| Background and aimSince the conception of "lipid nephrotoxicity" was raised in1982, increasing researches have suggested that dyslipidemia is the major risk for the development and progression of chromic kidney disease. This abnormal lipid metabolism mainly characteried by elevated plasma cholesterol and triglyceride levels, altered apolipoprotein profile. It is considered to causing the worsening of renal function in patients with pre-existing nephropathies. Recent studies also suggested a correlation between the pathogenesis of primary kidney diseases and dyslipidemia. This renal damage may start long before the appearance of hypertension or diabetes in patients with dyslipidemia. Nondiabetic patients with proteinuria, elevation in serum cholesterol and triglycerides had a nearly twofold increase in the rate of loss of renal function compared to normolipidemic patients.Recent studies shown that lipid deposition, glomerular mesangial matrix expansion, macrophage infiltration and collagen proliferation are recognized as early events in the development of glomerulosclerosis and interstitial fibrosis in this damage course. In addition, the early marker of impaired renal function is the presence of albuminuria. The onset of dyslipidemia-related renal injury is mainly associated with the renal hemodynamic changes, endothelium dysfunction, insulin resistance, chronic inflammation and oxidative stress. However, this pathophysiological mechanism is complicated and not yet fully understood. Along with the hyperlipidemia prevalence rate elevated year by year in worldwide, its complications including renal damage demand more attentions. Therefore, effective preventive and control measurement became one of the focus of clinical attention.Stagliptin, one of the new class of anti-diabetic agents dipeptidyl-peptidase-4(DPP-4) inhibitors, is mainly exert its effects through enhanced physiologic concentration of GLP-1(glucagon-like peptide-1). GLP-1is a peptide hormone secreted by L cell of the intestine, recognized as one of the strongest substances stimulating insulin release. Through combining the receptor, GLP-1regulates nutrient intake and maintances the energy banlance. However, GLP-1could be rapidly degraded by DPP-4, which extremely concentrated in kidney and widely expressed in multiple tissues and organs. Sitagliptin competitive inhibit the activation group of DPP-4and reduce the catalytic activity, consequently enhance physiologic concentration of GLP-1. Sitagliptin alone or in combination with other oral hypoglycemia drugs can effectively control blood glucose and decease the HbA1c. The main mechanisms of sitagliptin including:1. Glucose-dependently increase the bio-synthetic and secrete of insulin while the blood glucose concentration lower than4.5mmol/L could inhibit this promotion;2. GLP-1also has effects on pancreatic a cells, glucose-dependently inhibit the glucagon secretion, recduce glycogen degradation, increase the insulin sensitivity and the intake of blood glucose by peripheral tissues;3. Protect the function of pancreatic β cell, stimulate it proliferation and differentiation, inhibit apoptosis process;4. Furthermore, GLP-1has an effect on central nervous system to stimulate the satiety, consequently reduce food intake and lose weight;5. Improve the control of blood glucose by inhibiting intestine peristalsis, decreasing gastric juice secretion and delaying the gastric empting. Recent study suggested that sitagliptin also reduce the postprandial serum triglyceride and cholesterol. To our knonwledge, sitagliptin can reduce the albuminuria and ameliorate renal function of diabetic patients, postponeing the progression of diabetic nephropathy (DN). Besides improve left ventricular function and endothelial function, sitagliptin also has the glucose-independently protective effects on atherosclerosis, non-alcoholic cirrhosis, hypertension nephropathy, acute renal injury and even the end-stage kidney disease,Previous research has shown that TGF-β1(transforming growth factor-β1) plays an important rloe in the onset and progression of renal disease. The expression of TGF-β1could be induced by glucose metabolism disturbance and increment of hemoglobin Alc(HbAlc), glomerulus internal pressure and oxidant stress. This up-regulation of TGF-β1lead to the glomerular base-membrane thickness and glomerular mesangial matrix expansion, stimulate the expression of ECM components such as collegen, fibronectin (FN), lead to ECM accumulation. At the