Acetazolamide Attenuates Lithium–Induced Nephrogenic Diabetes Insipidus

BASIC RESEARCH www.jasn.org

Theun de Groot,* Anne P. Sinke,* Marleen L.A. Kortenoeven,* Mohammad Alsady,* † ‡ | Ruben Baumgarten, Olivier Devuyst, Johannes Lofﬁng,§ Jack F. Wetzels, and Peter M.T. Deen*

Departments of *Physiology and |Nephrology, Radboud University Medical Center, Nijmegen, The Netherlands; †Vivium Care Group, Huizen, The Netherlands; ‡Institute of Physiology, Zurich Centre for Integrative Human Physiology, Zurich, Switzerland; and §Anatomy, University of Zurich, Zurich, Switzerland

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.

J Am Soc Nephrol 27: 2082–2091, 2016. doi: 10.1681/ASN.2015070796

Lithium is the drug of choice for the treatment of prolonged lithium treatment might lead to cyst for- bipolar disorders, and it is also regularly used to treat mation and ESRD.4 However, cessation of lithium schizoaffective disorders and depression. Lithium is a frequently prescribed drug, because it is pro- Received July 21, 2015. Accepted September 30, 2015. vided to 0.1% of the Western population. Unfor- T.d.G. and A.P.S. contributed equally to this work. tunately, in 2%–85% of patients and depending on Published online ahead of print. Publication date available at age, lithium usage leads to nephrogenic diabetes www.jasn.org. insipidus (NDI), a disorder characterized by an im- Present address: Dr. Marleen L.A. Kortenoeven, Department of paired response of the kidney to vasopressin (argi- Biomedicine, Aarhus University, Aarhus, Denmark. nine vasopressin [AVP]), leading to polyuria and Correspondence: Prof. Peter M.T. Deen, 286 Department of – polydipsia.1 3 Patients with lithium–induced neph- Physiology, Radboud University Medical Center, 6525GA Nijmegen, rogenic diabetes insipidus (Li-NDI) are at risk for The Netherlands. Email: [email protected] dehydration–induced lithium toxicity, and Copyright © 2016 by the American Society of Nephrology

