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Identification of Acer2 as a First Susceptibility for Lithium-Induced Nephrogenic Diabetes Insipidus in Mice

Theun de Groot,1,2,3 Lena K. Ebert,1,2,4 Birgitte Mønster Christensen,5 Karolina Andralojc,6,7,8 Lydie Cheval,9,10 Alain Doucet,9 Cungui Mao,11,12 Ruben Baumgarten,13 Benjamin E. Low,1 Roger Sandhoff,14,15 Michael V. Wiles,1 Peter M.T. Deen ,2 and Ron Korstanje1

Due to the number of contributing authors, the affiliations are listed at the end of this article.

ABSTRACT Background Lithium, mainstay treatment for bipolar disorder, causes nephrogenic diabetes insipidus and hypercalcemia in about 20% and 10% of patients, respectively, and may lead to acidosis. These adverse effects develop in only a subset of patients treated with lithium, suggesting genetic factors play a role. Methods To identify susceptibility for lithium-induced adverse effects, we performed a genome- wide association study in mice, which develop such effects faster than . On day 8 and 10 after assigning female mice from 29 different inbred strains to normal chow or lithium diet (40 mmol/kg), we housed the animals for 48 hours in metabolic cages for urine collection. We also collected blood samples. Results In 17 strains, lithium treatment significantly elevated urine production, whereas the other 12 strains were not affected. Increased urine production strongly correlated with lower urine osmolality and elevated water intake. Lithium caused acidosis only in one mouse strain, whereas hypercalcemia was found in four strains. Lithium effects on blood pH or ionized calcium did not correlate with effects on urine production. Using genome-wide association analyses, we identified eight gene-containing loci, including a locus containing Acer2, which encodes a and is specifically expressed in the collecting duct. Knockout of Acer2 led to increased susceptibility for lithium-induced diabetes insipidus development. Conclusions We demonstrate that genome-wide association studies in mice can be used successfully to identify susceptibility genes for development of lithium-induced adverse effects. We identified Acer2 as a first susceptibility gene for lithium-induced diabetes insipidus in mice.

JASN 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018050549

Lithium is the drug of choice for the treatment of bipolar disorders and is also regularly used to treat schizoaffective disorders and depression. Lithium is Received May 26, 2018. Accepted August 7, 2019. therefore often prescribed and used by 0.1% of the T.d.G., L.K.E., P.M.T.D., and R.K. contributed equally to this population.1 Unfortunately, lithium treatment has work. different side effects, here called lithiopathies: Published online ahead of print. Publication date available at within a few years, patients may develop the urine www.jasn.org. concentration defect nephrogenic diabetes insipi- Correspondence: Dr. Ron Korstanje, The Jackson Laboratory, dus (NDI), metabolic acidosis, and hypercalcemia. 600 Main Street, Bar Harbor, ME 04609, or Dr. Peter M.T. Deen, 286 Department of Physiology, Radboud University Medical Center, Despite these side effects, lithium remains the drug Geert Grooteplein Zuid 30, 6525 GA Nijmegen, Nijmegen, The of choice because bipolar disorder has a larger effect Netherlands. E-mail: [email protected] or peterdeen11@gmail. on the patient’s quality of life than the lithiopathies, com and for many patients there is no good alternative Copyright © 2019 by the American Society of Nephrology

