Abnormal Fat Accumulation And Peroxisomal Fatty Acids β-oxidation In Kidney Of Diabetic Rats

Objective: Diabetic nephropathy (DN), a very common and serious chronic complication of diabetes mellitus(DM), is the main cause of death in DM. The abnormal content and deposition of lipid in the kidney has been thought as a major pathogenesis in DN.Diacylglycerol acyltransferase 1(DGAT1) is the rate-limiting enzyme in triglyceride synthesis. Whether lipid deposition relates to its expression in diabetes has been poorly characterized. It has been known that the lipid content in the tissue is not only associated with its synthesis, but also related to the velocity of its catabolism. The catabolism of fatty acid is regulated by the peroxisome proliferator activated receptorα(PPARα). After being activated by its ligand, PPARαcan up-regulate the expression of genes, involved in the uptake, transportation and oxidation of fatty acid, resulting in the increased oxidation of fatty acid. It was reported that the expression of key enzymes ofβ-oxidation of fatty acid, CPTⅠand ACOX1, was up-regulated in the myocardial mitochondria and peroxisome in DM mouse. It is presumed that the deposition of lipid in the DM kidney is due to the decreased velocity of lipid catabolism, and its deposition. Therefore, whether the decreased oxidation of fatty acid in the kidney of STZ induced DM rat, relates to the down regulation of CPTⅠ, LBP and DBP genes, involved in the oxidation of fatty acid, in addition to PPARαand ACOX1 genes, has not been reported.Ghebremeskel K etc. found fatty acids changes, and arachidonic acid (C20:4), docosahexaenoic acids (C22:6) increased in type 1 diabetes rats liver, which were induced to diabetes by streptozotocin (STZ). The changes of fatty acids in type 2 diabetes rats kidney have been not determined. The changes of enzymes gene expression which take part in peroxisomalβ-oxidation in type 2 diabetes rats has not been determined. In order to study these questions, rats were injected a low-dose of streptozotocin to induce type 2 diabetes. After that, we measured the activity ofβ-oxidation, enzymes gene expression which take part inβ-oxidation in peroxisome and mitochondrion and DGAT1, the rate-limiting enzyme in triglyeride synthesis. We also detected triglyeridethe and total free fatty acid in diabetic kidney, analyzed fatty acids by gas chromatography. All research was to investigate the role of fatty acidsβ–oxidation in peroxisome abnormal free fatty acids in diabetic kidney and mechanism of fatty degeneration.Methods:1 Animals and materialsMale Sprague-Dawley (SD) rats (weighing 200–250g) supplied by the Experimental Animal Center of Hebei Medical University were divided randomly into controls and high-fat group, which were fed high-fat diet to develop insulin-resistance. After overnight fasting, the latter group was injected intraperitoneally with 27 mg/kg of streptozotocin freshly dissolved in ice cold sodium citrate buffer (0.1 M, pH4.5). Diabetes was confirmed three days after the i.p. injection of streptozotocin by the presence of hyperglycemia. Six weeks after the induction of diabetes, 24 hours urine was collected for the detection of total amount of urine protein. Then rats were anesthetized under pentobarbital anesthesia (60mg/kg) and total RNA was extracted from freshly excised kidney (50–100mg pieces), which was stored at -80℃. Animals were exsanguinated. Prior to STZ injection, blood was obtained from the tail vein in tubes after overnight (12h) fasting. Samples were centrifuged at 3,000 rpm for 10 minutes and the supernatant plasma separated, aliquoted, and immediately stored at -20℃. At the time of sacrifice, blood was also collected from theaorta and plasma separated and stored as above.2 Observation of the pathological changes by light microscopeThe kidney tissue was routinely fixed by citromint, and prepared for light microscope observing.3 Detection of blood glucose and creatinineBlood glucose and creatinine were detected by Olympus Au2700 automatic biochemistry analysator.4 Detection of the total amount of 24h urine-protein by Lowry method. 