+4-PBA group was decreased significantly compared with CML group (111.67±18.93 μmol/L vs.150.00±10.00 μmol/L, P<0.05). Oil Red O staining showed no lipid droplets were found in the 4-PBA group, and less lipid droplets in CML+4-PBA group compared with CML group.3. Compared with Ctr, the mRNA and protein expression of HMG-CoAR, LDLr, SREBP-2, SCAP, GRP78 and CHOP were upregulated in CML treated HK-2 cells (P<0.05). However, the mRNA and protein expression of these molecules were downregulated in CML+4-PBA group (P<0.05).4-PBA alone did not change the mRNA and protein expression of HMG-CoAR, LDLr, SREBP-2, SCAP, GRP78 and CHOP (P>0.05).Conclusions:1. CML disturbs SCAP-SREBP-2-HMG-CoAR/LDLr pathway by triggering ER stress (ERS), resulting in lipid deposition in renal tubular epithelial cells.2. Inhibiting ERS is a potential novel approach to treat CML caused renal tubular lipid accumulation.Summary:During T2DM, the increased AGEs (CML) level may trigger ERS in renal tubular epithelial cells, which increases the expression of SCAP and SREBP-2, and disrupts the function of SCAP. Then, more SREBP-2 is carried to Golgi, where SREBP-2 is hydrolyzed and activated, thus upregulates the expression of HMG-CoAR and LDLr, causing increased intarcellular cholesterol synthesis and uptake. Finally, the deposition of lipid in the kidneys damages renal morphology and function, leading to DN. |
PDF Full Text Request