2082 ISSN : 1046-6673/2707-2082 JAmSocNephrol27: 2082–2091, 2016 www.jasn.org BASIC RESEARCH therapy is not an option for most patients with NDI, because downregulation in mpkCCD cells. Indeed, whereas lithium again bipolar disorder symptoms have a larger effect on the patient’s reduced the AQP2 abundance in mpkCCD cells, acetazolamide quality of life. significantly attenuated this downregulation (Figure 1, A and B). Fromstudiesinrats,itbecameclear thatLi-NDIdevelopsintwo Because our data suggest that both thiazide and acetazolamide stages. In the short term (10 days), Li-NDI coincides with influence lithium–reduced AQP2 abundances through CAs, we downregulation of aquaporin-2 (AQP2) water channels, which assessed whether the action mechanism of acetazolamide differs is caused by a reduced AQP2 transcription.5–7 Despite an increased from that of amiloride. If so, we anticipated that acetazolamide proliferation of the AQP2–expressing principal cells of the collect- and amiloride together should attenuate the lithium–induced ing duct, long–term lithium treatment (4 weeks) also results in a AQP2 downregulation better than cells treated with amiloride severe loss of AQP2–expressing principal cells, which might be only. Indeed, immunoblotting revealed a significantly higher attributed to a lithium–induced G2/M–phase cell cycle arrest.8,9 AQP2 abundance in cells treated with amiloride and acetazol- This principal cell loss is compensated for by an increased number amide compared with amiloride only (Figure 1, C and D). We of a-intercalated cells, which are involved in acid secretion.8 and others discovered that ENaC is the main cellular entry site for To reduce polyuria in patients receiving lithium, a low-sodium lithium and that amiloride strongly reduced the intracellular lith- diet together with thiazide and amiloride diuretics are prescribed.10 ium levels in mpkCCD cells.11,14 Determination of the intracel- Amiloride acts on the principal cell epithelial sodium channel lular lithium concentrations revealed that amiloride, indeed, (ENaC), and we and others found that amiloride blocks principal reduced the intracellular lithium concentration by 87%, whereas cell lithium entry through ENaC, thereby attenuating polyuria in this was only 30% with acetazolamide (Figure 1E). The mean rodents and humans.11–13 Thiazides are known to block sodium intracellular lithium concentration was nominally lower with and chloride reabsorption through the NaCl cotransporter (NCC) amiloride/acetazolamide; however, there was no significant differ- in the renal distal convoluted tubule, and the antidiuretic effect ence (P=0.23). Transcellular transport of sodium and potassium has been ascribed to a hypovolemia-induced activation of the through ENaC, renal outer medullary K+, and the Na/K-ATPase is renin-angiotensin-aldosterone system and a compensatory electrogenic and therefore, generates a transcellular voltage (Tv) increased uptake of sodium and water in proximal tubules. Re- over mpkCCD cell monolayers. Lithium slightly reduced the Tv, cently, however, we discovered that thiazide also has an NCC- which was not further decreased with acetazolamide (Figure 1F). independent effect, because NCC knockout mice with Li-NDI In contrast, amiloride completely blocked the Tv in lithium–treated showed a clear antidiuretic response on treatment with thiazide.14 mpkCCD cells. Together, these data reveal that the CA–specific Because urine of our thiazide-treated mice was alkalinized inhibitor acetazolamide attenuates lithium-induced downregula- and thiazides are derived from carbonic anhydrase (CA) tion of AQP2 in vitro andthatitsmechanismofactionisdifferent inhibitors,15 our data indicated that the antidiuretic effect of from that of amiloride. thiazide in Li-NDI may involve CA inhibition. CAs catalyze the hydration of carbon dioxide to form carbonic acid, which Acetazolamide Attenuates Development of Li-NDI in then rapidly dissociates to form protons and bicarbonate, and Mice they play major roles in pH balance regulation. Here, we show To investigate whether acetazolamide attenuates development thatCAsare,indeed,involvedinlithium–induced AQP2 of Li-NDI, mice were maintained on lithium chow only or lithium downregulation and that, by inducing a tubular glomerular combined with acetazolamide or thiazide/amiloride for 10 days.As feedback response and through direct action on collecting reported,11,16 mice treated with lithium developed severe polyuria duct cells, the CA–selective drug acetazolamide not only at- and polydipsia combined with a significantly reduced urine os- tenuates Li-NDI but yields superior in vivo effects compared molality (Figure 2). Interestingly, acetazolamide treatment with the presently used treatment for Li-NDI. induced a significant antidiuresis and increase in urine osmolality, which was slightly but not significantly better than that in mice treated with thiazide/amiloride. Consistent with the induced anti- RESULTS diuresis, water intake was significantly reduced with the acetazolamide treatment compared with lithium only. The Clinically Used Drug Acetazolamide Attenuates Because long–term lithium treatment coincides with reduced Lithium-Induced Downregulation of AQP2 in mpkCCD AQP2 and increased H+-ATPase abundance in the kidney,6 we Cells also analyzed their abundances. Immunoblot analysis revealed mpkCCD cells are mouse collecting duct cells showing dDAVP- that lithium reduced AQP2 abundance (Figure 3, A and B), which dependent expression of endogenous AQP2, and we have shown was significantly attenuated by both acetazolamide and thiazide/ that thiazide reduces lithium-induced downregulation of AQP2 in amiloride (Figure 3, A–D). With acetazolamide or thiazide/ami- these cells, whereas they lack NCC expression.7,11,14 Because our loride, however, AQP2 levels did not return to control levels. previous animal studies suggested that thiazides reduced polyuria H+-ATPase levels were similar for all groups (Supplemental in our NCC knockout mice by inhibiting CAs, we wanted to test Figure 1), which is in line with the notion that lithium–induced whether acetazolamide, a stable CA inhibitor that is commonly collecting duct remodeling is not present after 10 days of lithium used in patients, could also rescue lithium–induced AQP2 treatment in rodents.9,16

J Am Soc Nephrol 27: 2082–2091, 2016 Acetazolamide in Lithium-NDI 2083 BASIC RESEARCH www.jasn.org

AQP2 abundance was increased along the connecting tubule and entire collecting duct. Consistent with our immunoblot data, immunohistochemistry revealed no clear changes in H+-ATPase labeling between the different groups (Supplemental Figure 2).

