Ⅰ Effects And Mechanisms Of Cilostazol On Renal Inflammation Of Diabetic Rats Ⅱ Distribution Of Polymorphisms Of OCTN2 In The Han Chinese And The Related Studies

Backgrounds and aimsThe morbidity of diabetes has increased rapidly for the past few years. Diabetic nephropathy (DN) is one of the most common and severe chronic complications of diabetes. However, the pathogenesis of DN is still not clear. Recent studies indicate a critical role for the inflammatory response in the development of DN. The presence of various adhesion molecules, such as vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, and chemokines such as monocyte chemoattractant protein (MCP)-1 lead to the adhesion of monocytes to inflammatory sites, and are involved in DN. Vascular endothelial growth factor (VEGF), another proinflammatory cytokine, has been shown overexpression in serum, urine and kidney of diabetic rats. Expression of these proinflammatory molecules are always regulated by nuclear factors, such as nuclear factor-KB (NF-κB), peroxisome proliferator-activated receptors (PPARs). Thus, the repression of these proinflammatory molecules or their signaling pathways may be a strategy for the prevention and amelioration of DN.Cilostazol, a selective type 3 PDE inhibitor, has been widely used to improve ischemic symptoms of peripheral vascular diseases. Currently, an increasing number of studies have shown the potent anti-inflammatory actions of cilostazol. However, there have been few reports concerning effects of cilostazol on DN, much less about the impact of cilostazol on renal inflammation of diabetes.In the present study, therefore, we employed STZ-induced diabetic rats to investigate and characterize whether the oral administration of cilostazol is able to protect diabetic animals from the development of DN. The mechanism for the renoprotective effects of cilostazol was further explored.Materials and methodsMale SD rats were divided into a normal control group (n= 10) and diabetic group (n= 42). The latter were further divided randomly according to their respective BG levels 72 hours after streptozotocin (STZ) injection into three groups:diabetes mellitus (DM) group (n= 15), high dose of cilostazol group (n= 13,27 mg-kg-1·d"1), and low dose of cilostazol group (n= 13,9 mg-kg-1·d-1).At the end of the eight week cilostazol treatment, all animals were weighed, and a 24-h urine was collected for albumin excretion rate (UAE) determination using a sensitive fluorescence immunoassay. After an intraperitoneal injection of 10% chloral hydrate (3 ml-kg-1), all animals were sacrificed. The kidneys were rapidly isolated and weighed to obtain the relative kidney weight (g-kg-1 body weight), and then the cortices were saved. The kidney expression of VCAM-1, ICAM-1, PPARa andγwere determined by western blotting and RT-PCR/ real-time PCR, respectively. The renal MCP-1, VEGF and cAMP levels were examined by ELISA. The NF-κB-DNA binding activity was assessed by electrophoresis mobility shift assay (EMSA).Results1. The administration of cilostazol at both doses to diabetic rats for eight weeks did not alter body weight, BG or HbAlc levels compared with untreated diabetic rats.2. Diabetes resulted in a significant increase in the relative kidney weight (g-kg-1 body weight) compared to that of normal rats (p< 0.01). Although this ratio in diabetic rats treated with both doses of cilostazol were somewhat lower than the corresponding values for diabetes, these differences did not achieve statistical significance.3. Diabetes led to obvious glomerular hypertrophy and slight inflammatory cells infiltration. These changes could be ameliorated by a low dose of cilostazol treatment, while with a high dose of cilostazol, the glomerulus was almost retrieved to normal status.Glomerular hypertrophy was also evaluated by calculating glomerular volume. The glomerular volume was 2.1-times higher in diabetic rats than that in normal ones, while cilostazol treatment significantly reduced the glomerular hypertrophy by 51%(p< 0.01) with a high dose, and 38%(p< 0.01) with a low dose, respectively.4. The renal VCAM-1 expression was shown by Western blotting analysis to be elevated almost two-fold following diabetes induction, while the addition of a high-and low-dose of cilostazol lowered this expression by 40%(p< 0.01) and 28%(p< 0.01), respectively. In agreement with this, the ICAM-1 expression was also increased approximately one-fold in the renal cortex from diabetic rats, and this increase was reversed by cilostazol in a dose-dependent