Diet Significantly Influences the Immunopathology and Severity of Kidney Injury in Male C57Bl/6J Mice in a Model Dependent Manner.

Diet is a leading causative risk factor for morbidity and mortality worldwide, yet it is rarely considered in the design of preclinical animal studies. Several of the nutritional inadequacies reported in Americans have been shown to be detrimental to kidney health; however, the mechanisms responsible are unclear and have been largely attributed to the development of diabetes or hypertension. Here, we set out to determine whether diet influences the susceptibility to kidney injury in male C57Bl/6 mice. Mice were fed a standard chow diet, a commercially available "Western" diet (WD), or a novel Americanized diet (AD) for 12 weeks prior to the induction of kidney injury using the folic acid nephropathy (FAN) or unilateral renal ischemia reperfusion injury (uIRI) models. In FAN, the mice that were fed the WD and AD had worse histological evidence of tissue injury and greater renal expression of genes associated with nephrotoxicity and monocyte infiltration as compared to mice fed chow. Mice fed the AD developed more severe renal hypertrophy following FAN, and gene expression data suggest the mechanism for FAN differed among the diets. Meanwhile, mice fed the WD had the greatest circulating interleukin-6 concentrations. In uIRI, no difference was observed in renal tissue injury between the diets; however, mice fed the WD and AD displayed evidence of suppressed inflammatory response. Taken together, our data support the hypothesis that diet directly impacts the severity and pathophysiology of kidney disease and is a critical experimental variable that needs to be considered in mechanistic preclinical animal studies.

High-resolution methylation polymerase chain reaction for fragile X analysis: evidence for novel FMR1 methylation patterns undetected in Southern blot analyses.

PURPOSE: Fragile X syndrome is associated with the expansion of CGG trinucleotide repeats and subsequent methylation of the FMR1 gene. Molecular diagnosis of fragile X currently requires Southern blot analysis to assess methylation. This study describes the evaluation of a polymerase chain reaction-only workflow for the determination of methylation status across a broad range of FMR1 genotypes in male and female specimens. METHODS: We evaluated a novel method that combines allele-specific methylation polymerase chain reaction and capillary electrophoresis with eight cell line and 80 clinical samples, including 39 full mutations. Methylation status was determined using a three-step workflow: (1) differential treatment of genomic DNA using a methylation-sensitive restriction enzyme; (2) polymerase chain reaction with two sets of dye-tagged primers; and (3) amplicon sizing by capillary electrophoresis. All samples were analyzed by both methylation polymerase chain reaction and Southern blot analysis. RESULTS: FMR1 methylation status and CGG repeat sizing were accurately and reproducibly determined in a set of methylation controls and genomic DNA samples representing a spectrum of CGG repeat lengths and methylation states. Moreover, methylation polymerase chain reaction revealed allele-specific methylation patterns in premutation alleles that were unobtainable using Southern blot analysis. CONCLUSIONS: Methylation polymerase chain reaction enabled high throughput, high resolution, and semiquantitative methylation assessments of FMR1 alleles, as well as determinations of CGG repeat length. Results for all samples were concordant with corresponding Southern blot analyses. As a result, this study presents a polymerase chain reaction-based method for comprehensive FMR1 analysis. In addition, the identification of novel methylation mosaic patterns revealed after polymerase chain reaction and capillary electrophoresis may be relevant to several FMR1 disorders.