DNA methylation enzymes in the kidneys of male and female BTBR ob/ob mice

Abstract

Diabetic kidney disease (DKD) is the leading cause of the end-stage renal disease. Recent studies have shown that epigenetic modifications contribute to alterations in gene expression and the development of DKD. This study aimed to show an expression profile of key DNA (de)methylation enzymes (DNMT, TET proteins) and their differences between sexes under obesity and diabetic condition. Male and female black and tan brachyury (BTBR) ob/ob mice and their corresponding wild-type littermates (BTBR WT) were studied until 16 weeks of age. Metabolic parameters, kidney morphophysiology and the expression of fibrotic markers and epigenetic enzymes were studied in whole kidney tissue or specifically in the glomerulus. The results showed sexual dimorphism in the development of metabolic disease and in kidney morphophysiology. Female mice have a different profile of DNMTs expression in both WT and obese/diabetic condition. Furthermore, metabolic condition negatively modulated the glomerular expression of TET1 and TET3 only in females. To our knowledge, this is the first study that shows a kidney profile of the expression of key (de)methylation enzymes, DNMTs and TETs, in the BTBR ob/ob experimental model of DKD and its association with sex. The knowledge of this epigenetic profile may help future research to understand the pathophysiology of DKD in males and females.

Keywords: DNMT; TET; chronic kidney disease; epigenetics; obesity and diabetes.

Figure 1

The development of obesity and diabetes and their effect on kidney function and morphology is different between males and females. Effects of sex and metabolic condition in BTBR mice on body weight/week (A), body weight at 16^th week (B), body weight gain (C), blood glucose levels at 16^th week (D), urine albumin (E, F), albumin to creatinine ratio (G), kidney weight (H), glomerular area (I), PAS-positive glomerular area (J), and glomerular morphology (K). p values corresponding to the independent effect of metabolic condition or sex are specified in the graphs. p values of two-way ANOVA followed by Tukey’s multiple comparisons test are as follows: ^&&&&p<0.0001 versus the respective WT controls; ⁺p<0.05 versus male BTBR ob/ob; ^##p<0.01 versus male BTBR WT. The values are the mean ± SD (n = 4 - 7). (E): Representative full-length silver-stained gel of urine samples. (K): Kidney slices were stained with Periodic acid–Schiff for morphological analysis and images were captured using NIS-Elements software coupled to a light microscope equipped with a 40x objective. Bars = 50 μm. WT, wild type; kDa, protein size; BSA, bovine serum albumin; ACR, albumin to creatinine ratio. **p< 0.01; ***p< 0.001, ****p<0.0001.

Figure 2

Obesity and diabetes reduce WT1 positive cells in both males and females. Effect of sex and metabolic condition on WT1 positive cells per glomerulus. Representative images of WT1 and DAPI staining (A) and glomerular WT1 positive cells quantitation (B). The values are the mean ± SD (n = 3). Immunofluorescence images were captured using NIS-Elements software coupled to a light microscope equipped with a 40x objective at laser excitation of 488 nm (Eclipse 80i). Bars = 50 μm. WT, wild type. p values corresponding to the independent effect of metabolic condition or sex are specified in the graphs.

Figure 3

Obesity and diabetes increase whole kidney TGF-β1 content and glomerular expression of collagen IV in both males and females. Effects of sex and metabolic condition in BTBR mice on Tgfb1 (A), Tgfbr1 (B), and Tgfbr2 (C) transcript levels, TGF-β1 protein expression (D, E), collagen IV transcript levels (G), and glomerular collagen IV content (F, H). The values are the mean ± SD (n = 3 - 7). Immunofluorescence images were captured using NIS-Elements software coupled to a light microscope equipped with a 40x objective at laser excitation of 488 nm (Eclipse 80i). Bars = 50 μm. WT, wild type; AU, arbitrary units; Tgfb1, TGF-β1; Tgfbr1, TGF-β receptor 1; Tgfbr2, TGF-β receptor 2; Col4a1, collagen IV. p values corresponding to the independent effect of metabolic condition or sex are specified in the graphs.

Figure 4

Female BTBR ob/ob mice exhibit decreased glomerular expression of epigenetic enzyme DNMT3A. Effects of sex and metabolic condition in BTBR mice on Dnmt1 (A), Dnmt3a (B), and Dnmt3b (C) transcripts levels and on total glomerular expression of DNMT3a (D, E). The values are the mean ± SD (n = 3 - 7). Immunohistochemistry images were captured using NIS-Elements software coupled to a light microscope equipped with a 40x objective (Eclipse 80i). Bars = 50 μm. WT, wild type; Dnmt1, DNA methyltransferase 1; Dnmt3a, DNA methyltransferase 3 alpha; Dnmt3b, DNA methyltransferase 3 beta. p values corresponding to the independent effect of metabolic condition or sex are specified in the graphs. ^*p<0.05 versus male BTBR WT; ^**p<0.05 versus female BTBR WT.

Figure 5

Obesity and diabetes negatively modulate TET protein expression in females. Effects of sex and metabolic condition in BTBR mice on Tet1 (A), Tet2 (B), and Tet3 (C) transcript levels and on total glomerular fluorescence of TET1 (D, F) and TET3 (E, G). The values are the mean ± SD (n = 5 - 7). Immunofluorescence images were captured using NIS-Elements software coupled to a light microscope equipped with a 40x objective at laser excitation of 543 nm (Eclipse 80i). Bars = 50 μm. WT, wild type; AU, arbitrary units; Tet1, Tet Methylcytosine Dioxygenase 1; Tet2, Tet Methylcytosine Dioxygenase 2; Tet3, Tet Methylcytosine Dioxygenase 3. p values corresponding to the independent effect of metabolic condition or sex are specified in the graphs. ^**p<0.05 versus male BTBR WT; ^*p<0.05 versus female BTBR WT.