manner.5. RT-PCR experiments were performed to directly evaluate the contents of VCAM-1 and ICAM-1 mRNA in the renal cortex. mRNA expression of both VCAM-1 and ICAM-1 increased significantly in the kidney of diabetic rats. Cilostazol down-regulated the gene expression, more evident with high dose.Results from real-time PCR confirmed the above data. the VCAM-1 mRNA levels in diabetic rats were elevated 2-fold as compared to that of normal control rats (p< 0.01). A similar 1.7-fold increase in ICAM-1 mRNA was also seen (p< 0.01). Treatment with cilostazol resulted in the suppression of the mRNA expression of these two adhesion molecules in a dose-dependent manner; the VCAM-1 mRNA levels decreased by 43%(p< 0.01) and 40%(p< 0.05) with a high-and a low-dose of cilostazol, respectively, and the ICAM-1 mRNA contents were decreased by 38%(p< 0.01) and 25%(p< 0.05) with a high-and a low-dose of cilostazol, respectively. 6. Diabetes was associated with a nearly two-fold elevation of renal MCP-1 after eight weeks when compared with normal control rats (p< 0.01). The diabetes-induced increase in MCP-1 was significantly attenuated by cilostazol and to a greater extent with high dose. There was also a 1.6 fold increase in renal VEGF expression in diabetes (p< 0.01), which was ameliorated by cilostazol therapy, more evident in high dose than in low dose group7. The NF-κB activation in the diabetes-induced rats increased the intensity of gel-retarded bands. In rats treated with cilostazol, however, NF-κB response to a high dose of cilostazol was markedly decreased (p< 0.01), while to a low dose less decreased (p< 0.05).8. Correlation analysis showed that there was a significant positive correlation between the NF-κB-DN A binding activity and the VCAM-1 mRNA expression (r = 0.967, p= 0.000). A similar correlation also existed between NF-κB-DNA binding activity and ICAM-1 mRNA expression (r= 0.880, p= 0.000).9. The mRNA expression of PPARa and y decreased significantly in the kidney of diabetic rats, compared with normal controls (both p< 0.01). Administration of cilostazol up-regulates the gene expression in a dose-dependent manner.10. Cilostazol did not cause a statistically significant difference of cAMP levels in the kidney of diabetic rats (p> 0.05).ConclusionsTaken together, the renoprotective effects of cilostazol may be mediated by its anti-inflammatory actions, including inhibition of NF-κB activation, PPARs expression and the subsequent decrease in proinflammatory factors, such as VCAM-1, ICAM-1, MCP-1 and VEGF expression in kidneys of diabetic rats. Moreover, the anti-inflammatory effcts of cilostazol is cAMP independent.The modulation of inflammation with cilostazol might provide a new approach for the prevention and treatment of DN. Our study renders support to future prospective clinical use of cilostazol on DN. In addition, the pleiotropic effects of cilostazol have made it an interesting drug to explore. Backgrounds and aimsPharmacogenomics studies indicate that inherited variation in activities of drug-metabolizing enzymes, receptors or transporters is fundamental causes involved in interindividual differences in drug reactions. Following the studies of polymorphisms of drug-metabolizing enzymes and drug-target, more attentions have been evoked on the role of polymorphism of transporters in drug disposition.Novel organic cation transporter (OCTN) 2, distributed widespread in humans, are newly found transporters of organic cations and endogenous substances. OCTN2 physiologically transports carnitine to participate theβ-oxidation of fatty acid in mitochondria, thus plays critical role in maintaining normal functions of cardiovascular, nervous and reproductive systems. OCTN2 are still involved in the transporting multiple drugs, such asβlactam antibiotics, verapamil, spironolactone, valproate and fluoroquinolones antibacterial drugs. Variation of OCTN2 gene may (1) alter the pharmacokinetics, and then the pharmacodynamics of some drugs; (2) result in full functional loss of OCTN2. Thus, the excretion of carnitine from urine increases, and fatal systemic carnitine deficiency happens; (3) change the transcription and function of OCTN2 partially, and add the ratio of Crohn's disease and ulcerative colitis, solely or interacted with other inflammatory bowel diseases associated genes.To date, a number of single-nucleotide polymorphisms (SNPs) have been identified in the Western races and Japaneses, some of which were proved to be associated with abnormal transporter activities. However, no corresponding data