Acute and Chronic Effects of Hyperosmolality on mRNA and Protein Expression and the Activity of Na-K-ATPase in the IMCD

Abstract

We investigated acute and chronic effects of hyperosmolality on mRNA and protein expressions of Na-K-ATPase α and β isoforms and Na-K-ATPase activity in the rat inner medullary collecting duct (IMCD). Incubation of IMCD in hypertonic medium for 30 min reduced the Na-K-ATPase activity by 50%. The Na-K-ATPase activity of dehydrated rats measured in isotonic medium was decreased, and incubation in hypertonic medium did not further decrease the activity. Incubation of IMCD in hypertonic medium for 6 h did not change α₁ mRNA. In contrast, dehydration decreased α₁ subunit mRNA and protein and β₁ protein expressions without changing β₁ mRNA. These data show (1) that acute hyperosmolality decreases Na-K-ATPase activity in IMCD without changing α₁ and β₁ mRNA and (2) that 2 days of dehydration decreased Na-K-ATPase activity by reducing α₁ and β₁ proteins. Thus, the mechanisms for the inhibition of the Na-K-ATPase activity in IMCD is defferent between acute and chronic exposure to hyperosmolality.

Differential Effects of Hyperosmolality on Na-K-ATPase and Vasopressin-Dependent cAMP Generation in the Medullary Thick Ascending Limb and Outer Medullary Collecting Duct

“…The expression of the α-1, β-1, and β-2 subunits was observed at 100, 50−60, and 62 kD, respectively, as previously reported (17). No expression of α-2 or a-3 protein was observed in the membrane fraction from the outer medulla.…”

“…The expression of the α -1, β -1, and β -2 subunits was observed at 100, 50 − 60, and 62 kD, respectively, as previously reported ( 17 ) (Fig. 4).…”

“…The synthetic oligonucleotide probes were end-labelled with digoxigenin (DIG), as previously described. Pre-hybridization/hybridization washes were also performed as previously described (17).…”

“…The expression of the α-1, β-1, and β-2 subunits of Na-KATPase was examined using the membrane fraction from the outer medulla and Western blot analysis, as previously described (17,24). The antibodies against the α-1, β-1, and β-2 subunits of Na-K-ATPase were obtained from Upstate Biotechnology (Lake Placid, USA) and were diluted to 1:1000 with buffer.…”

“…AVP stimulates sodium reabsorption through the Na-K-2Cl cotransporter (NKCC2) in the MAL, which stimulates water reabsorption in the collecting ducts ( 23 ). We previously reported that hyperosmolality decreases Na-KATPase activity in the inner medullary collecting duct (IMCD), where the interstitial osmolality is highest in the body ( 17 ). However, it remains unknown whether Na-KATPase activity in the MAL and OMCD is stimulated by dehydration or hyperosmolality.…”