Searching for new biomarkers of diabetic nephropathy in Malaysian subjects with type 2 diabetes

Abstract

According to the latest National Health and Morbidity Survey reports in year 2011, the prevalence of type 2 diabetes (T2D) in Malaysia has risen to 20.8%, much faster than expected. Of concern, 57% of T2D patients in Malaysia had diabetic nephropathy (DN). T2D and DN are complex diseases, in which genetic and environmental factors have been reported to play an important role. In this thesis, we collected a total of 1142 blood samples from a cohort of Malaysian subjects, including normal glucose tolerance (NGT) subjects and T2D patients with or without DN and conducted genetic, epigenetic and protein analyses. The objectives of this thesis were to improve our knowledge for better understanding of the pathogenesis of T2D and DN, and to search for new biomarker of the disease.

We investigated two members of solute carrier (SLC) family. SLC12A3 is a sodium/chloride transporter in kidneys. In Study I, we found that Arg913Gln polymorphism in the SLC12A3 gene was associated with T2D and DN in a Malaysian cohort. The allele Arg913 conferred the susceptibility risk to the diseases. We also found that slc12a3 led to structural abnormality of kidney pronephric distal duct at 1-cell stage by knockdown of zebrafish ortholog. Slc12a3 mRNA and protein expression levels were upregulated in kidneys of db/db mice from 6, 12, and 26 weeks at the age. This study provided evidence suggesting that SLC12A3 may be a new biomarker for DN. To evaluate this biomarker, analyzing plasma or serum SLC12A3 levels with advanced protein analysis technique and prospective study in the patients with T2D and DN have been taken into our future research plan. SLC30A8 is a zinc efflux transporter and is highly expressed in the pancreas, particularly in alpha, beta and PP cells of the islets of Langerhans. In Study II, we replicated the genetic association of SLC30A8 polymorphisms with T2D and further analyzed DNA methylation of this gene. Results demonstrated that increased DNA methylation levels of this gene were associated with T2D but not with DN. This study thus provided evidence that SLC30A8 has epigenetic effects in T2D.

Recent research has been implicated that the inflammation may be a key pathophysiological mechanism in DN. Intercellular adhesion molecule 1 (ICAM-1) is an acute phase marker of inflammation. In Study III, we found that the ICAM1 K469E(A/G) polymorphism was significantly associated with DN. The plasma ICAM-1 levels were increased in T2D patients with DN compared with the patients without DN. Among T2D patients with DN, the carriers with heterozygous genotype had higher plasma ICAM-1 levels than both homozygous carriers. Therefore, analyzing ICAM-1 protein according to the ICAM1 K469E(A/G) polymorphism genotypes may be useful for predicting susceptibility to DN. Pentraxin 3 (PTX3) is an acute-phase glycoprotein and a soluble receptor acting as an opsonin. This protein is expressed in vascular endothelial cells and macrophages and its level may reflect more directly the inflammatory status of the vasculature. In Study IV, we investigated the plasma PTX3 levels and found that decreased plasma PTX3 levels were associated with T2D and DN in Malay men.

Taking together, we have analyzed four candidate genes of SLC12A3, SLC30A8, ICAM1 and PTX3 in Malaysian subjects with T2D and DN. Results may provide useful information for better understanding of the pathogenesis of the diseases. We shall add our effort to analyze SLC12A3 protein variation in diabetes patients and to further evaluate this molecule as a new biomarker for DN.