same time, the distributed expression of TGF-β1in tubulointerstitial tissue of injuried kidney contributed to tubulointerstitial fibrosis, which correlated with the declined glomerular filtration rate (GFR), increased urinary albumin excretion and renal dysfunction.Whilst MAPK (mitogen activated protein kinase) is the major cellular signal transduction pathway and also is a important composition of TGF-β1pathway. P38is the stress pathway of MAPK in renal tissues, mediating cell proliferation, hypertrophy and apoptosis principally. Many studies proved that blocking the p38MAPK signal pathway can decrease the accumulation of mesangial matrix and the expression of collagen and FN, sequentially ameliorate the renal fibrosis and postpone the development of glomerulosclerosis. Meanwhile, Erk (extracellular signal-regulated kinase) as another component of MAPK family has the effect of increasing the collagen synthesis and involving in the ECM reconstruction. It has proved that TGF-β1, FN and collagen, as the down-stream signal events of AMPK, could be inhibited by the phosphorlation of AMPK in the rat glomerular mesangial cells. This renoprotective effect is independent of the blood glucose level.Furthermore, as a sensor of cellular energy regulation, the activity of AMP-activated protein kinase (AMPK) is closely related to metabolic stress such as glycometabolic and lipometabolic disorders. Growing evidence demonstrated that declined AMPK phosphorylation by HFD-induced energy metabolism disorder also paly an important role in the progression of kidney damage. Considering its well-documented contribution to increasing glucose uptake and fatty acid oxidation, decreasing abnormal lipid deposition and to maintaining the cellular homeostasis, AMPK is now recognized as a new target for the treatment of type2diabetes mellitus and obesity. What’s more, AMPK phosphorylation activated by increment of GLP-1was observed in the researches of DN and hepatic fibrosis. Whether sitagliptin has the similar effect on dyslipidemia-related renal injury is not clear.Therefore, we designed this research to investigating the renoprotective role of sitagliptin in the dyslipidemia. Select and built the ideal animal model are the important parts of research process. ApoE gene knockout (apoE-/-) mice as the classic hyperlipidemia model were widely used in the researches of atherosclerosis, non-alcoholic cirrhosis and other organs injury. Studies have reported that due to the defect of apoE gene, lipid-lowing agents have no effect to improving the HFD-induced dislipidemia in apoE-/-mice. It also observed that there were no impaired fasting glucose in the whole16weeks experimental stage. High fat diet which contain a certain amount of fat and cholesterol could accelerate the onset of dislipidemia and lead to the damage of kidneys.Taking the apoE gene knockout mice as experimental objects, we firstly established the models with dyslipidemia-related renal damage to observing the influence of sitagliptin on metabolic disturbance, renal morphological and functional damage. Furthermore, we detected the changes in AMPK, TGF-β1and p38/Erk MAPK signaling pathway, allowed us to hypothesis the potential mechanism of this renal protective action, eventually afforded the experimental evidence for sitagliptin’s clinical significance.On the strength of above-mentioned research background and theoretical foundation, the content of this study including:1. Built and identify the apoE-/-mice.2. Given the HFD for16weeks to inducing the renal damage by hyperlipidemia.3. Observe the effects of sitagliptin on mice metabolic disturbance, kidney morphology and function injury.4. Detect the mRNA and protein expression of related indexes after sitagliptin treatment, investigating its poteintal mechanism.Methods(1) All animal experiments were performed according to Institutional Animal Care Guidelines. C57BL/6J mice and apoE-/-mice were purchased from the animal center of Southern Medical University and Joslin Diabetes Center (Bostin, MA) respectively. Mice were maintained on a12h light/dark cycle and were fed a diet of standard chow with water ad libitum in a room controlled for temperature and humidity. Six weeks old male apoE-/-mice and C57/BL mice with the same age and gender were given the standard chow. After2weeks of adjustable feeding, C57/BL mice were fed with a HFD (21.8%fat,1.25%cholesterol,76.95%carbohydrate, WT-control, n=8) while apoE-/-mice were randomized into a HFD (apoE-/-control, n=10) or HFD mixed with0.3%sitagliptin (Merck&Company Inc,200mg/kg/d, sig+apoE-/-, n=8). The feeding time was16-week.