Acetazolamide Shows an Improved Overall Electrolyte Balance over Thiazide/Amiloride Some patients on thiazide/amiloride therapy havebeenreportedtodevelophyponatremia, hyperkalemia, metabolic acidosis, and/or hypercalcemia.17–20 Moreover, initiating thiazide/amiloride treatment in patients with Li-NDI often leads to elevated blood lithium levels, necessitating adjustment of the lithium dose.21 Therefore, to assess and compare the effect of acetazolamide and thiazide/amiloride on these parameters, we analyzed blood and urine for lithium and electrolyte levels (Table 1). Indeed, although mice treated with thiazide/amiloride had a reduced body weight and developed hyponatremia, hyperkalemia, and a metabolic acidosis, these parameters were not affected in acetazolamide-treated mice. Also, thiazide/ amiloride treatment induced hypercalcemia, fi Figure 1. Acetazolamide (Acz) reduces lithium (Li+) -induced downregulation of AQP2 which was signi cantly reduced with acet- abundance in mpkCCD cells. Native mpkCCD cells were grown to confluence for 4 azolamide, although not to control levels. days and subsequently exposed to 1 nM dDAVP for another 4 days. During the last 2 Moreover and consistent with patients, se- days, cells were incubated in the absence (control [Ctr]) or presence of Li+ only or with rum lithium concentrations were signifi- Li+ and 100 mMAcz,10mM amiloride (Am), 100 mM hydrochlorothiazide and Am cantly increased in our thiazide/amiloride (T+Am), or Am and Acz (Am+Acz). At the basolateral and apical sides, final concen- mice but unchanged in our acetazolamide trations of 1 and 10 mM Li+ were used, respectively. (F) After measurements of Tv, (A mice compared with lithium controls. 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 The Antidiuretic Effect of values6SEMs of normalized AQP2 abundance are given relative to Ctr. (E) Intracellular Acetazolamide Coincides with a Li+ concentrations were determined, corrected for contamination with extracellular Lowered GFR and a Reduced + + Li , and normalized for the amount of protein ([Li ]6SEM in picomoles per microgram Prostaglandin E2 Release protein). Data from three independent experiments (one-way ANOVA and Bonferroni Paradoxically, the clear antidiuresis found in , multiple comparison test). *P 0.05. our acetazolamide group (Figure 2) coincided with a significantly increased creatinine clearance (Figure 4A). However, because the cre- To examine segment-specific effects of the different therapies atinine clearance (especially in mice) highly depends on both on AQP2 abundance, immunohistochemistry was performed. creatinine secretion and reabsorption in the proximal tubule, Consistent with our immunoblot data, lithium treatment strongly the segment mainly influenced by acetazolamide,22,23 we hypoth- reduced AQP2 staining, which was clearly attenuated in kidneys of esized that acetazolamide may affect proximal tubular creatinine mice treated with lithium and acetazolamide or thiazide/amiloride reabsorption/secretion in our mice and thus, that the creatinine (Figure 3E). Interestingly, although lithium abolished AQP2 ex- clearance did not properly reflecttheGFR.Therefore,weused pression in the entire kidney, the increased AQP2 abundance in FITC-inulin to determine the GFR in an identically performed the thiazide/amiloride-treated group mainly localized to the inner animal experiment. Although acetazolamide again significantly medulla of the kidney, whereas in the acetazolamide-treated mice, attenuated Li-NDI (not shown), the clearance of FITC-inulin

