Aquaporin-2 mRNA Levels Research Articles

Changes of Rat Kidney AQP2 and Na,K-ATPase mRNA Expression in Lithium-Induced Nephrogenic Diabetes insipidus

Background/Aim: In a rat model, lithium treatment is associated with polyuria and severe downregulation of aquaporin-2 (AQP2) protein in the inner medulla (IM) or in the whole kidney. However, it is not known (1) to what extent this downregulation occurs at the mRNA level; (2) whether the main sodium transporter of the nephron, Na,K-ATPase, is regulated in parallel at the mRNA level, and (3) whether lithium treatment induces zonal or segmental differences in AQP2 and Na,K-ATPase mRNA levels. Method: We examined the changes in mRNA expression levels for AQP2 and Na,K-ATPase in kidney cortex, inner stripe of the outer medulla (ISOM), and IM of rats treated with lithium orally using semiquantitative Northern blot analyses and in situ hybridization at the light and electron microscopic levels. Results: The AQP2 mRNA levels decreased significantly (p < 0.01) in lithium-treated rats to 37 ± 4% in the cortex, to 17 ± 4% in the ISOM, and to 23 ± 5% in the IM, while the Na,K-ATPase mRNA levels were not altered in the cortex, but were significantly (p < 0.05) altered in the ISOM (144 ± 15% after 10 days, but 68 ± 4% after 4 weeks) and in the IM (63 ± 8% after 10 days, but normalized after 4 weeks). In situ hybridization showed reduced levels of AQP2 mRNA in all zones of the kidney, but the Na,K-ATPase mRNA expressions were slightly decreased only in IM collecting ducts. At the ultrastructural level, principal cells in the IM collecting ducts showed slight hypertrophy, but no cell damage after 4 weeks of lithium treatment. The results demonstrate substantial downregulation of AQP2 at the mRNA level throughout the collecting duct in experimental lithium-induced nephrogenic dabetes insipidus and moderately decreased Na,K-ATPase mRNA levels in the ISOM and in the IM. Conclusion: The results suggest that decreased mRNA expressions of AQP2 and Na,K-ATPase contribute to the development of lithium-induced nephrogenic diabetes insipidus.

Dehydration reverses vasopressin antagonist-induced diuresis and aquaporin-2 downregulation in rats.

To examine the involvement of vasopressin and dehydration in the regulation of aquaporin-2 (AQP2) expression in rat kidney, we investigated the effects of treatment for 60 h with the specific V2-receptor antagonist OPC-31260 (OPC), alone and in conjunction with dehydration for the last 12 h. Changes in AQP2 protein and mRNA expression in kidney inner medulla were determined by Western and Northern blotting, and AQP2 distribution was analyzed by immunocytochemistry and immunoelectron microscopy. Treatment with OPC increased urine output fourfold, with a reciprocal decrease in urine osmolality. AQP2 expression decreased to 52 +/- 11% of control levels (n = 12, P < 0.05), and AQP2 was found predominantly in intracellular vesicles in collecting duct principal cells. This is consistent with efficient blockade of the vasopressin-induced AQP2 delivery to the plasma membrane and with the observed increased diuresis. Consistent with this, AQP2 mRNA levels were also reduced in response to prolonged OPC treatment (30 +/- 10% of control levels, n = 9). Five days of treatment with furosemide, despite producing even greater polyuria than OPC, was not associated with downregulation of AQP2 levels, demonstrating that AQP2 downregulation is not secondary to increased urine flow rate or loss of medullary hypertonicity. During 12-h thirsting in the continued presence of OPC, urine output dropped dramatically, to levels not significantly different from that seen in (nonthirsted) control animals. In parallel with this, AQP2 levels rose to control levels. Control experiments confirmed continued effective receptor blockade. These results indicate that the V2-receptor antagonist causes a modest decrease in AQP2 expression that is not a consequence of increased urine flow rate or washout of medullary hypertonicity. However, this decrease is much less marked than that seen in some forms of acquired nephrogenic diabetes insipidus. In conjunction with the effects of thirsting, this suggests that modulation of AQP2 expression is mediated partly, but not exclusively, via V2 receptors.

Acute effects of vasopressin V2-receptor antagonist on kidney AQP2 expression and subcellular distribution.

The acute effect of treatment with the vasopressin V2-receptor antagonist OPC-31260 (OPC) on aquaporin-2 (AQP2) distribution and expression in rat kidney was examined. Immunofluorescence and semi-quantitative immunoelectron microscopy revealed that 15 and 30 min of OPC treatment resulted in significant reduction in apical plasma membrane labeling of AQP2, with a concomitant increase in labeling of vesicles and multivesicular bodies. In parallel, OPC treatment induced a large increase in urine output [0.6 +/- 0.2 vs. 8.3 +/- 1.0 ml/h (n = 4)]. Northern blotting using a 32P-labeled AQP2 cDNA probe and a digoxigenin-labeled AQP2 RNA probe revealed a band of approximately 1.6 kb corresponding to the predicted size of AQP2 mRNA. In control experiments, thirsting increased, whereas water loading decreased AQP2 mRNA levels. Treatment of rats with OPC caused a significant reduction in AQP2 mRNA within 30 min (52 +/- 21%, n = 8, P < 0.025) and 60 min (56 +/- 7%, n = 4, P < 0.001) of treatment compared with intravenous saline-injected controls. Thus a very rapid reduction in AQP2 mRNA was observed in response to vasopressin-receptor antagonist treatment. The reduction in AQP2 mRNA persisted after 24 h (40 +/- 17%, n = 5, P < 0.05) of OPC treatment. There was a parallel increase in diuresis and reduction in urine osmolality. In conclusion, V2-receptor blockade produced a rapid internalization of AQP2 parallel with a rapid increase in urine output. Furthermore, OPC treatment caused a rapid and significant reduction in AQP2 mRNA expression, demonstrating that for rapid regulation of AQP2 expression, modulation of AQP2 mRNA levels is regulated via vasopressin-receptor signaling pathways.