JASN 30: ccc–ccc,2019 ISSN : 1046-6673/3012-ccc 1 BASIC RESEARCH www.jasn.org medication.2 To lower the burden currently associated with Significance Statement lithium use, it is essential to understand the pathophysiology of, and the susceptibility for, the diverse lithiopathies. Lithium causes nephrogenic diabetes insipidus andhypercalcemia in Lithium-induced NDI (Li-NDI) develops in approximately 20% and 10% of patients, respectively, and may lead to metabolic 20% of patients and is thereby the most common cause of NDI. acidosis. To determine the potential role of genetic predisposition in these adverse effects, the authors fed mice from 29 genetically Li-NDI is caused by the inability of the kidney to concentrate its different inbred strains a normal or a lithium-supplemented diet. – prourine and is characterized by polyuria and polydipsia.3 5 Urine Some strains developed adverse effects, whereas others did not. concentration is mediated by collecting duct principal cells that Genome-wide association studies revealed eight loci containing take up water from the prourine via the luminal water channel different candidate genes that were associated with development of Acer2 Aquaporin-2 (AQP2), which then exits through AQP3 and AQP4 lithium-induced nephrogenic diabetes insipidus. Of these, is specifically expressed in the collecting duct; mice lacking Acer2 at the basolateral membrane. Urine concentration is tightly reg- were more likely to develop lithium-induced nephrogenic diabetes ulated via the hormone arginine vasopressin, which is released insipidus. These findings demonstrate the importance of genetic from the pituitary in response to hypovolemia or hypernatremia variation in susceptibility for lithium-induced adverse effects in and binds its type-2 receptor in the basolateral membrane of mice, and the genes identified may facilitate subsequent identifi- principal cells, leading to redistribution of AQP2 from intracel- cation of susceptibility genes. lular vesicles to the apical membrane. In Li-NDI, AQP2 is down- regulated in the short term, whereas prolonged treatment METHODS also reduces the ratio of principal cells to intercalated cells.6,7 Lithium-induced AQP2 downregulation is a consequence of Experimental Animals Strain Survey principal cell lithium entry through the epithelial sodium chan- Female mice (7–9 weeks old) from 29 different inbred mouse nel (ENaC).8–10 Inside principal cells, lithium inhibits the activity strains (n=18 per strain) were obtained from The Jackson of glycogen synthase kinase type 3.11 Moreover, Li-NDI coincides Laboratory (Bar Harbor, ME). Mice were housed in a climate- with elevated urinary prostaglandin E 2 (PGE2) levels, which controlled facility with a 12-hour light/dark cycle. All mice are known to reduce AQP2 abundance and elevate diuresis.12 had free access to food, water, and a salt (sodium chloride) Lithium also affects acid-base balance. Within days, lithium lick throughout the experiment. At 10 weeks of age, 8 mice administration impairs acid excretion in rats, dogs, and hu- from each strain received normal rodent diet (7013, NIH-31 mans.13–16 Development of metabolic acidosis is mostly ob- Modified; Harlan Laboratories, Madison, WI), while the other served in animal models using acute supraclinical lithium ten mice received the same diet supplemented with 40 mmol doses,13,14 whereas clinically relevant lithium doses have no lithium chloride/kg. During a 20-week interval, the control/ or very mild effects on blood pH.15–20 Rats treated with lith- lithium diet was initiated for nine mice per strain (four con- ium for 1 month enhanced the number of acid-secreting trol, five lithium) for a maximal number of three strains per a-intercalated cells and the abundance of its proton pump week, while the second cohort of nine mice (four control, ATPase (H+-ATPase).6,15 This is likely a compensatory mech- five lithium) of a strain started in a different week. At days anism to enhance acid excretion and may explain the en- 8–10 and 26–28, mice were individually housed in metabolic hanced net acid excretion in both rats and humans observed cages (3600M021; Tecniplast) and urine was collected dur- with long-term lithium treatment.18 ing the last 24 hours of their stay. After their stay in the met- In addition to effects on the kidney, lithium causes hyper- abolic cages, blood was obtained through submandibular parathyroidism and hypercalcemia in approximately 10% of bleeding. At day 18, blood was collected in Microvette tubes patients.21 Interestingly, hypercalcemia is an independent risk (Sarstedt, Nürmbrecht, Germany) through submandibular factor for the development of NDI.22 The molecular cause of bleeding, centrifuged at 3000 3 g for 5 minutes to sediment lithium-induced hyperparathyroidism and hypercalcemia is the red blood cells, and serum was stored at 280°C. not well understood, but ex vivo studies demonstrated lithium All animals were housed at The Jackson Laboratory, which is directly increased the secretion of parathyroid hormone from approved by the American Association for Accreditation of isolated bovine parathyroid cells.23,24 Laboratory Animal Care. All animal studies were approved The fact that not all lithium-using patients develop the by The Jackson Laboratory’s Institutional Animal Care and above-described lithiopathies indicates that humans vary in Use Committee. their susceptibility for the development of lithiopathies, which is likely partly determined by genetic factors. Genome-wide Acer2 Knockout Mice association (GWA) could be used to identify these factors, but C57BL/6J-Acer2em1Mvw/MvwJ mice (Acer2 knockout) large and long-term cohort studies are needed to perform such (JR#026793) were obtained from The Jackson Labora- an approach in humans. Because commonly used rodents de- tory, where they were generated as described.28 After crossing 2 2 2 velop these lithiopathies consistently and in a much shorter Acer2+/ mice, 10- to 12-week-old Acer / (n=19) and their time,6,13,25–27 we used a GWA approach in mice to investigate heterozygous (n=20) and wild-type (n=20) littermates were the potential role of genetic factors in the development of treated with a control or lithium diet. Each control or lith- lithiopathies and identify lithiopathy susceptibility genes. ium treatment group consisted of five female and five male