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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. was signiﬁcantly reduced with acetazolamide (Figure 4B), indicating The observed hyperkalemia is likely caused by inhibition of that acetazolamide reduced the GFR. Urinary prostaglandin E2 ENaC by amiloride, because renal secretion of potassium (PGE2), which extensively contributes to AQP2 downregula- occurs only in exchange of ENaC–mediated sodium reabsorption in Li-NDI,24 was signiﬁcantly increased in our Li-NDI tion.27 Lithium itself can lead to metabolic acidosis,28 which mice but fully attenuated in our mice treated with lithium/ would even be increased with inhibition of bicarbonate uptake acetazolamide (Figure 4C). Moreover and consistent with its by acetazolamide, but we only found metabolic acidosis in our CA inhibitory action in proximal tubules, acetazolamide fur- thiazide/amiloride-treated mice. Whereas metabolic acidosis ther increased urinary pH (Figure 4D) and strongly reduced in our acetazolamide group may have been compensated for the abundance of NHE3 in the renal cortex compared the by increased ventilation, metabolic acidosis in the thiazide/ cortex of mice treated with lithium only (Figure 4, E and F). amiloride group may be secondary to the observed hyperkalemia, because mammals attenuate hyperkalemia at the ex- pense of development of metabolic acidosis.29 DISCUSSION Our Li-NDI mice developed hypercalcemia, which was sus- tained in our thiazide/amiloride-treated mice but not in our Acetazolamide Is Superior to Thiazide/Amiloride to acetazolamide mice. Hyperparathyroidism is common with Attenuate Li-NDI lithium-using patients, and the occurrence of hypercalcemia in Our mouse studies revealed that acetazolamide attenuates devel- Li-NDI has been ascribed to inhibition of calcium–sensing recep- opment of Li-NDI to the same extent as thiazide/amiloride, but in tor signaling by lithium in the parathyroid.30–32 The corrected contrast to acetazolamide, our thiazide/amiloride-treated mice blood calcium levels with acetazolamide, however, may be unre- developed hyponatremia, hyperkalemia, and metabolic acidosis. lated to the parathyroid effect of lithium, because plasma calcium In humans, hyponatremia is mostly a consequence of upregulated is increased by bone resorption, a process that involves CA2 ac- AQP2 expression by high circulating AVP levels. Although AVP tivity in osteoclast and is strongly inhibited by acetazolamide.33,34 levels are elevated in Li-NDI, this cannot explain hyponatremia in Another important advantage of the use of acetazolamide over our thiazide/amiloride-treated mice, because AQP2 is down- thiazide/amiloride is that plasma lithium concentrations remained regulated here. Instead, our data indicate that the hyponatremia is unchanged with acetazolamide. Blood lithium is mainly set by the becauseoftheinducednatriuresiscausedby thiazideandamiloride amount reabsorbed in proximal tubules, a process in which the in a status of polyuria and polydipsia, because our Li-NDI mice apical NHE3 is highly involved, and it is stimulated by thiazide.35,36 werenormonatremicandourthiazide/amiloridemicewerehighly Considering this, the unaltered blood lithium levels with acetazol- natriuretic compared with the other groups. Note, however, that amide are best explained by combinatory effects of the reduced part of the increased natriuresis must be because of an increased NHE3 abundance in proximal tubules and the reduced GFR. consumption of salt from the provided salt block, because food intake was not increased. The mice apparently drank water to Acetazolamide Attenuates Li-NDI by a Dual Mode of satiety, because the hematocrit was not different between the Action groups (data not shown). Similarly, patients with congenital NDI Our data indicate that the observed antidiuresis and reduced also sometimes develop hyponatremia when treated with thiazide GFR with acetazolamide is because of a tubular glomerular combinations.25,26 feedback response caused by inhibition of CAs in the proximal

