Salt Loading Promotes Kidney Injury via Fibrosis in Young Female Ren2 Rats

Abstract

Background/aims: It is increasingly recognized that there is sexual dimorphism in kidney disease progression; however, this disparity is lost in the presence of diabetes where women progress at a similar rate to men. The renin-angiotensin-aldosterone system (RAAS) is known to regulate diabetes-induced kidney injury, and recent literature would suggest that gender differences exist in RAAS-dependent responses in the kidney. In this regard, these gender differences may be overcome by excessive salt intake. Thereby, we hypothesized that salt would promote proteinuria in transgenic female rats under conditions of excess tissue angiotensin (Ang) II and circulating aldosterone.

Materials and methods: We utilized young female transgenic (mRen2)27 (Ren2) rats and Sprague-Dawley (SD) littermates and fed a high-salt diet (4%) over 3 weeks.

Results: Compared to SD and Ren2 controls, female Ren2 rats fed a high-salt diet displayed increases in proteinuria, periarterial and interstitial fibrosis as well as ultrastructural evidence of basement membrane thickening, loss of mitochondrial elongation, mitochondrial fragmentation and attenuation of basilar canalicular infoldings. These findings occurred temporally with increases in transforming growth factor-β but not indices of oxidant stress.

Conclusions: Our current data suggest that a diet high in salt promotes progressive kidney injury as measured by proteinuria and fibrosis associated with transforming growth factor-β under conditions of excess tissue Ang II and circulating aldosterone.

Keywords: Angiotensin II; Fibrosis; Proteinuria; Reactive oxygen species; Transgenic (mRen2)27 rat.

Figures

Fig. 1

Salt induces protein excretion in female transgenic Ren2 rats. Proteinuria as determined by the urine protein:creatinine ratio is shown. Values are means ± SE. * p † p < 0.05 when compared to Ren2 controls.

Fig. 2

Salt induces kidney periarterial and interstitial fibrosis in female transgenic Ren2 rats. Representative bright field images of immunostaining with picrosirius red of a periarterial fibrosis and b interstitial fibrosis with corresponding measures of intensity to the right. Values are means ± SE. * p < 0.05 when compared to SD controls; † p < 0.05 when compared to Ren2 controls. Scale bars = 50 µm.

Fig. 3

Salt induces ultrastructural remodeling of PTCs in female transgenic Ren2 rats. There is a similar morphology of the S1 basilar region of the proximal tubules in control SD rats (SD-C) as well as in Ren2 (Ren2-C) rats with elongation of mitochondria and canalicular infoldings (arrows). Salt had little effect in the SD; however, in the Ren2 (Ren2-HS), we observed increases in basement membrane thickening (BM), loss of mitochondrial elongation, and attenuation of canalicular infoldings compared to the other groups. Scale bars = 1 µM; magnification ×2,000.

Fig. 4

Salt does not induce ROS generation and 3-NT formation in female transgenic Ren2 rats. a ROS generation as determined by chemiluminescence. b Representative sections for 3-NT, a marker of peroxynitrite (ONOO−) formation with corresponding measures of intensity. RLUs = Relative light units. Values are means ± SE. * p < 0.05 when compared to SD controls. Scale bars = 50 µm.

Fig. 5

Salt induces TGFβ-1 in female transgenic Ren2 rats. Representative images of immunohistochemical analysis with corresponding measures of intensity to the right. Values are means ± SE. * p † p < 0.05 when compared to Ren2 controls. Scale bars = 50 µm.