is available in the Han Chinese, which is almost 1/4 of total population worldwide. As almost all the variations of transporters is dependent of races, it is hard to empoly the results from one race to another.Nowadays, the common approaches to detect the allele discrimination of polymorphisms in OCTN2 gene include RFLP, SSCP, DNA sequencing, et al. Each of these approaches has advantages and limitations, and are not routine in most laboratories. Denaturing high performance liquid chromatography (DHPLC) is a high-throughput, sensitive, specific and robust technology for the detection of SNPs or DNA variants, especially suitable in detecting unknown mutations from large samples. Recently this approach has been well used in researches of human functional SNPs.Taking into account the important roles of OCTN2 in physiology and pharmacology, it is essential to clarify the distribution characteristics of OCTN2 genetic polymorphisms in the Han Chinese. Our study may help to expand the human polymorphism database of SLC22A5, and to provide molecular explanation of interindividual differences of drug reactions and guidelines for rational administration. This may also help to predict the onset risk of some related diseases.Another aim of this study is to develope a platform for detecting OCTN2 variation with DHPLC. Combined with DNA sequencing, we wish to analyze the polymorphisms of OCTN2 in the Han Chinese, and to provide a quick, automatic pathway for the detecting of OCTN2 variants.MethodsGenomic DNA samples were collected from peripheral blood of 92 unrelated healthy Han Chineses (including 49 males and 43 females, average ages 23±1.2). Primers were designed manually to span the exons, and 50 to 200 base pairs of flanking intronic sequence per exon. OCTN2 variants were identified by DHPLC with the products amplied by PCR. With the mixed unkown and wile type samples (2:1), the PCR-DHPLC platform was set up to determine OCTN2 variants by DHPLC profiles.To validate the genotype of samples and the accuracy of DHPLC, a number of samples from each of the genotype groups detected by DHPLC profiling were selected to perform DNA sequencing.Results1. Total of 1012 fragments were amplified by PCR reaction effectively and qualitatively. The size of all fragments were in line with expectation.2. DHPLC detected both heterozygous and homozygous profiles in amplicons. The single peak indicated homoduplexes while double-peak shows heteroduplexes which means mutation. Totally,111 amplicons showed heteroduplex peak.3. Of all the 92 specimen,38 were found mutation in exon 1, with the frequency 41.3%. DNA sequencing showed 285T>C (rs2631365) mutation, which is synonymous.4. Of all the 92 specimen,40 were found mutation in exon 4, with the frequency 43.5%. DNA sequencing showed 807A>G (rs274558) mutation, which is synonymous.Of all the 92 specimen,38 were found mutation in the junction region of exon 4 /intron 4, with the frequency 41.3%. DNA sequencing showed 824+13 T>C (rs274557).16 subjects were found these two mutations simultaneously, with the frequency 17.4%.5. Of all the 92 specimen,11 were found mutation in exon 10, with the frequency 12%. DNA sequencing showed deletion of A in 3'UTR (*92delA, rs60522531).6. With the exploration and optimization of related conditions, we initially set up the platform for detecting variations of SLC22A5 with DHPLC. The accuracy of SNPs detection by DHPLC analysis was 100% as varied by DNA sequencing. Conclusions1. This is the first study conducted on systematically screening the polymorphisms of the OCTN2 gene in the Han Chinese with such large sample. Our study established the profile (variants and frequencies) of OCTN2 polymorphisms in the Chinese Han population. These data can serve as a baseline for larger studies on the mechanism and effects of OCTN2 polymorphism in Asian populations. It should also provide important instructions for the advance of personalized medicine, and facilitate further drug and detecting chip discovery.2. The PCR-DHPLC platform for detection of OCTN2 gene provides a good and feasible methods for corresponding examination. Identification of mutation with DHPLC shows relatively high accuracy and sensitivity, thus can improve the efficiency of DNA sequencing. The present PCR/DHPLC method is a semi-automated, fast, effective, economic and straightforward assay, which provides a routine tool in genetic screening and investigation.3. Screening are being performed in larger samples. Moreover, functional analysis and clinicla trials are to be carried out.