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secretion of protons by NHE3 is important. By blocking these CAs, acetazolamide pre- vents the intracellular generation of H+, which is needed for NHE3 to reabsorb fil- tered Na+.40–43 The consequently increased tubular salt and water load in the proximal tubule leads to an increased fluid delivery 2 and tubular [Cl ]atthemaculadensa, which induces a tubular glomerular feedback response (i.e., a reduced GFR).38 In- deed, acetazolamide in our Li-NDI mice led to an increased urinary pH, reduced NHE3 abundance, and reduced GFR, which is in agreement with reported data on NHE3 knockout mice.38,41,44 Other than intercalated cells (see below), the increased fluid delivery to the macula densa with acetazolamide may also partially explain the observed lower urinary levels of PGE2, thereby attenuating Li-NDI. By act- ing on EP1/3 receptors, increased urinary PGE2 levels in Li-NDI reduce principal cell AQP2 expression and thus, water reabsorption.45 In Li-NDI, a fraction of the elevated urinary PGE2 levels is thought to be derived from macula densa and surrounding cTAL cells, which produce PGE2 to increase renin synthesis and release in response to a reduced fluid delivery to the TAL/hypovolemia.43,44 As such, the increased fluid delivery to the TAL with acetazolamide will reduce the cortical release of PGE2 and therefore, Li-NDI. However, our in vitro data indicate that acetazolamide also directly protects collecting duct cells from lithium, but it is at present unclear whether in vivo acetazol- Figure 3. Thiazide/amiloride (T+Am) and acetazolamide (Acz) reduce lithium (Li+) amide acts directly on principal cells or in- -induced downregulation of AQP2 in Li-NDI mice. (A–D) Immunoblot and corre- directly through intercalated cells. Support sponding densitometric analyses of AQP2 of mouse kidneys that are untreated for the first is that mpkCCD cells endoge- + + (control [Ctr]), treated with Li only, or treated with Li together with Acz or T+Am. (B nously express and show proper regulation fi 6 and D) The signals for AQP2 are densitometrically quanti ed. Mean values SEMs of of the typical principal cell proteins AQP2 normalized AQP2 abundance are given relative to Ctr. Equal loading of the samples and ENaC. Moreover, mpkCCD cells ex- 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. press high CA2 mRNA levels (http://esbl. Paraffin sections of immersion-fixed kidneys from (A, E, and I) Ctr, (B, F, and J) Li+- nhlbi.nih.gov/mpkCCD-transcriptome/), treated, (C, G, and K) Li++T+Am-treated, and (D, H, and L) Li++Acz-treated mice were which are also expressed in principal cells – incubated with a rabbit polyclonal AQP2 antibody followed by a Cy3–coupled goat in vivo.39,46 48 Also, the in vivo activity of anti–rabbit IgG. (A–D) Overviews and high magnifications of representative (E–H) ENaC, the lithium entry site of principal connecting tubules (CNTs) and (I–L) cortical collecting ducts (CCDs). cells, has been reported to be functionally paired with CA activity, because CA inhibition by acetazolamide reduced the 37,38 2 tubule. Ninety percent of renal HCO3 is reabsorbed in intracellular pH and ENaC activity in sweat duct cells and proximal tubules, which is strongly facilitated by CA4/14 colon.49,50 (luminal/apical), CA2 (intracellular), and CA4/12 (basolateral) However, mpkCCD cells may not fully represent principal cells, 39 hydrating CO2 and dehydrating H2CO3. In this process, and because intercalated cells express abundant levels of CA2, -4,