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2 2 2 2 2 Acer2+/+, Acer2+/ ,orAcer2 / mice, except for the Acer2 / Bio-Rad Laboratories GmbH, München, Germany). Equal control group, which consisted of four female and five male loading of the samples was confirmed by staining blots with mice. To determine whether male mice reacted differently to Coomassie blue. lithium compared with female mice, a regression analysis was performed but no sex effects were found. After 8 days of treat- PGE2 Assay and Analysis of Levels ment,micewerehousedinmetaboliccagesandurineand PGE2 metabolites (PGEMs) were measured in urine samples blood were collected as described above. At day 10, mice (day 9–10, stored at 280°C) using the PG E Metabolite EIA were euthanized by cervical dislocation and the kidneys were kit (Cayman Chemical, Ann Arbor, MI) according to the man- rapidly removed. One kidney was sliced in two; one half was ufacturer’s instructions. Control samples were diluted by a processed for immunohistochemistry (IHC), while the other factor of 300. The dilution rate of the lithium samples was be- half was stored at 280°C together with cortex and medulla tween 40 and 1000, depending on the urine production of the material obtained from the second kidney. different strains. The absorbance was measured on a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA) at Urine and Blood Analyses a wavelength of 405 nm. Renal ceramide levels were determined Collected urine was centrifuged at 1000 3 g for 5 minutes as described.32 to remove sediment. Urine osmolality was measured using a Micro-Osmometer Model 3320 (Advanced Instruments, Analysis of mRNA Expression along the Mouse Norwood, MA) and pH was measured using an Oakton pH Nephron Spear (Eutech Instruments). Blood obtained in the strain survey Different nephron segments were dissected from collagenase- was immediately analyzed for sodium ions (Na+), ionized blood treated kidneys of six 8- 10-weeks-old C57BL/6J males calcium (iCa2+), hematocrit, hemoglobin, pH, base excess, (Charles River Breeding Laboratories), fed ad libitum with a 33 partial pressure of carbon dioxide (pCO2), partial pressure of standard diet (A04, SAFE; Epinay, France) as described. To- 2 oxygen, bicarbonate (HCO3 ), total carbon dioxide, and oxy- tal RNA was extracted, using the RNeasy Micro Kit (Qiagen, gen saturation (sO2) using EG7+ cartridges and the i-STAT Hilden, Germany) from pools of approximately 50 nephron Clinical Analyzer (Abbott BV, Hoofddorp, The Netherlands). segments, and reverse transcribed using a first strand cDNA Serum lithium concentration was measured using The Medi- synthesis kit for RT-PCR (Roche Diagnostics). Real-time PCR mate MiniLab (Enschede, The Netherlands). was performed using a cDNA quantity corresponding to 0.1 mm of nephron segments using the LightCycler 480 IHC SYBR Green I Master qPCR kit (Roche Diagnostics). In each Kidneys were immersion fixed in 3.5% paraformaldehyde in experiment, a standardization curve was made using serial 0.1 M phosphate buffer for 3 days. Tissues were then dehy- dilutions of a standard cDNA stock solution made from drated in ethanol, incubated in xylene, and embedded in par- whole-kidney RNA. The amount of PCR product was calcu- affin. Paraffin sections (approximately 2 mm) were labeled lated as the percentage of standard DNA and gene expression with antibodies recognizing either AQP229 (dilution 1:8000) was normalized as a function of that of the housekeeping gene or H+-ATPase7 (dilution 1:1500). Labeling was visualized us- Rpl26, as done previously. The use of Rpl26 as a housekeeping ing peroxidase-conjugated goat anti-rabbit secondary anti- gene was validated using housekeeping gene Ppia, demonstrat- body (Dako, Glostrup, Denmark) and 3,39-diaminobenzidine. ing highly similar relative expression levels for the different Cell counting was performed on the microscope in the inner nephron segments (data not shown). medulla on sections labeled for H+-ATPase and was blinded to the group. The numbers of positive (labeled) and negative GWA Mapping Analysis (unlabeled) cells were counted in tubules containing at least Genome-wide analysis wasperformed using the Efficient Mixed one positive cell and with a visible lumen. Model Association (EMMA; http://mouse.cs.ucla.edu/emma) method, which uses a linear mixed model algorithm to control Immunoblotting for population structure and genetic relatedness.34 Groups Whole-kidney material (from half kidney) and medulla were with a sample size of less than three were not included in the homogenized in 1 ml and 300 ml of ice-cold homogenization analysis because the reliability of such data were considered as buffer and diluted in Laemmli buffer, respectively, as de- not sufficient. We used urine volume and osmolality ratios scribed. SDS-PAGE, blotting, and blocking of the polyvinyli- from day 10 and 28 as phenotype input, which consisted of dene difluoride membranes were done as described,30 using the absolute urine volume/osmolality values of the control ver- affinity-purified rabbit precarboxy tail AQP2 antibody recog- sus lithium treatment group, and an NDI data set containing nizing amino acids 236–255 (1:2000) and goat anti-rabbit binary data comprising both phenotypes. Strains that devel- IgGs coupled to horseradish peroxidase (Sigma, St. Louis, oped NDI, defined by a more than twofold increased ratio MO) as secondary antibodies.31 Densitrometric analysis of urine output and .2.5-fold decreased ratio of urine osmo- was done using Bio-Rad quantification equipment (690c densi- lality, were marked with “1;” whereas strains without NDI, de- tometer, Chemidoc XRS; Bio-Rad) and software (QuantityOne; fined by a less than twofold increased ratio of urine output

