CXCL4L1 Promoter Polymorphisms Are Associated with Improved Renal Function in Type 1 Diabetes.

Inflammation is a recognized mechanism underlying the pathogenesis of renal dysfunction in type 1 diabetes. Evidence suggests that genetic factors modulate the expression of inflammatory genes, which may lead to an enhanced predisposition to developing renal complications in patients with diabetes. In this study, we examined 55 genetic variants from 16 human candidate inflammatory genes for associations with renal function expressed as the estimated glomerular filtration rate in 1540 participants from the Genetics of Kidneys in Diabetes study. We observed protective associations between three variants in the CXCL4L1 promoter (rs872914/A, rs941757/G, and rs941758/A) and renal function in patients with type 1 diabetes. In reporter gene assays, all three variants increased CXCL4L1 promoter activity in HEK293 cells stimulated with IL-1 and TNF-α. We performed overexpression and knockdown experiments in primary human mesangial cells to examine the glucose-mediated regulation of endogenous CXCL4L1 gene expression and signaling pathways. The mRNA and protein levels of CXCL4L1 increased in response to high glucose (30 mM) treatment. Overexpression of CXCL4L1 increased the endogenous expression of SMAD7 and IκBα, which are key inhibitory factors in renal inflammation. Knockdown of CXCL4L1 expression also resulted in reduced levels of SMAD7 and IκBα. Our findings suggest that CXCL4L1 promoter variants may protect against the development of renal inflammation in diabetes by increasing CXCL4L1 expression, which in turn activates the anti-inflammatory SMAD7 and IκBα factors in mesangial cells.

RNA mapping protocols: Northern blot and amplification of cDNA ends.

Mapping of transcribed mRNA and protein coding sequences is the initial step in functional characterization of genomic sequences. There are several techniques to identify transcribed sequences; however, Northern blot analysis remains a standard method for detection and quantification of mRNA levels despite the relatively recent availability of other methods, such as RT-PCR (reverse transcriptase polymerase chain reaction), which are also sensitive, accurate, cost-effective, and simple to use. Northern blot analysis provides a direct relative comparison of message abundance between samples on a single membrane. It is also the preferred method for determining transcript size and detecting alternatively spliced isoforms. Other methods utilizing RT-PCR allow detection and cloning of isolated complementary DNA (cDNA) domains and ends from cellular RNA or commercially available cDNA libraries.