Btg2 mutation induces renal injury and impairs blood pressure control in female rats

Abstract

Hypertension (HTN) is a complex disease influenced by heritable genetic elements and environmental interactions. Dietary salt is among the most influential modifiable factors contributing to increased blood pressure (BP). It is well established that men and women develop BP impairment in different patterns and a recent emphasis has been placed on identifying mechanisms leading to the differences observed between the sexes in HTN development. The current work reported here builds on an extensive genetic mapping experiment that sought to identify genetic determinants of salt-sensitive (SS) HTN using the Dahl SS rat. BTG antiproliferation factor 2 (Btg2) was previously identified by our group as a candidate gene contributing to SS HTN in female rats. In the current study, Btg2 was mutated using transcription activator-like effector nuclease (TALEN)-targeted gene disruption on the SSBN congenic rat background. The Btg2 mutated rats exhibited impaired BP and proteinuria responses to a high-salt diet compared with wild-type rats. Differences in body weight, mutant pup viability, skeletal morphology, and adult nephron density suggest a potential role for Btg2 in developmental signaling pathways. Subsequent cell cycle gene expression assessment provides several additional signaling pathways that Btg2 may function through during salt handling in the kidney. The expression analysis also identified several potential upstream targets that can be explored to further isolate therapeutic approaches for SS HTN.

Keywords: TALEN; hypertension; renal; salt sensitivity.

Figure 1.

Transcription activator-like effector nuclease (TALEN) targeted exon 1 on rat Btg2 gene. The 10- to 14-day-old pups were ear-punched and DNA was screened for TALEN-induced mutations. Among several mutant founders, one founder animal (m7) harbored a 44-bp deletion (green text) in exon 1 of rat Btg2. Representative sequences showing TALEN target (gray highlight) and editing outcome along with corresponding translated peptide sequences.

Figure 2.

Blood pressure and heart rate measurements by radiotelemetry. Twenty-four measurements of heart rate in female (A) and male (B) Btg2^+/+ and Btg2^−/− rats during low-salt diet (LS) and 20 days of high-salt diet (HS). Bottom figures depict 24-h measurements of mean arterial pressure (MAP) in female (C) and male (D) Btg2^+/+ and Btg2^−/− rats during low-salt diet (LS) and 20 days of high-salt diet (HS). Graphs represent recordings obtained once every 2 min continuously for 24 h. Data are presented as mean shown in circles and SE shown as bars (only depicted in positive direction). Levels of statistical significance were analyzed by two-way ANOVA. *P < 0.05 (strain comparison). BPM, heart rate in beats per minute; F, female; HR, heart rate; M, male.

Figure 3.

Day-night difference in blood pressure and heart rate measurements. Telemetry blood pressure measurements were recorded for 15 s every 2 min and were binned into 12-h light and dark cycle bins for diurnal comparison. Differences between adjacent light and dark cycle 12-h bins were normalized to the mean of the same adjacent 12-h segments. The protocol shown covers 3 days of low-salt (LS) and 20 days of high-salt (HS) diet. Heart rate is plotted for female (A) and male (B) Btg2^+/+and Btg2^−/− rats along with MAP for females (C) and males (D). The plots depict recordings obtained once every 2 min continuously for 12 h. Data are presented as mean shown in circles and SE shown as bars (only depicted in positive direction). Levels of statistical significance were analyzed by two-way ANOVA. *P < 0.05 (strain comparison), #P < 0.05 (strain by time comparison), ‡P < 0.05 (Sidak post hoc). F, female; HR, heart rate; M, male.

Figure 4.

Decreased body weight in female Btg2^−/− compared with Btg2^+/+ rats. Nine-week-old animals were switched to high-salt diet and measured after 2 (12 wk) and 3 (13 wk) wk. Individual data presented with means ± SE in red. Statistical significance was analyzed separately within each sex by two-way ANOVA. #P < 0.05 (strain comparison), *P < 0.05 (Sidak post hoc). BW, body weight; F, female; M, male; n = 6–8/group.

Figure 5.

Impaired renal function in female Btg2^−/− compared with Btg2^+/+ rats. Urinary protein and creatinine were measured in male and female Btg2^+/+ and Btg2^−/− rats following 17 days of high-salt diet. Figures depict female and male urinary protein excretion (A) and urinary protein-to-creatinine ratio (B). Individual data are presented with means ± SE shown in red. Statistical significance was analyzed by unpaired t test within each sex separately. *P < 0.05. F, female; M, male; UCr, Urinary creatinine excretion; UPro, urinary protein excretion.

Figure 6.

Female Btg2^−/− rats exhibit lower nephron density with protected glomeruli compared with Btg2^+/+ rats. Following 3 wk of high-salt diet, rat kidneys were processed and imaged for assessment of glomerulus damage and are presented for comparison within female and male rat strains shown in A. Cortical area normalized glomeruli counts in female and male rats were also determined and results are depicted in B. Individual data are presented with means ± SE shown in red. Statistical significance was analyzed by unpaired t test within each sex separately. *P < 0.05. F, female; M, male; gl, glomeruli.

Figure 7.

Btg2 zygosity distribution in genotyped litters. Heterozygous breeders produced 49 litters with 371 pups that were genotyped before wean. Pup genotype distribution is presented in the chart. Data presented as % of total litter count. Statistical significance was determined by χ²-test. *P < 0.05.