JASN 30: ccc–ccc,2019 GWAS in Mouse Lithium-Induced NDI 3 4 acontrol( 1. Figure ubro tan,icuigWB Z,adPD twsntpsil ocletbodi h eurdnme fmc o h statistical the for mice of number (Ctr), required Control the (B). in on blood based collect is to small D) possible a and In not levels. (C was lithium serum on it determine strains to PWD, of collected order and was blood The NZO, analysis. 18 WSB, day including at (D) strains, and of determined, was number production urine 24-hour 28, (C) and 10 AI RESEARCH BASIC JASN C A B D Li n h feto ihu nuiepouto ifr mn os tan.()Mc rm2 ifrn nrdsriswr rae with treated were strains inbred different 29 from Mice (A) strains. mouse among differs production urine on lithium of effect The Ctr (n=8) 8 rltim(n lithium or =8) + Serum Li+ levels (mM) Urine output from day 9-10 (n=10)

Urine output (mL/20 g bodyweight/24h) (29 strains) 0.0 0.2 0.4 0.6 0.8 start diets day 0 12 (mL/20 g bodyweight/24h) 12 0 3 6 9 0 3 6 9 * C3H C3H * C3H www.jasn.org * SM SM * SM 1)de o 0ad2 asadwr osdi eaoi ae rmdy o1 n 6t 8 t()day (B) At 28. to 26 and 10 to 8 days from cages metabolic in housed were and days 28 and 10 for diet =10) * BUB BUB * BUB x * xs()i ae nltimefc nuiepouto satn ihlretaslt nrae;teodrin order the increase); absolute largest with (starting production urine on effect lithium on based is (B) axis SWR SWR SWR * B6 B6 * B6 10 9 8 metabolic cage * * n FVB FVB FVB =3 * * * – SJL SJL SJL ;Ltimtetd(Li treated Lithium 8; (ions andgasses) * * NOD NOD NOD blood sampling C57L C57L C57L

