A C Girardi1, S M Titan, G Malnic, N A Rebouças. 1. Instituto de Ciências Biomédicas, Departamento de Fisiologia e Biofísica, Universidade de São Paulo, São Paulo, Brazil.
Abstract
BACKGROUND: The most abundant Na+/H+ exchanger in the apical membrane of proximal tubules is the type 3 isoform (NHE3), and its activity is acutely inhibited by parathyroid hormone (PTH). In the present study, we investigate whether changes in protein abundance as well as in mRNA levels play a significant role in the long-term modulation of NHE3 by PTH. METHODS: Three groups of animals were compared: (1) HP: animals submitted to hyperparathyroidism by subcutaneous implantation of PTH pellets, providing threefold basal levels of this hormone (2.1 U. h-1); (2) control: sham-operated rats in which placebo pellets were implanted; (3) PTX: animals submitted to hypoparathyroidism by thyroparathyroidectomy followed by subcutaneous implantation of thyroxin pellets, which provided basal levels of thyroid hormone. After eight days, we measured bicarbonate reabsorption in renal proximal tubules by in vivo microperfusion. NHE3 activity was also measured in brush border membrane (BBM) vesicles by proton dependent uptake of 22Na. NHE3 expression was evaluated by Northern blot, Western blot and immunohistochemistry. RESULTS: Bicarbonate reabsorption in renal proximal tubules was significantly decreased in HP rats. Na+/H+ exchange activity in isolated BBM vesicles was 6400 +/- 840, 9225 +/- 505, and 12205 +/- 690 cpm. mg-1. 15 s-1 in HP, sham, and PTX groups, respectively. BBM NHE3 protein abundance decreased 39.3 +/- 8.2% in HP rats and increased 54.6 +/- 7.8% in PTX rats. Immunohistochemistry showed that expression of NHE3 protein in apical BBM was decreased in HP rats and was increased in PTX rats. Northern blot analysis of total kidney RNA showed that the abundance of NHE3 mRNA was 20.3 +/- 1.3% decreased in HP rats and 27. 7 +/- 2.1% increased in PTX. CONCLUSIONS: Our results indicate that the chronic inhibitory effect of PTH on the renal proximal tubule NHE3 is associated with changes in the expression of NHE3 mRNA levels and protein abundance.
BACKGROUND: The most abundant Na+/H+ exchanger in the apical membrane of proximal tubules is the type 3 isoform (NHE3), and its activity is acutely inhibited by parathyroid hormone (PTH). In the present study, we investigate whether changes in protein abundance as well as in mRNA levels play a significant role in the long-term modulation of NHE3 by PTH. METHODS: Three groups of animals were compared: (1) HP: animals submitted to hyperparathyroidism by subcutaneous implantation of PTH pellets, providing threefold basal levels of this hormone (2.1 U. h-1); (2) control: sham-operated rats in which placebo pellets were implanted; (3) PTX: animals submitted to hypoparathyroidism by thyroparathyroidectomy followed by subcutaneous implantation of thyroxin pellets, which provided basal levels of thyroid hormone. After eight days, we measured bicarbonate reabsorption in renal proximal tubules by in vivo microperfusion. NHE3 activity was also measured in brush border membrane (BBM) vesicles by proton dependent uptake of 22Na. NHE3 expression was evaluated by Northern blot, Western blot and immunohistochemistry. RESULTS:Bicarbonate reabsorption in renal proximal tubules was significantly decreased in HPrats. Na+/H+ exchange activity in isolated BBM vesicles was 6400 +/- 840, 9225 +/- 505, and 12205 +/- 690 cpm. mg-1. 15 s-1 in HP, sham, and PTX groups, respectively. BBM NHE3 protein abundance decreased 39.3 +/- 8.2% in HPrats and increased 54.6 +/- 7.8% in PTXrats. Immunohistochemistry showed that expression of NHE3 protein in apical BBM was decreased in HPrats and was increased in PTXrats. Northern blot analysis of total kidney RNA showed that the abundance of NHE3 mRNA was 20.3 +/- 1.3% decreased in HPrats and 27. 7 +/- 2.1% increased in PTX. CONCLUSIONS: Our results indicate that the chronic inhibitory effect of PTH on the renal proximal tubule NHE3 is associated with changes in the expression of NHE3 mRNA levels and protein abundance.
Authors: Luciana C Veiras; Adriana C C Girardi; Joshua Curry; Lei Pei; Donna L Ralph; An Tran; Regiane C Castelo-Branco; Nuria Pastor-Soler; Cristina T Arranz; Alan S L Yu; Alicia A McDonough Journal: J Am Soc Nephrol Date: 2017-08-03 Impact factor: 10.121
Authors: Wanling Pan; Jelena Borovac; Zachary Spicer; Joost G Hoenderop; René J Bindels; Gary E Shull; Michael R Doschak; Emmanuelle Cordat; R Todd Alexander Journal: Am J Physiol Renal Physiol Date: 2011-09-21