Effect of thyroid hormone on the postnatal renal expression of NHE8

Abstract

We previously demonstrated that there are developmental changes in proximal tubule Na(+)/H(+) exchanger (NHE) activity. There is a maturational increase in postnatal brush-border membrane (BBM) vesicle NHE3 protein abundance and decrease in NHE8 protein abundance. The purpose of this study was to determine whether thyroid hormone plays a role in the rat renal maturational isoform switch from NHE8 to NHE3 and whether thyroid hormone regulates NHE8. Administration of thyroid hormone to neonatal rats, before the normal postnatal increase in serum thyroid hormone levels at 3 wk of age, resulted in a premature increase in NHE3/beta-actin BBM protein abundance and mRNA abundance. Thyroid hormone also caused a premature decrease in BBM NHE8/beta-actin protein abundance, whereas there was no change in mRNA expression (standardized to 28s). Rats made hypothyroid from birth were studied at 28 days, after the normal maturational increase in thyroid hormone. In these hypothyroid adult rats, the maturational increase in BBM NHE3 protein abundance and NHE3 mRNA expression was prevented. In contrast, the developmental decrease in BBM NHE8 protein abundance was prevented in hypothyroid adults, but mRNA expression was unchanged in hypothyroid rats. To determine whether the effect of thyroid hormone was due to a direct epithelial effect, we studied normal rat kidney cells in culture. We recently showed that this cell line expresses NHE8, but does not express NHE3. Thyroid hormone caused a decrease in surface expression of NHE8, determined by biotinylation, but total cellular abundance remained unchanged. NHE8 activity, measured as the sodium-dependent rate of intracellular pH recovery from an acid load, was less with thyroid treatment than control. In conclusion, thyroid hormone plays a potential role in the developmental isoform change from NHE8 to NHE3 and decreases NHE8 activity.

Fig. 1

Effect of 3,5,3′-triiodothyronine (T₃) on Na⁺/H⁺ exchanger (NHE)8 and NHE3 protein abundance on brush-border membrane vesicles (BBMV) from 7-day-old rats. Rats were administered T₃ (10 μg/100 g body wt) by intraperitoneal injections daily for 4 days starting on day 4 of life. Rats were studied on day 7 of life, 2 h after the last injection. Administration of T₃ before the developmental increase in thyroid hormone resulted in the premature decrease in NHE8 and increase in NHE3 brush-border membrane protein abundance studied by immunoblot analysis.

Fig. 2

Effect of T₃ on NHE8 and NHE3 mRNA abundance from 7-day-old rats. Rats were treated as in Fig. 1. T₃ administration caused an increase in NHE3 but no change in NHE8 mRNA abundance measured using quantitative real-time PCR.

Fig. 3

Effect of hypothyroidism (HypoT) on NHE3 and NHE8 brush-border membrane protein abundance. To prevent the normal developmental increase in thyroid hormone, pregnant rats, nursing mothers, and neonates were given water containing propyl-thiouracil (PTU; Sigma) to prevent the maturational increase in thyroid hormone at the time of weaning. The hypothyroid rats and controls (Cont) were studied at 28 days, a time after the normal maturational increase in thyroid hormone. The hypothyroid rats had a lower brush-border membrane NHE3 and higher NHE8 protein abundance compared with controls. Protein abundance was assessed by immunoblot analysis.

Fig. 4

Effect of hypothyroidism on NHE3 and NHE8 mRNA abundance. Rats were made hypothyroid and studied at 28 days of life as described in Fig. 3. Hypothyroid rats had reduced NHE3 mRNA abundance but comparable NHE8 mRNA to control rats using RT-PCR.

Fig. 5

Effect of T₃ on NHE8 mRNA abundance in normal rat kidney (NRK) cells. NRK cells express NHE8 on their apical membrane. We examined the effect of 10⁻⁷ M T₃ for 24 h on NHE8 mRNA abundance using RT-PCR. As can be seen, T₃ did not affect NHE8 mRNA abundance.

Fig. 6

Effect of T₃ on NHE8 surface protein abundance in NRK cells. Incubation of NRK cells with T₃ (10⁻⁷ M) for 24 h resulted in reduced surface NHE8 abundance determined by biotinylation and immunoblotting.

Fig. 7

Effect of T₃ on NHE8 activity in NRK cells. NRK cells express NHE8 but not NHE3 on their apical membrane (42). T₃ (10⁻⁷ M for 24 h) reduced NHE activity assayed as the sodium-dependent pH recovery from cell acidification using the K⁺-nigericin technique.