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7.0 A P B6 PL LP D2 KK SM 129 SJL B10 FVB C3H CBA BUB MRL NON NOD RIIIS NZW SWR C57L BLKS BALB BTBR

Figure 2. The effect of lithium on blood pH differs among mouse strains. Mice from 29 different inbred strains were treated with a control or lithium diet for 10 and 28 days. At (A) day 10 and (B) 28, blood was sampled by cheek puncture and pH was determined for 25 strains at both time points. In the remaining strains it was not possible to collect blood in the required number of mice for the statistical analysis. Order strains on x axis (A) is based on lithium effect on blood pH (starting with largest relative decrease); the order in (B) is based on (A). Control (Ctr), n=3–8; lithium treated (Li+), n=3–10. *P,0.002, significantly different from control. and ,2.5-fold decreased ratio of urine osmolality, were threshold resulted in few significant associations by compar- marked with “0.” These ratio thresholds were based on the ing GWA analyses from different data sets with similar vari- definition of diabetes insipidus in patients, in which similar ation. Because of the lack of many GWA studies (GWAS) in ratios are used for urine volume and osmolality as compared mice, it remains difficult to implement a commonly accepted with normal levels.35 All strains that did not fall into the threshold, however this P value is also implemented in other above-mentioned criteria were not included in the analysis. studies.38,39 Associations exceeding the threshold were ex- For the exact number of mice per strain for each analysis, see cluded from further analysis if only one or two strains shared Supplemental Table 1. The analysis was carried out using a the associated haplotype. Loci were established by putting panel of 4,016,612 single-nucleotide polymorphisms (SNPs), together all associations with the same haplotype within which were previously identified in four wild-derived and 11 a distance of 1.0 Mb of each other, whereas SNPs with 2 classic strains36 and subsequently imputed with high confi- P values ,1310 5 were also included to determine the dence for the less densely typed classic inbred strains.37 Each boundaries of a locus. Loci that only contained one or two SNP was evaluated individually and those without variation SNPswereexcluded from furtheranalysis.Genome sequences in the set of inbred strains were automatically filtered out by within the candidate regions were compared between the dif- the EMMA server. P values were recorded as the strength of ferent strains based on their haplotype distribution using the the genotype-phenotype association and were transformed Sanger institute Mouse Genomes database (www.sanger.ac.uk/ using 2log10 (P value) in the scan plot. We only considered resources/mouse/genomes/), which also contains whole- 2 P values exceeding the threshold of 1310 6,becausethis genome sequences of ten strains that were included in the

JASN 30: ccc–ccc,2019 GWAS in Mouse Lithium-Induced NDI 5 BASIC RESEARCH www.jasn.org

A 1.5 Ctr Li+

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1.1 A P PL B6 LP D2 KK SM 129 SJL B10 FVB C3H BUB CBA MRL NON NOD RIIIS NZW SWR C57L BALB BLKS BTBR

Figure 3. The effect of lithium on blood Ca2+ differs among mouse strains. Mice from 29 different inbred strains were treated with a control or lithium diet for 10 and 28 days. At (A) day 10 and (B) 28, blood was sampled by cheek puncture and blood calcium ions (Ca2+) was determined, as depicted for 26 and 25 strains at day 10 and 28, respectively. In the remaining strains it was not possible to collect blood in the required number of mice for the statistical analysis. Order strains on x axis (A) is based on lithium effect on blood Ca2+ (starting with largest relative increase); the order in (B) is based on (A). Control (Ctr), n=3–8; lithium (Li+), n=3–10. (A) *P,0.002 (A) and (B) *P,0.002, significantly different from control.