5 Determination of oral glucose tolerance test (OGTT) and insulin sensitivity index (ISI)The rats were perfused with glucose on empty stomaches. The blood glucose was measured respectively at 0, 30, 60, 120 minutes, then blood glucoses of the two groups were analyzed. Fasting blood were collected for detections of insulin and FBG, then the ISI was calculated.6 Detection of TG accumulations by Oil red staining.7 Detection of the relative expression of related gene in the kidneyTotal RNA was extracted with Trizol method. The relative expression of PPARα, CPT I, ACOX1, ACOX3, LBP and DBP mRNA was evaluated by RT-PCR, withβ-actin as a"housekeeping gene".8 Measurement of peroxisomeβ-oxidation activityPeroxisomeβ-oxidation activity were performed by the conversion of Palmitoyl-CoA to Palmitate.9 Detection of kidney triglycerideKidney was homogenated with isopropanol. Supernatant was imbibed after centrifugalization. Then the content of triglyceride was detected.10 Detection of total free fatty acids in kidney with the kit11 Detection of kidney fatty acids with gas chromatographDetect the content of C16:0, C18, C20:0, C24:0, C26:0 in kidney. Results:1 The changes of general state, blood and urine biochemical indicatorThe DM rats were emaciated obviously, with torpidity, and their fur lost luster and became rare faction. Blood sugar, CRE, 24h urine-protein were obviously higher than these of Con group (P<0.01), ISI was lower than Con group (P<0.05).2 Morphological changes in kidneyThe DM kidneys went bigger obviously, and punctiform yellow brown fatty degeneration and congestion patch were seen, which were not integrated or accretio peplos. The darker cortex was seen in cross section. The observation with HE dyeing shows: distal renal tubule swelled and cell liked vacuolus; renal glomerulus went bigger with sublobe, some renal glomerulus became hyalinization. In contrast to minimal or absent oil red O staining in the kidney of the control mice, there was very strong staining of oil red O in diabetic mice.3 The expression of genes3.1 The relative expression of PPARαmRNACompared with Con group (0.11±0.01), the changes of relative expression of PPARαmRNA in kidney (0.14±0.03) had no statistics significance (P>0.05).3.2 The relative expression of CPT I mRNACompared with Con group (0.19±0.01), the change of relative expression of CPT I mRNA in DM kidney (0.34±0.02) increased significantly(P<0.01). 3.3 The relative expression of ACOX1 mRNACompared with Con group (1.12±0.22), the change of relative expression of ACOX1 mRNA in DM kidney (1.51±0.10) increased significantly(P<0.01).3.4 The relative expression of ACOX3 mRNACompared with Con group (0.17±0.02), the change of relative expression of ACOX3 mRNA in DM kidney (0.30±0.04) increased significantly(P<0.01).3.5 The relative expression of LBP mRNACompared with Con group (0.25±0.02), the change of relative expression of LBP mRNA in DM kidney (0.47±0.03) increased significantly (P<0.01).3.6 The relative expression of DBP mRNACompared with Con group (0.09±0.03), the change of relative expression of DBP mRNA in DM kidney(0.21±0.02) increased significantly (P<0.05).3.7 The relative expression of DGAT1 mRNACompared with Con group (0.13±0.01), the change of relative expression of DBP mRNA in DM kidney(0.25±0.04) increased significantly (P<0.05).4 Peroxisomeβ-Oxidation activityCompared with Con group(1.13±0.27 mU/mg prot), DM kidney peroxisomeβ-oxidation activity(2.12±0.37 mU/mg prot) increased significantly (P<0.05).5 Kidney triglycerideCompared with Con group (0.84±0.16mmol/L), DM kidney triglyceride(2.98±1.62 mmol/L) increased significantly (P<0.05).6 Total free fatty acid in kidneyDifferences of total free fatty acid in kidney between DM group(166.57±40.55μmol/g) and Con group(162.52±32.65μmol/g) were no statistical significance (P>0.05).7 Kidney fatty acids contentsDifferences of percentage contents of C16:0, C18, C20:0, C24:0 between DM group and Con group were no statistical significance. Compared with Con group (1.1±0.1)%, DM kidney percentage contents of C26:0 (2.6±0.4)% increased significantly(P<0.05).Conclusion:1 In high-fat-induced diabetic rats with kidney impaired, it was increased that the expression of peroxisome fatty acidsβ-oxidation relative gene ACOX1, ACOX3, LBP and DBP, and resulted in the enhancement of peroxisomeβ-oxidation activity.2 In high-fat-induced diabetic rats with kidney impaired, triglyceride synthesis increased and kidney triglyceride accumulated. It may be associated with the increased of DGAT1 mRNA expression.