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Table 1. Metabolic parameters of mice treated for 10 days with standard chow the lack/inhibition of b-intercalated cells only or together with lithium; lithium, thiazide, and amiloride; or lithium and H+-ATPase led, through flow–stimulated lu- acetazolamide minal ATP release, to autocrine and para- Metabolic Parameters Ctr Li+ Li+ +Am+T Li+ + Acz crine release of PGE2, which reduced the Serum osmolality (mosM/kg) 32061 31961 31160.5a 32163b cortical and medullary ENaC activity and Serum sodium (mmol/L) 15060.3 14960.4 13960.8a 15060.5b AQP2 abundance. Importantly, thiazides Serum potassium (mmol/L) 5.360.1 5.660.2 7.660.5a 5.460.2b inhibited NCC–independent NaCl reabsorp- Serum lithium (mmol/L) — 0.6360.04c 2.1160.12a 0.6960.04b tion through NDCBE/pendrin,59 and acet- Serum creatinine (mg/dl) 0.0860.01 0.0960.00 0.0660.01a 0.0460.01a,b azolamide reduced pendrin abundance,63 c a,b Blood ionized calcium (mmol/L) 1.2460.00 1.3260.01 1.3460.01 1.2760.01 drugs that we showed to attenuate Li-induced 6 6 6 a 6 b Blood pH 7.34 0.01 7.32 0.01 7.24 0.02 7.35 0.02 downregulation of AQP2 and Li-NDI 6 6 c 6 a 6 b Urine sodium (mmol/L) 352 40 46 6 484 68 112 12 through a similar mechanism. As such, the Urine potassium (mmol/L) 870683 131615c 154619 271631a,b c attenuated Li-NDI with acetazolamide, which Urine lithium (mmol/L) — 2063 24633868 Urine creatinine (mg/dl) 70671061c 14621862a was given during the entire lithium treatment, Total sodium excretion (mmol/24 h) 0.1760.02 0.1660.01 1.2660.12a 0.2260.02b may be caused by an impaired functioning of Total potassium excretion (mmol/24 h) 0.4260.05 0.5560.02c 0.4060.03a 0.5460.05 pendrin, resulting in increased ATP/PGE2 re- Total lithium excretion (mmol/24 h) — 8463c 646672611 lease, reduced ENaC activity in principal cells, Body weight (g) 18.960.4 18.060.2c 16.060.5a 17.460.4b and thus, reduced influx of lithium from Food intake (mg/g per 24 h) 21468 198610 18865 22865a,b prourine. The finding that acetazolamide is Feces production (mg/g per 24 h) 1116798688262 12369b beneficial chiefly in the cortical segments Values are means6SEMs. Ctr, control (standard chow only); Li+, lithium; Am, amiloride; T, thiazide; Acz, that contain intercalated cells is consistent acetazolamide; —, below detection limit. aP,0.05 compared with lithium treatment. with the possibility that intercalated cell CAs bP,0.05 compared with lithium, thiazide, and amiloride treatment. could be involved. A prime candidate here is cP,0.05 compared with control treatment. CA12, because it is highly sensitive to acetazolamide, and patients with reduced CA12 -12, and -15,39 acetazolamide may increase principal cell AQP2 activity have a preponderance to hyponatremic dehydration.64 expression and water uptake indirectly by inhibiting ACs in Whether one of these mechanisms underlies the beneficial effect intercalated cells. Indeed, long–term lithium treatment leads to of acetazolamide in Li-NDI remains to be studied. metabolic acidosis, which underlies the increased number of Taken together, we have shown that CA activity contributes a-intercalated cells,11,51 and because lithium inhibits intercalated to Li-NDI development, that acetazolamide attenuates Li-NDI cell H+-ATPase and H+/K+-ATPase activity,52,53 it has been sugges- by inducing a tubular glomerular feedback response and ted that acidosis-induced proliferation of a-intercalated cells may through a direct action on the collecting duct, and that contribute to Li-NDI.16 It is unlikely, however, that attenuation of acetazolamide attenuates Li-NDI development similar to Li-NDI in our mice is caused by direct action of acetazolamide on thiazide/amiloride but with fewer side effects. a-intercalated cells or collecting duct remodeling for several rea- sons. First, acetazolamide increases the number of a-intercalated cells in rodents, because it causes acidosis itself.54,55 Second and CONCISE METHODS consistent with the unchanged H+-ATPase expression in our mice, collecting duct remodeling is not observed within 10 days of lith- Cell Culture ium treatment but only starts at about 4 weeks of treatment.9,56 mpkCCD cells were cultured as described.65 Cells were seeded at a density An effect of acetazolamide on b-intercalated cells, however, of 1.53105 cells per centimeter2 on semipermeable filters (Transwell; 0.4-mm may be more likely. Although lithium treatment did not reduce pore size; Corning Costar, Cambridge, MA) and cultured for 8 days. Unless the number of these cells,57 exciting recent studies revealed the stated otherwise, the cells were exposed to 1 nM dDAVP at the basolateral existence of extensive cross-talk between b-intercalated and prin- side for the last 96 hours to induce AQP2 expression. Lithium and com- cipal cells in the regulating collecting duct function.58–61 Although pounds were administered as indicated. At the end of the experiment, Eladari and coworkers62 elegantly showed that, in rodents, the transcellular electrical resistance and voltage were measured using a sodium–dependent chloride bicarbonate exchanger (NDCBE; Millicell-ERS Meter (Millipore Corp., Bedford, MA). On day 8, cells SLC4a8) and chloride bicarbonate exchanger protein pendrin al- were harvested and lysed in Laemmli bufferforWesternblottingorstored low for NaCl reabsorption through b-intercalated cells, chloride in Trizol Reagent (Invitrogen, Carlsbad, CA) at 280°C for RNA isolation. permeation through pendrin also seemed necessary for ENaC– mediated sodium reabsorption and expression. Also, in mice lack- Lithium Assays ing functional intercalated cell–specificH+-ATPase, the observed Determination of intracellular lithium concentrations was done as 11 2 natriuresis and aquaresis were caused by dysfunctional and lower described. Shortly, mpkCCDcl4 cells were grown on 4.7-cm filters. abundances of ENaC and pendrin/NDCBE and reduced AQP2 To determine the extent of lithium contamination from the extracel- levels, respectively.60 Eladari and coworkers62 further showed that lular side, FITC-dextran was added to the lithium-containing medium

