Acetazolamide Attenuates Lithium-Induced Nephrogenic Diabetes Insipidus

Abstract

To reduce lithium-induced nephrogenic diabetes insipidus (lithium-NDI), patients with bipolar disorder are treated with thiazide and amiloride, which are thought to induce antidiuresis by a compensatory increase in prourine uptake in proximal tubules. However, thiazides induced antidiuresis and alkalinized the urine in lithium-NDI mice lacking the sodium-chloride cotransporter, suggesting that inhibition of carbonic anhydrases (CAs) confers the beneficial thiazide effect. Therefore, we tested the effect of the CA-specific blocker acetazolamide in lithium-NDI. In collecting duct (mpkCCD) cells, acetazolamide reduced the cellular lithium content and attenuated lithium-induced downregulation of aquaporin-2 through a mechanism different from that of amiloride. Treatment of lithium-NDI mice with acetazolamide or thiazide/amiloride induced similar antidiuresis and increased urine osmolality and aquaporin-2 abundance. Thiazide/amiloride-treated mice showed hyponatremia, hyperkalemia, hypercalcemia, metabolic acidosis, and increased serum lithium concentrations, adverse effects previously observed in patients but not in acetazolamide-treated mice in this study. Furthermore, acetazolamide treatment reduced inulin clearance and cortical expression of sodium/hydrogen exchanger 3 and attenuated the increased expression of urinary PGE2 observed in lithium-NDI mice. These results show that the antidiuresis with acetazolamide was partially caused by a tubular-glomerular feedback response and reduced GFR. The tubular-glomerular feedback response and/or direct effect on collecting duct principal or intercalated cells may underlie the reduced urinary PGE2 levels with acetazolamide, thereby contributing to the attenuation of lithium-NDI. In conclusion, CA activity contributes to lithium-NDI development, and acetazolamide attenuates lithium-NDI development in mice similar to thiazide/amiloride but with fewer adverse effects.

Keywords: cell and transport physiology; diabetes insipidus; diuretics; osmolality; pathophysiology of renal disease and progression; water transport.

Figure 1.

Acetazolamide (Acz) reduces lithium (Li⁺) -induced downregulation of AQP2 abundance in mpkCCD cells. Native mpkCCD cells were grown to confluence for 4 days and subsequently exposed to 1 nM dDAVP for another 4 days. During the last 2 days, cells were incubated in the absence (control [Ctr]) or presence of Li⁺ only or with Li⁺ and 100 μM Acz, 10 μM amiloride (Am), 100 μM hydrochlorothiazide and Am (T+Am), or Am and Acz (Am+Acz). At the basolateral and apical sides, final concentrations of 1 and 10 mM Li⁺ were used, respectively. (F) After measurements of Tv, (A and C) cells were lysed and subjected to AQP2 immunoblotting. Molecular masses (in kilodaltons) are indicated. (B and D) The signals for nonglycosylated (29 kD) and complex-glycosylated (40–45 kD) AQP2 were densitometrically quantified. Mean values±SEMs of normalized AQP2 abundance are given relative to Ctr. (E) Intracellular Li⁺ concentrations were determined, corrected for contamination with extracellular Li⁺, and normalized for the amount of protein ([Li⁺]±SEM in picomoles per microgram protein). Data from three independent experiments (one-way ANOVA and Bonferroni multiple comparison test). *P<0.05.

Figure 2.

Acetazolamide (Acz) attenuates lithium (Li⁺)-induced NDI in mice. (A) Urine volume, (B) water intake, and (C) urine osmolality of untreated mice (control [Ctr]) or mice treated for 10 days with Li⁺ or Li⁺ combined with thiazide/amiloride (T+Am) or Acz. During the last 48 hours, mice were housed in metabolic cages; during the last 24 hours, water intake was measured, and urine was collected to determine urine volume and osmolality (n=8 mice per group; one-way ANOVA and Bonferroni multiple comparison test). *P<0.05.

Figure 3.

Thiazide/amiloride (T+Am) and acetazolamide (Acz) reduce lithium (Li⁺) -induced downregulation of AQP2 in Li-NDI mice. (A–D) Immunoblot and corresponding densitometric analyses of AQP2 of mouse kidneys that are untreated (control [Ctr]), treated with Li⁺ only, or treated with Li⁺ together with Acz or T+Am. (B and D) The signals for AQP2 are densitometrically quantified. Mean values±SEMs of normalized AQP2 abundance are given relative to Ctr. Equal loading of the samples was confirmed by staining of the blots with Coomassie blue (Cm). One-way ANOVA and Bonferroni multiple comparison test. *Significant differences (P<0.05) from Ctr. Paraffin sections of immersion-fixed kidneys from (A, E, and I) Ctr, (B, F, and J) Li⁺-treated, (C, G, and K) Li⁺+T+Am-treated, and (D, H, and L) Li⁺+Acz-treated mice were incubated with a rabbit polyclonal AQP2 antibody followed by a Cy3–coupled goat anti–rabbit IgG. (A–D) Overviews and high magnifications of representative (E–H) connecting tubules (CNTs) and (I–L) cortical collecting ducts (CCDs).

Figure 4.

Acetazolamide (Acz) reduces the GFR and abolishes the elevated PGE2 levels in lithium-treated mice. Mice were treated for 10 days with control (Ctr) diet or diet containing lithium (Li⁺) only or Li⁺ combined with Acz. During the last 48 hours, mice were housed in metabolic cages, and during the last 24 hours, urine was collected to determine (A) urinary pH, (D) creatinine clearance, and (F) PGE2 levels. (C) At day 10, mice were euthanized, and blood and kidneys were isolated, enabling the analysis of renal NHE3 abundance. In B, the arrow indicates the approximately 85-kD band of NHE3. (E) To measure GFR using FITC-inulin, the above-mentioned experiment was repeated; however, at day 4, osmotic minipumps containing FITC-inulin were implanted, and at day 10, FITC-inulin levels were measured in 24-hour urine and serum (n=8 mice per group). One-way ANOVA and Bonferroni multiple comparison test. Cm, Coomassie blue. *P<0.05.