GWA analysis. Whole-genome sequences of these strains, Bonferroni multiple-comparisons procedure by dividing also representing other strains with the same haplotype, 0.05 by the number of included strains or genes. were then assessed for potential deleterious effects of nonsy- nonymous variations on the structure or function of the protein using this website via the Variant Effect Predic- RESULTS tor software from ENSEMBL.40 A SIFT score of ,0.05 was considered deleterious.40 Li-NDI Develops Differently among Inbred Mouse Strains Statistical Analysis To identify susceptibility genes for different lithiopathies, fe- Data are presented as mean with SEM. t tests (one-tailed test male mice from 29 different inbred strains (for abbreviations of with unequal variances) were used to evaluate the differences the strains and number of mice per strain, see Supplemental between control and lithium-treated mice of 29 strains. Only Table 1) were treated with a control or lithium diet, starting at strains with a sample size of three or more were included in the 10 weeks of age. Following lithium treatment, mice were analysis. A one-way ANOVA with Bonferroni correction was housed in metabolic cages at days 8–10 and 26–28 to collect applied for the mRNA expression data and the data on the urine during the last 24 hours of each period, whereas blood Acer2 mutant mice. A threshold of P,0.05 was used to test was collected at three different time points (Figure 1A). The for significance which, if required, was corrected using the wild-derived strains CAST, PWD, and WSB demonstrated a

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Table 1. Urine volume and osmolality ratios with included strains for binary analysis Day 10 NDI Day 28 NDI Strains Urine Volume Urine Osmolality NDI Urine Volume Urine Osmolality NDI (Li/Ctr) (Ctr/Li) (Binary Data) (Li/Ctr) (Ctr/Li) (Binary Data) 129 1.9 1.4 0 2.2 2.0 — A 1.2 1.5 0 1.7 2.0 0 B10 6.1 3.6 1 4.0 2.5 1 B6 7.8 5.7 1 4.6 4.2 1 BALB 1.4 1.4 0 1.1 1.0 0 BLKS 2.4 2.5 — 1.6 1.7 0 BTBR 1.8 1.9 0 2.9 2.7 1 BUB 4.6 3.9 1 3.1 3.0 1 C3H 10.5 9.3 1 14.2 11.9 1 C57L 2.9 2.6 1 1.9 1.3 0 CBA 3.0 2.0 — 2.0 1.5 — D2 1.3 1.6 0 1.3 1.5 0 FVB 10.4 8.6 1 6.7 4.8 1 KK 3.4 2.4 — 2.3 2.6 1 LP 1.2 1.5 0 2.4 1.9 — MRL 2.2 2.3 — 2.6 2.3 — NOD 3.0 2.8 1 2.3 2.4 — NON 7.2 3.7 1 15.8 4.8 1 NZO 1.6 2.1 0 2.2 1.7 — NZW 1.6 1.2 0 1.3 1.1 0 P 1.9 1.9 0 2.2 2.9 1 PL 2.7 2.4 — 2.2 1.8 — RIIIS 2.5 2.3 — 0.8 1.7 0 SJL 3.6 3.1 1 6.9 6.6 1 SM 15.6 10.2 1 11.1 4.2 1 SWR 6.2 5.0 1 5.5 4.1 1 Ctr, control; Li, lithium treated. lot of food spoilage in the urinary collection system of the blood hematocrit and Na+ levels after 10 and 28 days of lith- metabolic cages. Therefore, our read out of urine volume ium treatment. Lithium did not significantly alter hematocrit and osmolality for the wild-derived strains are less reliable. in any of the strains; but after treatment for 28 days, Na+ At 10 days of lithium treatment, urine output was significantly concentrations were increased in LP mice and decreased increased in 17 strains (e.g.,C3H,SM,BUB,andSWR), in C57L mice (Supplemental Table 2). Hematocrit and blood whereas urine output in other strains (e.g., BALB, LP, and A) Na+ levels were unaltered in the vast majority of strains. As was not affected by lithium (Figure 1B). Consistent with the PGE2 has been described as one of the most important in- NDI phenotype, most mice with an increased urine volume dependent factors in Li-NDI development, we measured uri- exhibited a decreased urine osmolality (Supplemental Figure 1A) nary PGEM levels from day 9 to 10. However, none of the and increased water intake (Supplemental Figure 2A). Prolong- strains demonstrated a significant difference in urinary ing lithium treatment of up to 28 days also resulted in large PGEM content (Supplemental Figure 5). differences in urine output (Figure 1C), urine osmolality (Sup- plemental Figure 1B), and water intake (Supplemental Figure 2B) The Effect of Lithium on the Acid-Base Balance between strains. Serum lithium levels varied from 0.34 to To investigate whether lithium affected the acid-base balance, 2 0.71 mM among the strains (Figure 1D) and did not correlate blood pH, pCO2,andHCO3 , urinary pH was measured af- with the lithium effect on urine output or urine osmolality at ter 10 and 28 days of lithium treatment. With lithium treat- day 10 or 28 (Supplemental Figure 3, A–D). Moreover, in most ment, blood pH was only significantly decreased in BUB strains, the extent of Li-NDI on days 10 and 28 was similar and P mice after 10 and 28 days, respectively (Figure 2). The (Supplemental Figure 4). effects of lithium on blood pH per strain from day 10 corre- lated with day 28 (Supplemental Figure 6A), but did not cor- Lithium Does Not Affect Blood Hematocrit, Na+,or relate with the extent of Li-NDI (Supplemental Figure 6, B and PGE2 Excretion in Most Strains C). Lithium did not affect blood pCO2 in any strain at 10 or To investigate whether the extent of Li-NDI affected volume 28 days (Supplemental Figure 7) and only decreased blood 2 status or blood electrolyte concentrations, we determined HCO3 in the BUB strain after 10 days of lithium treatment