(2) During the experimental period, weighting and recoeding mouse of each group by a electronic autobalance weely from8-week-old. Intra-peritoneal glucose tolerance test (IPGTT) and intra-peritoneal insulin tolerance test (IPITT) were measured respectively to detect the glucose levels and insulin sensitivity at15th week and16th week of experiment.(3) At the end of experiment, mice were housed in metabolic cages with access to water and food to collect the24h-urine for subsequent measurements.24h urinary albumin and8-OHdG excretion were measured by using the mouse ELISA kit. These assays were performed according to the manufacturer’s protocol.(4) After16wks, mice were anesthetized by an intraperitoneal injection of0.8%pentobarbital sodium (40mg/kg body wt). Blood samples was collected by eyeball extirpating under anesthesia, and plasma were stored at-80℃after centrifugation for future analyses. High-density lipoprotein (HDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), total triglyceride (TG) and cholesterol (CHOL) were measured by automatic biochemical analyzer.(5) All mice were decapitated after blood collecting, quick weighing mouse kidney and recording it. One of the mouse kidneys was incision along the sagittal plane to two parts, fixed in4%paraformaldehyde or snap frozen in liquid nitrogen respectively to prepare for renal cortex paraffin sections, frozen section and histology of the tissue assessed. To examine the glomerular messangial matrix area, tissue paraffin sections were stained with PAS (Periodic Acid-Schiffstain) staining and analysis performed. Tissue paraffin sections were stained with Sirius red and the area of collagen fibers assessed. Neutral lipid accumulation was evaluated on frozen sections using Oil-red O staining. Another kidney was stored at-80℃for further studies. All stained sections were observed under the light microscope,5visions were randomly selected of each kidney section and were evaluated by a researcher blind to the experimental groups. The areas of glomerular mesangial matrix, collagen proliferation and glomerular lipid deposition were measured by Image-Pro Plus software, then performed the statistic analysis.(6) Real-time PCR was performed on cortex homogenates using the primers for GLP-1R, DPP-4, TGF-β1, FN-land GAPDH as the housekeeping gene. Renal cortex tissues were grinded in the RNA lysis solution in order to extract RNA. After the extraction, RNA concentration was measured,2μg of total RNA added to the reaction system of reverse transcription for cDNA synthesis which participate in the PCR reaction. The mRNA levels of the target gene were normalized to the GAPDH mRNA levels.(7) Renal cortex tissues were grinded in the protein lysis solution in order to extract total protein according to the manufacturer’s instructions. After the extraction, protein concentration was measured by microplate system in order to conculated and determined the sampling amount. Protein samples were diluted in5x loading buffer and heated for5min at95℃. The proteins were separated on an10%polyacrylamide gel using SDS-PAGE and transferred to a polyvinylidene fluoride membrane to evaluating the expression of protein for p-AMPK, TGF-β1, FN and p38/Erk MAPK signals. The protein levels expressed as relative intensity units normalized to P-actin were averaged for each group.(8) Statistics:normal distributed measursement data are presented as mean±standard deviation(s.d.). Analyses and description were performed using the SPSS Software version13.0and Graph Pad Prism Software version5.0. Differences between the mean of groups under completely randomized design were analysed using a one-way analyses of variance (ANOVA). When homogeneity of variances is >0.05, Least-significant Difference (LSD) post hot test was used for multiple comparisons; while when homogeneity of variances is<0.05, Dunnett T3post hot was used for multiple comparisons. The level for statistical significance was defined as P<0.05Results(1) There was a significant increase of body weight in the apoE-/-and sig+apoE-/-group compared to the control group from the1th week. This weight gain was continued through the rest experimental period and the difference have statistical sense (F=14.871, P<0.001). However, there were no significant difference between apoE-/-group and sig+apoE-/-group (P>0.05).After16-week, the levels of TG, CHOL, LDL, VLDL in apoE-/-group and sig+apoE-/-group were all prominently increased compared with control group (P<0.01) there were no significant difference between these two groups (P>0.05). While the level of HDL was significantly enhanced after sitagliptin treatment in the sig+apoE-/-group compared to apoE-/-group (P<0.001).IPGTT showed that the fasting blood glucose levels have no significant differences between three groups (F=1.947, P=0.177). IPITT test showed that blood glucose levels at15,30,60min were all significantly higher in apoE-/-group compared with control group (P<0.05). This increased blood glucose level was reduced by sitagliptin treatment at30,60min (P=0.023, P=0.012).