Hypothyroidism Increases Osmotic Water Permeability (Pf) in the Developing Renal Brush Border Membrane

Abstract

The osmotic water permeability (P f ) of the rabbit proximal tubule brush border membrane vesicles (BBMV) increases during maturation and is mediated by an increase in aquaporin-1 (AQP1) protein expression. Serum thyroid hormone levels increase after birth and have been shown to play a role in the maturation of other renal transport functions. We examined the hypothesis that thyroid hormone plays a role in the maturational increase in osmotic water permeability. Hypothyroidism was induced by addition of 0.1% propylthiouracil (PTU) to the drinking water of pregnant rabbits (starting 9 d before delivery) and was continued until the rabbits were studied as adults (9-11 wk). Some animals received thyroid hormone replacement by daily injection with triiodothyronine (T 3 ; 10 g/100 g body weight) for three days before study. P f was found to be higher in BBMV from hypothyroid (82.7 Ϯ 5.5 m/s) than from euthyroid (60.6 Ϯ 4.0 m/s) and T 3 -replacement rabbits (69.0 Ϯ 5.0 m/s) (p Ͻ 0.05). The activation energy (E a ; in kcal/deg⅐mol) of P f was not different among the three experimental groups (euthyroid 5.6 Ϯ 0.9, hypothyroid 4.9 Ϯ 0.8, T 3 -replacement 5.0 Ϯ 1.0; p ϭ NS), nor was the percentage mercury inhibition of P f (euthyroid 66.5 Ϯ 5.3, hypothyroid 74.2 Ϯ 3.2 and T 3 -replacement 73.1 Ϯ 4.3; p ϭ NS). AQP1 expression, measured by immunoblotting, was highest in BBMV from hypothyroid rabbits (p Ͻ 0.05). Membrane fluidity, measured as steady-state generalized polarization (GP) of Laurdan, which is inversely related to membrane fluidity, was significantly different between the three groups (GP: euthyroid 0.307 Ϯ 0.004, hypothyroid 0.271 Ϯ 0.004 and T 3 -replacement 0.287 Ϯ 0.003; for all p Ͻ 0.05). These data demonstrate that the maturational increase in thyroid hormone levels is not responsible for the maturational increase in water transport. Surprisingly, congenital hypothyroidism in rabbits is associated with an increased P f when rabbits are studied as adults. The higher P f in hypothyroid adult rabbits is due to a higher expression of AQP1 protein as well as a greater membrane fluidity than in euthyroid rabbits. The proximal tubule reabsorbs approximately two-thirds of the glomerular ultrafiltrate (1). This occurs in a nearly isoosmotic manner due to the high water permeability of this segment. We have previously demonstrated that the osmotic water permeability of both apical (brush border) and basolateral membranes increased during postnatal development (2, 3). In both membranes, this was accompanied by an increase in aquaporin-1 (AQP1) expression; the predominant water channel in proximal tubular membranes (4).The factors that are responsible for the maturational change in proximal tubule water transport and AQP 1 expression remain unknown. Thyroid hormone levels rise postnatally (5, 6) and have been shown to be a factor in developmental changes in proximal tubular function, including NaCl transport (7), Na ϩ /H ϩ -antiporter activity (5) and phosphate transport (6). The role of thyroid hormone with regard to ...

Increased Renal Proximal Convoluted Tubule Transport Contributes to Hypertension in Cyp4a14 Knockout Mice

“…The minced cortex was then homogenized with a Power Gen 125 (Fisher Scientific, Pittsburgh, Pa., USA) homogenizer at 4°C. After addition of 230 µl of 1.0 M MgCl 2 to precipitate cell debris, the homogenate was shaken vigorously for 10 s, every 5 min for 20 min, as previously described [12,13,14]. Subsequently, the homogenate was centrifuged at 2,500 g for 15 min at 4°C.…”

“…Protein was determined in the crude homogenate and brush border membrane vesicles (BBMV) using a BCA protein assay (Pierce, Rockford, Ill., USA). Alkaline phosphatase activity was used to determine the enrichment as described previously [12,13,14]. …”

“…BBMV protein was denatured and separated on a 7.5% polyacrylamide gel as previously described (50 µg/lane) [12,13,14]. The separated proteins within the gel were then transferred to polyvinylidone difluoride membrane overnight at 140 mA at 4°C.…”

Renal Tubular Development

“…Administration of glucocorticoids to neonatal rabbits resulted in an increase in brush border membrane water permeability and aquaporin 1 expression [226]. Of interest, the developmental increase in thyroid hormone was not shown to be a factor mediating these postnatal maturational changes in water transport [227].…”

Ontogeny of water transport in the rabbit proximal tubule

“…In subsequent studies, we have directly measured the neonatal and adult BBMV fluidity using the fluorophore, laurdan [20,21]. These results indicate that the membrane fluidity of the neonatal renal BBMV is indeed higher than the adult BBMV.…”