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Table 2. Genetic peak locations associating with development of Li-NDI Duration Treatment Locus (Mb)a P Value Data File Genes in Intervalb 2 10 and 28 d 19 18.56–18.67 1.7310 07 Binary Ostf1 2 10 d 2 143.86–145.38 5.2310 08 Binary Banf2, Snx5, 8430406107Rik, Ovol2, Csrp2bp, Dzank1, Polr3f, Rbbp9, Sec23b, Gm561, Dtd1, 1700010M22Rik, Slc24a3 2 475.64–76.97 1.0310 266 Binary, urine osmolality Ptprd 2 485.03–86.57 4.2310 07 Binary AK044374, Adamtsl1, Fam154a, AK015482, Rraga, Haus6, Scarna8, Plin2, Dennd4c, Rps6, Acer2 2 676.49–77.07 5.2310 07 Binary Ctnna2 2 8 126.36–126.93 5.2310 07 Binary 1700054N08Rik, Acta1, Nup133, AK013187, Abcb10, Taf51, mKIAA0133, Mir1967, Urb2, Galnt2, AK085459, Pgbd5 2 19 44.78–45.07 4.4310 08 Binary Pax2, Fam178a, Sema4g 2 19 48.57–48.61 2.3310 239 Binary, urine osmolality Sorcs3 aNational Center for Biotechnology Information m37 assembly. bBased on University of California Santa Cruz Genome Browser (genome.ucsc.edu).