(2) Mice urinary tests showed that the excretion of urinary albumin in apoE-/-group was apparently increased1.5fold and1.3fold than sig+apoE-/-group (24.074±2.940vs16.510±3.583, P<0.001) and control group (24.074±2.940vs18.475±2.859, P=0.003), the difference has statistical sense (F=10.613, P=0.001). Consistent with this, urinary8-OH-dG excretion were higher in the apoE-/-group compared to sig+apoE-/-group (39.127±7.064vs28.883±3.359, P=0.003) and control group (39.127±7.064vs26.842±4.656, P=0.001) at16th week (F=10.613, P=0.001). There was no significant difference between the sig+apoE-/-and control group in urinary albumin and urinary8-OH-dG levels (P>0.05).(3) Histological sections staining showed an overall increment of glomerular messangial matrix area in apoE-/-group compared to the control group (27.174±2.333vs8.341±1.079, P<0.001). This was alleviated by sitagliptin treatment (P<0.001). Oil-red O staining showed an remarkably increased lipid area in the glomerulus of apoE-/-group (20.130±1.678vs4.041±0.889, P<0.001) and siginificantly reduced in sig+apoE-/-group (P<0.001). Similarly, there was an increasing collagen area, as determined by Sirus red staining, in the apoE-/-group compared to control group (19.428±1.796vs3.221±0.775, P<0.001) and siginificantly reduced in sig+apoE-/-group (P<0.001). There was no significant difference between the sig+apoE-/-and the control group (P>0.05).(4) The mRNA expression of renal cortical FN-1(P=0.028) and pro-fibrotic marker TGF-β1(P<0.001) of apoE-/-group were obviously enhanced compared with control group. Sitagliptin treatment significantly decreased the mRNA expression of TGF-β1(P<0.001) and FN-1(P=0.004) in sig+apoE-/-group. At the sametime, DPP-4(P=0.609), GLP-1R (P=1.178) were also evaluated, however there was no significant difference between the three groups.(5) Western blot analysis showed that dyslipidemia markedly decreased the phospho-AMPK Thr121/total AMPK protein ratio in apoE-/-mice compared to control mice (P<0.001). In contrast, the protein levels of TGF-β1(P<0.001), FN (P<0.001), phospho-p38/total p38(P=0.014), phospho-Erk/total Erk (P<0.001) in the kidney were significantly higher in the apoE-/-group compared with control group. After sitagliptin treatment, the phospho-AMPK Thr121/total AMPK protein ratio were restored in sig+apoE-/-group (P=0.001). Along with the changes of phospho-AMPK Thr121/total AMPK, the levels of TGF-β1(P<0.001), FN (P=0.002) were significantly decreased with sitagliptin treatment. Consistent with these findings, the protein expression of phospho-p38/total p38(P=0.037) and phospho-Erk/total Erk (P<0.001) also obviously decreased in sig+apoE-/-group.ConclusionsOur study dicussed the protective role of sitagliptin against the mice renal damage induced by dyslipidemia and investigated its potential mechanism at the molecular level. Summary, we may safely arrive at the following conclusion:1. There was no obviously effects on the body weight, fasting blood glucose and blood lipid of apoE-/-mice after16weeks of sitagliptin treatment. Sitagliptin enhanced mice insulin sensitivity, ameliorated renal function, reduced oxidative stress and significantly decreased the glomerular mesangial matrix expansion, lipid deposition and collagen proliferation. It is suggested its renal protective effect on dyslipidemia-related renal injury, which independent of improving fast blood glucose and blood lipids.2. Sitagliptin alleviated intracellular energy imbalance by AMPK phosphorylation. In addition, it also inhibited TGF-β1and p38/Erk MAPK signal pathway to postponing the onset and progression of glomerulosclerosis and interstitial fibrosis. This might be the underlying mechanism of sitagliptin protects against the dyslipidemia-related kidney injury. |
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