Studies of renal aquaporin-2 expression during renal escape from vasopressin-induced antidiuresis.

In animal models of the syndrome of inappropriate antidiuresis (SIADH), sustained administration of vasopressin and water results in free-water retention and progressive hyponatremia for several days, which is then followed by escape from the vasopressin-induced antidiuresis. With the onset of vasopressin escape, water excretion increases despite sustained administration of vasopressin, allowing water balance to be re-established and the serum sodium to be stabilized at a steady, albeit decreased, level. Studies from our laboratories have investigated whether this escape phenomenon can be attributed to altered regulation of aquaporin water channels. After four-day pre-treatment with 1-deamino-[8-D-arginine]-vasopressin (dDAVP) by osmotic minipump, rats were divided into control (continued dDAVP) and water-loaded (continued dDAVP plus a daily oral water load) groups. A significant increase in urine volume in the water-loaded rats was observed by the second day of water loading, indicating escape from antidiuresis. The onset of escape coincided temporally with a marked decrease in renal aquaporin-2 protein (measured by semi-quantitative immunoblotting), which began at day 2 and fell to 17% of control levels by day 3. In contrast, there was no decrease in the renal expression of aquaporins 1, 3, or 4. The marked suppression of whole kidney aquaporin-2 protein was accompanied by a concomitant suppression of whole kidney aquaporin-2 mRNA levels. Immunocytochemical localization and differential centrifugation studies demonstrated that trafficking of aquaporin-2 to the plasma membrane remained intact during vasopressin escape. Additional studies have indicated that the observed down-regulation of aquaporin-2 expression also occurs in the renal cortex as well as the inner and outer medullas, and can be reversed simply by water restriction despite maintenance of hyponatremia. Our results therefore suggest that escape from vasopressin-induced antidiuresis is attributable, at least in part, to a vasopressin-independent and osmolality-independent decrease in aquaporin-2 water channel expression in the renal collecting duct. Similar mechanisms likely contribute to the phenomenon of escape from antidiuresis seen clinically in patients with SIADH as well.

Downregulation of aquaporin-2 parallels changes in renal water excretion in unilateral ureteral obstruction.

In bilateral ureteral obstruction, both aquaporin-2 (AQP2) levels and urinary concentrating capacity are markedly reduced. However, the mechanisms involved in AQP2 downregulation are unknown. In rats with unilateral ureteral obstruction (UUO) the relative role of intrarenal and systemic factors can be evaluated. Semiquantitative immunoblotting revealed a marked decrease in AQP2 in obstructed kidneys to 23 +/- 7% (n = 9) of sham levels. This downregulation persisted 24 h after release of UUO. Furthermore, there was a significant but less extensive downregulation of AQP2 in the nonobstructed kidneys to 75 +/- 7% (n = 9) of sham levels. Consistent with impairment of collecting duct water reabsorption, free water clearance was greatly elevated in the obstructed kidneys (-2 +/- 1 microliter-min-1.kg-1, determined immediately after release) and only moderately elevated in nonobstructed kidneys (-44 +/- 5 microliters.min-1.kg-1) compared with sham-operated controls (-59 +/- 3 microliters.min-1.kg-1). Also AQP2 mRNA levels were reduced in obstructed kidneys. Immunocytochemistry confirmed the marked decrease in AQP2 expression in obstructed kidneys. In nonobstructed kidneys AQP2 was predominantly found in intracellular vesicles, which together with the reduced expression and elevated free water clearance strongly suggests a role of AQP2 in the observed compensatory diuresis from nonobstructed kidneys. The much lower AQP2 protein and mRNA levels in obstructed vs. nonobstructed kidneys are consistent with intrarenal factors playing a major role for downregulation of AQP2.

Development of urinary concentrating capacity: role of aquaporin-2.

The capacity to concentrate urine develops progressively during postnatal life in most mammalian species. Here we have examined whether low expression of the arginine vasopressin (AVP)-activated water channel aquaporin-2 (AQP-2) may be a limiting factor for the concentrating capacity in the infant rats. Urine osmolality in response to 24-h dehydration increased significantly from 10 to 40 days of age. The most rapid increase occurred during the weaning period, i.e., days 15-20. A similar developmental pattern was observed for AQP-2 mRNA levels in the renal medulla. AQP-2 protein levels also increased markedly from day 10 to 40. Immunohistochemistry revealed that AQP-2 was exclusively located in collecting duct principal cells both in infant and adult rats but that the signal was much weaker in infants. To further examine the relationship between urinary concentrating capacity and AQP-2 expression, we treated rats with a single injection of betamethasone, which is known to accelerate maturation in several organs. Twenty-four hours after treatment, there was an increase in urine osmolality, renal medullary AQP-2 mRNA, and AQP-2 protein levels in infant but not in adult rats. A single injection of a specific V2 agonist caused within 6 h significant increase of AQP-2 mRNA in both infant and adult. The expression of the mRNA of three other transporters involved in the concentrating process, medullary Na(+)-K(+)-ATPase alpha-subunit, Na-K-2Cl cotransporter, and epithelial chloride channel also increased during the weaning period and were upregulated by glucocorticoids. We conclude that there is a well-synchronized development of the many of the components that determine the concentrating capacity and that the low expression of AQP-2 is one of the limiting factors for low concentrating capacity in infants.