Abstract
To assess whether metabolic acidosis per se regulates rBSC-1, the rat medullary thick ascending limb (MTAL) apical Na+-K+(NH4+)-2Cl- cotransporter, rat MTALs were incubated for 16 h in an acid 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's medium. Cotransport activity was estimated in intact cells and membrane vesicles by intracellular pH and 22Na+ uptake measurements, respectively; rBSC-1 protein was quantified by immunoblotting analysis and mRNA by quantitative reverse transcription-polymerase chain reaction. As compared with incubation at pH approximately 7.35, acid incubation (pH approximately 7.10) up-regulated by 35-100% rBSC-1 transport activity in cells and membrane vesicles, and rBSC-1 protein and mRNA abundance. In contrast, acid incubation did not alter alkaline phosphatase and Na+/K+-ATPase enzyme activities or beta-actin protein abundance. After 3 h of in vivo chronic metabolic acidosis (CMA) rBSC-1 mRNA abundance increased in freshly harvested MTALs, which was accompanied after 1-6 days of CMA with enhanced rBSC-1 protein abundance. These results demonstrate that both in vivo and in vitro CMA stimulate rBSC-1 expression, which would contribute to the adaptive increase in MTAL absorption and urinary excretion of NH4+ in response to CMA.
Highlights
Increased urinary NH4ϩ excretion, which augments acid excretion, has long been recognized to be quantitatively the major compensatory response of the kidney against chronic metabolic acidosis (CMA)1 [1]
Present results demonstrate for the first time that a long term in vitro incubation of freshly harvested medullary thick ascending limb (MTAL) in an acid medium increases transport activity and protein and mRNA abundance of rBSC-1, the apical Naϩ-Kϩ(NH4ϩ)-2ClϪ cotransporter of the rat MTAL
This is confirmed in the present study in which rBSC-1 protein and mRNA were detected in this preparation
Summary
In Vivo CMA—CMA was induced in male Sprague-Dawley rats in two ways. In a first series, experimental rats were given 0.28 M NH4Cl in distilled drinking water (8 –11 mmol/day), whereas control rats from the same shipment drank distilled water for 6 days; rats were housed two or three per cage and were allowed free access to standard rat chow and drinking solution up to the time of the experiments, at which times the rats were anesthetized by intraperitoneal injection of sodium pentobarbital. 0.5 group, or 7.5 mM Tris, 15 mM NaHCO3, pH ϳ7.10 when gassed with 95% O2, 5% CO2 for the acid group (we have checked that a difference of 25 mosmol/liter with mannitol at pH 7.35 had no significant effect on rBSC-1 mRNA abundance by the method described below (quantitative reverse transcription-PCR; rBSC-1 mRNA was 1.27 Ϯ 0.14 amol/100 ng of RNAtot in control and 1.31 Ϯ 0.11 with additional 25 mM mannitol; n ϭ 3 for both)). Control experiments with affinity-purified antirBSC-1 antibody pre-adsorbed with fusion protein gave no labeling for Both the ϳ150-kDa and high molecular weight bands (Fig. 1); the rBSC-1 bands were not observed with the pre-immune rabbit serum (not shown). Statistical significance between experimental groups was assessed by Student’s paired or unpaired t test or by one-way ANOVA completed by a t test using the within-groups residual variance of ANOVA, as appropriate
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