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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+)onlyorLi+ 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. to a final concentration of 10 mM just before harvesting, after which the with lights on from 8:00 AM to 8:00 PM. They received normal diet (ssniff medium was mixed. Then, the filterswerewashedthreetimeswithiso- R/M-H V1534; ssniff Spezialdiaten GmbH, Soest, Germany) with osmoticsucrose(pH7.3)at4°C,andcellswerelysedbysonicationin1ml additions (see below) and water ad libitum for 10 days. For the experi- milli-Q Water. Of the 800-ml sample, the amount of lithium was deter- ments, mice were divided into four groups (n=8), which were treated as mined by flame photometry, from which the total amount of lithium in follows: group 1: control mice given a normal diet; group 2: normal diet the sample was calculated. with 40 mmol LiCl per 1 kg dry food66; group 3: diet of group 2 with 200 Of the 100-ml sample, the amount of FITC-dextran was measured mg amiloride11 and 350 mg hydrochlorothiazide per 1 kg dry food36;and using spectrofluorophotometry (RF-5301; Shimadzu, Tokyo, Japan) at group 4: diet of group 2 with 180 mg acetazolamide per 1 kg dry food.67 492-nm (excitation) and 518-nm (emission) wavelengths. By comparing LiCl,amiloride,hydrochlorothiazide,andacetazolamideweresolubilized the obtained values with a 2-fold FITC-dextran dilution series, the FITC- in water and mixed with the chow, after which it was dried. All mice had dextran concentration in each sample was determined, from which the free access to water, food, and a sodium-chloride block. extent of extracellular lithium contamination was calculated. This was For the last 48 hours of the experiment, mice were housed in metabolic subtracted from the total amount to obtain the intracellular lithium cages to measure water intake and urine output during the last 24 hours. amount. With the used FITC-dextran concentration, a contamination Mice were anesthetized with isofluorothane, after which their blood was .1:5000 would be detected. Tocorrect for differences in cellular yield, the removed by orbita extraction. Then, mice were killed by cervical intracellular lithium amounts were normalized for the protein amount in dislocation, and the kidneys were rapidly removed. One kidney was each sample, which was determined using the Bio-Rad Protein Assay processed for immunohistochemistry, whereas the other kidney was used (Bio-Rad, Munich, Germany). for immunoblotting, both as described below. For immunoblotting, the tissue was homogenized using a Polytron Homogenizer (VWR Interna- Experimental Animals tional, Amsterdam, The Netherlands) in 1 ml ice–cold homogenization Eight- to 10-week-old female C57Bl6/JOlaHsd mice (Harlan Laboratories buffer A (20 mM Tris, 5 mM MgCl2,5mMNa2HPO4,1mMEDTA,80 Inc., Frederick, MD) were maintained in a temperature-controlled room mM sucrose, and protease inhibitors [1 mM PMSF, 5 mg/ml pepstatin A,

