Effects of Cacna1d D307G Mutation on Blood Pressure and Kidney Function in Rats with Salt Loading

Fig. 1.. Establishment of Cacna1d D307G and Cacna1d G307G mutant rats. a Genomic and amino acid sequences in Rattus norvegicus and Homo sapiens; the mutant nucleotides and amino acids are marked yellow. bCacna1d exon 1–51 and the target locus of Cacna1d D307G located on exon 7. c Identification of WT, Cacna1d D307G, and Cacna1d G307G by the Sanger sequencing of tail tissue samples.

Fig. 2.. SBP, plasma ET-1, and plasma Ald levels and kidney pathological staining in three different genotype groups fed with normal-salt diet (NSD). a SBP changes from 10 weeks to 26 weeks in WT, D307G, and G307G groups. Data are presented as mean ± SD, n = 25. *p < 0.05 vs. D307G+NSD group; #p < 0.05 vs. G307G+NSD group. b Plasma ET-1 level in WT, D307G, and G307G groups fed with NSD. c Plasma Ald level in WT, D307G, and G307G groups fed with NSD. d H&E staining (original magnification, ×200) and Masson’s trichrome staining (original magnification, ×400) to assess the structural changes, glomerular sclerosis, and collagen deposition in the three different genotype groups fed with NSD. Total kidney histology scores in the three different genotype groups are displayed in the bar graph. Data are presented as mean ± SD, n = 10; ns, p > 0.05, *p < 0.05, **p < 0.01 vs. WT+NSD group. SD, standard deviation.

Fig. 3.. Measurement of SBP, △SBP, plasma vasoactive substances, and urinary ET-1 excretion level in the three different genotype groups fed with HSD. a Changes in the SBP in rats from 10 weeks to 26 weeks. *p < 0.05 vs. D307G+HSD, #p < 0.05 vs. G307G+HSD. b Mean △SBP changes between the HSD group and NSD group with age-matched rats in the three different genotype groups. c Plasma ET-1 level in WT, D307G, and G307G groups fed with HSD. d Urinary ET-1 excretion level in the three genotype groups fed with HSD. e, f Plasma Ald and plasma renin level in the three genotype groups fed with HSD. All data are shown as mean ± SD, n = 6; ns, p > 0.05, *p < 0.05, **p < 0.01 vs. WT+HSD. SD, standard deviation.

Fig. 4.. Effects of Cacna1d D307G mutation on the renal function and kidney injury in the three different genotype groups fed with HSD. a Kidney and bodyweight ratios in the three different genotype groups fed with HSD. b CCr in the three different genotype groups fed with HSD. c Level of 24-h urinary protein in the three different genotype groups fed with HSD. d Urinary albumin/creatinine ratio in the three different genotype groups fed with HSD. e, f Serum and urine KIM-1 level. Total kidney pathological scores in the three different genotype groups fed with HSD. All data are presented as mean ± SD, n = 6; ns, p > 0.05, *p < 0.05, **p < 0.01 vs. WT+HSD. SD, standard deviation.

Fig. 5.. Effects of Cacna1d D307G mutation on renal tubular epithelial cell apoptosis, CD3+ T lymphocyte infiltration, and inflammatory cytokines expression. a TUNEL staining for renal tubular epithelial cell apoptosis in the three different genotype groups; bright green cells represent apoptotic cells; original magnification, ×400. b Immunohistochemistry staining for CD3+ T lymphocyte infiltration in the renal cortex; dark brown cells marked by black arrows represent CD3+ cells; original magnification, ×400. c Immunohistochemistry staining for CD3+ T lymphocyte infiltration in the renal medulla; dark brown cells marked by black arrows represent CD3+ cells; original magnification, ×400. d Percentage of TUNEL-positive cells. e, f Number of CD3+ cells in the renal cortex and renal medulla, respectively. g, h Monocyte chemoattractant protein (MCP)-1 and IL-6 mRNA expression in the renal cortex and renal medulla, respectively. All data are presented as mean ± SD, n = 6; ns, p > 0.05, *p < 0.05, **p < 0.01 vs. WT+HSD. SD, standard deviation.

Fig. 6.. Effects of Cacna1d D307G mutation on sodium homeostasis and protein expression of ETA and ET-1 in the renal artery; protein expression of ETA and ETB in the renal cortex; protein expression of ETB in the renal medulla; and protein expression of αENaC in the kidney. a Plasma sodium level in the three different genotype groups fed with NSD and HSD. b Urinary sodium excretion in the three different genotype groups fed with NSD and HSD. c Western blot analysis of ETA and ET-1 in the renal artery; GAPDH was used as the internal control. d Western blot analysis of ETA and ETB in the renal cortex; GAPDH was used as the internal control. e Western blot analysis of ETB in the renal medulla; GAPDH was used as the internal control. f, g Western blot analysis of αENaC in the renal cortex and the medulla; β-actin was used as the internal control. All data are presented as mean ± SD, n = 6; ns, p > 0.05, *p < 0.05, **p < 0.01 vs. WT+HSD. SD, standard deviation.

Fig. 7.. Effects of specific ETA receptor blocker, ABT-627, on SBP, CCr, urinary sodium excretion, serum KIM-1 level, and protein expression of the three subunits of ENaC in D307G rats fed with HSD. a SBP changes in WT+HSD and D307G+HSD groups with or without treatment with ABT-627 for 6 weeks. b CCr alterations in WT+HSD and D307G+HSD groups with or without treatment with ABT-627 for 6 weeks. c Urinary sodium excretion level in WT+HSD and D307G+HSD groups with or without treatment with ABT-627 for 6 weeks. d Serum KIM-1 level in WT+HSD and D307G+HSD groups with or without treatment with ABT-627 for 6 weeks. e, f Protein expression level of the three subunits of ENaC in the cortex and medulla for WT+HSD and D307G+HSD with or without treatment with ABT-627 for 6 weeks. All data are presented as mean ± SD, n = 6; ns, p > 0.05, *p < 0.05, **p < 0.01 vs. WT+HSD. SD, standard deviation.