Thyroid hormone deficiency alters expression of acid-base transporters in rat kidney

Abstract

Hypothyroidism in humans is associated with incomplete distal renal tubular acidosis, presenting as the inability to respond appropriately to an acid challenge by excreting less acid. Here, we induced hypothyroidism in rats with methimazole (HYPO) and in one group substituted with l-thyroxine (EU). After 4 wk, acid-base status was similar in both groups. However, after 24 h acid loading with NH(4)Cl HYPO rats displayed a more pronounced metabolic acidosis. The expression of the Na(+)/H(+) exchanger NHE3, the Na(+)-phosphate cotransporter NaPi-IIa, and the B2 subunit of the vacuolar H(+)-ATPase was reduced in the brush-border membrane of the proximal tubule of the HYPO group, paralleled by a lower abundance of the Na(+)/HCO(3)(-) cotransporter NBCe1 and a higher expression of the acid-secretory type A intercalated cell-specific Cl(-)/HCO(3)(-) exchanger AE1. In contrast to control conditions, the expression of NBCe1 was increased in the HYPO group during metabolic acidosis. In addition, net acid excretion was similar in both groups. The relative number of type A intercalated cells was increased in the connecting tubule and cortical collecting duct of the HYPO group during acidosis. Thus thyroid hormones modulate the renal response to an acid challenge and alter the expression of several key acid-base transporters. Mild hypothyroidism is associated only with a very mild defect in renal acid handling, which appears to be mainly located in the proximal tubule and is compensated by the distal nephron.

The Anion Exchanger Pendrin (SLC26A4) and Renal Acid-base Homeostasis

“…Moreover, the relative percentage of pendrin positive intercalated cells was lower in human kidney (approx. 30 %) [24] than in rodent kidney where a majority of intercalated cells in the CNT and CCD are pendrin positive [5][6][25][26].…”

“…Inappropriately high expression of pendrin was observed in rats treated with the calcineurin inhibitor FK506 (tacrolimus) and loaded with NH 4 Cl [26]. In contrast, the susceptibility to develop a more pronounced metabolic acidosis in hypothyroid rats is not associated with dysregulated pendrin expression [25]. The interpretation of these results is complicated by the fact that most conditions are associated with hyperchloremia and increased urinary chloride excretion causing downregulation of pendrin expression per se.…”

Induction of Metabolic Acidosis with Ammonium Chloride (NH₄Cl) in Mice and Rats – Species Differences and Technical Considerations

“…50 μg of crude membrane proteins were solubilised in Laemmli sample buffer and SDS-PAGE was performed on 10 % polyacrylamide gels. Immunoblotting and image analysis was performed as described previously [23,28]. The primary antibodies used were: rabbit anti aquaporin 2 (1:5000) (kindly provided by J. Loffing, Institute of Anatomy, University of Zurich, Switzerland [29], rabbit anti aquaporin 3 (1:200) (Alomone Labs, Jerusalem, Israel), rabbit anti pSer256-AQP2 1:3000 (kindly provided by Dr. Soren Nielsen, University of Aarhus, Denmark), rabbit anti pSer261-AQP2, 1:2000 (Lifespan Biosciences, Seattle, WA, USA), and mouse monoclonal anti--actin antibody (42 kD; Sigma, St. Louis, MO; 1:5000); secondary antibodies used were: donkey anti-rabbit or sheep anti-mouse antibodies linked to horseradish peroxidase (1:10000) (GE Healthcare, Little Chalfont Buckinghamshire, UK) or goat anti-rabbit antibody (1:5000) linked to alkaline phosphatase (Promega, USA).…”

Haploinsufficiency of the Mouse Atp6v1b1 Gene Leads to a Mild Acid-Base Disturbance with Implications for Kidney Stone Disease

“…Sections were then treated with 1% SDS/PBS during 5 min, washed with PBS, and blocked with 1% bovine serum albumin/PBS. After blocking, sections were incubated with primary antibodies (guinea pig anti-AE1 1:500 [33] [35], and goat anti AQP-2 (Santa Cruz Biotechnology), 1:1000) diluted in PBS for incubation over night at 4°C. Then, samples were washed 3 times with PBS/NaCl (18g NaCl/PBS), and incubated with the secondary antibodies (donkey anti-rabbit 594, donkey anti-goat 488 and donkey anti-guinea pig 647 (Molecular Probes) at 1:1000 and DAPI, 1:400) during 2 hours at room temperature.…”

“…For immunoblotting, proteins were transferred electrophoretically to polyvinylidene fluoride membranes (Immobilon-P, Millipore Corp., Bedford, MA, USA). After blocking with 5 % milk powder in Tris-buffered saline/0.1% Tween-20 for 60 min, blots were incubated with primary antibodies: rabbit anti-mouse Atp6v1b1 (B1) (1:5, 000) [37], rabbit anti-ATP6v1b2 (B2) (1:5, 000) [35], rabbit anti-ATP6v0a4 [34], (1:5, 000), rabbit anti-ATP6v1A 1:2, 000, rabbit anti-pendrin (1:2, 000) [29], rabbit anti-AE1 (1:2, 000) [33], rabbit anti-AQP2 (1:2, 000) and rabbit anti-NKCC2 (1:5, 000) (kind gift of J. Loffing, University of Zurich) [38], rabbit anti-NBCe1 (1:1, 500) (Proteintech, United Kingdom), rabbit anti-NHE3 (1:2, 000) (StressMarq Biosciences, Inc, Victoria, British Columbia) and mouse monoclonal anti-β-actin antibody (Sigma, St. Louis, MO; 1:20, 000) overnight at 4°C. After washing and blocking with 5 % milk powder for 60 min, membranes were incubated for 2 h at room temperature with secondary goat anti-rabbit or donkey anti- …”