2088 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 2082–2091, 2016 www.jasn.org BASIC RESEARCH

5 mg/ml leupeptin, and 5 mg/ml a-protinin]), cleared from nuclei and visualized using enhanced chemiluminescence (ECL; Pierce, Rockford, unbroken cells by centrifugation at 40003g for 15 minutes, and diluted in IL). Densitrometric analyses were performedusingBio-Radquantifica- Laemmli buffer to a final protein concentration of 1 mg/ml. tion equipment (Bio-Rad 690c Densitometer; Chemidoc XRS) and software (QuantityOne; Bio-Rad). Equal loading of the samples was con- Determination of GFR Using FITC-Inulin firmed by staining of the blots with Coomassie blue. Todetermine the GFR by the FITC-inulin clearancemethod,68,69 we used mice as described above, and these mice were also treated as before (n=8 Immunohistochemistry per group). Four days after the start of the diet, minipumps (Model 2001; Kidneys were fixed by immersion for 24 hours in 4% paraformaldehyde Alzet, Cupertino, CA) containing 3% FITC-inulin were subcutaneously in 0.1M phosphate buffer at 4°C, embedded in paraffin, and cut into 3- to implanted in the isofluorane-anesthetized mice. At treatment days 9 and 4-mm-thick sections. After deparaffinization, sections were placed into a 10, mice were housed in metabolic cages, and 24-hour urine was collected microwave oven and heated for 10 minutes at 98°C in 0.01 M sodium- in amber tubes at day 10. During this 24 hours, metabolic cages and urine citrate buffer (pH 6.0) for antigen retrieval. Subsequently, sections were collection tubes were covered with aluminum foil to prevent exposure to incubated overnight at 4°C with 1:80,000–diluted rabbit polyclonal AQP2 light. Traces of left FITC-inulin urine in metabolic cages were added to the antibodies or 1:2000–diluted rabbit polyclonal H+-ATPase antibodies as collected urine by washing the cage with 5 ml 500 mM HEPES buffer. On described.72,73 The bound primary antibodies were revealed with Cy3– day 10, mice were anesthetized with isofluorane, blood was collected by coupled goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories, retro-orbital bleeding, and mice were killed by cervical dislocation. Urine West Grove, PA). To check for unspecific binding of primary or secondary fluorescence was determined using a Cytofluor II Fluorescence Multiwell antibodies, incubations with nonimmune sera or without any primary Plate Reader (PerSeptive Biosystems, Framingham, MA) with 485-nm antibodies were performed. All control experiments were negative. Cry- excitation and 538-nm emission. The excretion rate of inulin (24-hour osections were studied by epifluorescence using a Leica Microscope (Leica urinary fluorescence counts/plasma fluorescence counts per ml) was Microsystems, Buffalo Grove, IL). Connecting tubules and cortical col- taken as the GFR. lecting ducts were distinguished on the basis of their specific localization in the cortical labyrinth and the medullary rays, respectively. Images were Blood and Urine Analyses – Whole blood was analyzed immediately for sodium, potassium, hemat- acquired with a charge coupled device camera. For overviews, single ocrit, and pH using the EG7+ Cartridge and the I-Stat Clinical Analyzer images were taken with the automated scanning mode of the microscope (Abbott BV, Hoofddorp, The Netherlands). The remaining blood was and afterward, stitched using the Leica Application Suite. Digital images collected in a BD Microtainer SST Tube (REF 365968; Becton Dickinson were processed electronically with Adobe Photoshop (Adobe Systems, BV, Breda, The Netherlands) for serum and centrifuged at 10,0003g for 3 Inc., San Jose, CA) and Microsoft Powerpoint (Microsoft, Redmond, minutes to sediment the red blood cells. Serum and urine samples were WA) software. Adjustments for brightness and contrast were kept con- analyzed for osmolality using an osmometer (Fiske, Needham Heights, stant for each kidney section. MA), and electrolyte concentrations were measured on a Synchron CX5 Analyzer (Beckman Coulter, Inc., Brea, CA) following the manufacturer’s Statistical Analyses protocols. Urine PGE2 levels were determined by measuring stable pros- One-way ANOVAwith Bonferroni correction was applied. A P value taglandin E2 metabolite (PGEM) after chemical derivation of PGE2 and of ,0.05 was considered significant. Data are presented as means and its primary metabolites 13,14-dihydro-15-keto PGE2 and 13,14-dihydro- SEMs. 15-keto PGA2 to the single PGEM compound. PGEM concentrations were determined with the Prostaglandin E Metabolite EIA Kit (Cayman Study Approval Chemicals, Ann Arbor, MI) according to the manufacturer’s instructions. All animal studies (DEC no. 2011–010) were approved by the Animal Ethical Committee of the Radboud University Medical Center. Immunoblotting mpkCCD cells from 1.13-cm2 filters were lysed in 200 mlLaemmli buffer and sonicated. mpkCCD lysate and 5–10 mgkidneymaterialin Laemmli were denatured for 30 minutes at 37°C. Protein concentration ACKNOWLEDGMENTS was determined using the Bio-Rad Protein Assay (Bio-Rad) according to manufacturer’s instructions. SDS-PAGE, blotting, and blocking of the We thank Marthe Minderman, Marcel Jaklofsky, and Monique Carrel PVDFmembranesweredoneasdescribed.70 Membranes were incubated for their expert help. The H+-ATPase antibody was a gift from Dr. for16hoursat4°Cwith1:2000affinity–purified rabbit pre–CtailAQP2 Carsten Wagner. antibody recognizing amino acids 236–25571 in Tris-Buffered Saline This project received support from a Niels Stensen Fellowship (to T.d. Tween-20 supplemented with 1% nonfat dried milk. In an identical G.), Marie Curie Fellowship PIOF-GA-2012-332395 (to T.d.G.), grants way, other blots were incubated with a rabbit CA12 antibody (gift from from the European Community’s Seventh Framework Programme William S. Sly, St. Louis University School of Medicine, St. Louis, MO) FP7/2007-2013 (agreement 305608 [EURenOmics]; to O.D.), Swiss and a rabbit CA2 antibody (Abcam, Inc., Cambridge, MA). After washing National Science Foundation Grants 310030_146490 (to O.D.) and in Tris-Buffered Saline Tween-20, all blots were incubated for 1 hour with 310030_143929/1 (to J.L.), grants from the Rare Disease Initiative Zurich 1:5000-diluted goat anti–rabbit IgGs (Sigma-Aldrich, St. Louis, MO) as from the University of Zurich (to O.D.), VICI Grant 865.07.002 (to P.M.T. secondary antibody coupled to horseradish peroxidase. Proteins were D.) from The Netherlands Organization for Scientific Research, Radboud

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