(Supplemental Figure 8). After both 10 and 28 days, lithium treatment group, and an NDI data set containing binary data increased urine pH in most strains, including the BUB strain composed of both phenotypes (Table 1). Genome-wide scans (Supplemental Figure 9, A and B), suggesting a decreased abil- from day 10 and 28 demonstrated various associations exceed- 2 26 ity to excrete protons/reabsorb HCO3 . However, the pH of a ing our threshold of 1310 (Figure 4). Haplotypes of all solution is affected by dilution. Indeed, the lithium-induced significant associations were determined. Although the increase in urine pH strongly correlated with NDI develop- genome-wide scan of the urine volume analysis of day 28 re- ment (Supplemental Figure 9, A and B). Correcting urine pH vealed many loci exceeding the threshold, all were excluded for urine output by analyzing total free hydrogen ion (H+) from further analysis due to the presence of only one or two excretion revealed there were no significant differences at strains in a haplotype block. This led to the identification of day 10; whereas after 28 days of lithium treatment, free H+ eight gene-containing loci with up to 13 genes in the interval excretion was increased in eight strains (Supplemental on chromosome 2 (Chr 2), Chr 4, Chr 6, Chr 8, and Chr 19 Figure 10). (Table 2). Of these genome-wide scans, QQ plots were gener- ated and genomic inflation was calculated (Supplemental Lithium Induces Hypercalcemia in SWR, A, C3H, and Figure 12). CBA Mice To determine whether lithium induced hypercalcemia in one Expression of Candidate Genes in Renal Tubule or more strains, iCa2+ was analyzed. After 10 days, lithium To determine which candidate genes are involved in the devel- significantly increased blood iCa2+ levels in SWR, A, C3H, opment of Li-NDI, we narrowed the candidate list by selecting and CBA mice; whereas after 28 days no significant changes for genes that are involved in the regulation of glycogen were observed (Figure 3). The effects of lithium on iCa2+ were synthase kinase type 3, osmoregulation in the collecting not correlated with serum lithium levels nor with the devel- duct, or cell proliferation via literature search in PubMed. opment of Li-NDI (Supplemental Figure 11, A–D). This resulted in the identification of Ovol2, Rbbp9, Ptprd, Plin2, Acer2, Pax2, Urb2,andGalnt2.41–45 Because Li-NDI GWAS is a disorder of the renal connecting tubule and collecting We thus identified many strains that developed Li-NDI, duct, we then determined their segment-specific expression whereas only a few strains developed metabolic acidosis or using reverse transcription quantitative PCR on RNA isolated hypercalcemia with lithium. As GWA requires variation in from different segments of adult C57BL/6J mice. Although all the parameter of interest between most of the strains in the genes were expressed in connecting tubules and collecting study, GWA was only possible for Li-NDI. The wild-derived duct, only Acer2 expression was enriched in the collecting strains (CAST, WSB, PWD) were excluded from this analysis duct because its levels were significantly higher in these tu- due to the increased food spoilage in the urinary collection bules than in all other segments (Figure 5). Convincingly, this system of the metabolic cages. To identify loci associated with segment-specific distribution of Acer2 expression was similar Li-NDI, genome-wide analysis was performed for urine vol- to that found in rat.46 The locus containing Acer2 harbored ume and osmolality ratios, consisting of the absolute numerous variations among the strains. By analyzing whole- urine volume/osmolality values of the control versus lithium genome sequence data from ten strains (representing both

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0.20

0.15

0.10

*

mRNA (units/mm) 0.05 *

0.00 PST PST PST PST PCT PCT PCT PCT DCT CNT DCT CNT DCT CNT DCT CNT CCD CCD CCD CCD CTAL CTAL CTAL CTAL MTAL MTAL MTAL MTAL OMCD OMCD OMCD OMCD

Ovol2 Plin2 Acer2 Pax2

0.03

0.02

0.01 mRNA (units/mm)

0.00 PST PST PST PST PCT PCT PCT PCT DCT CNT DCT CNT DCT CNT DCT CNT CCD CCD CCD CCD CTAL CTAL CTAL CTAL MTAL MTAL MTAL MTAL OMCD OMCD OMCD OMCD

Rbbp9 Ptprd Urb2 Galnt2

Figure 5. Expression of Ovol2, Rbbp9, Ptprd, Plin2, Acer2, Pax2, Urb2,andGalnt2 differs in the mouse kidney. mRNA was isolated from different nephron segments from C57BL/6J mice, and subjected to reverse transcription quantitative PCR to determine mRNA levels of Ovol2, Rbbp9, Ptprd, Plin2, Acer2, Pax2, Urb2,andGalnt2 along the nephron. The determined mRNA levels were normalized to that of housekeeping gene Rpl26. Values are mean6SEM from six mice. Indicated segments are proximal convoluted (S1) and straight (S3) tubule (PCT, PST), medullary (MTAL) and cortical (CTAL) thick ascending limb of the loop of Henle, distal convoluted tubule (DCT), connecting tubule (CNT), and the cortical (CCD) and outer medullary (OMCD) collecting duct. *P,0.006, significantly different from PCT, PST, MTAL, CTAL, and DCT.

2 haplotypes) via the Sanger institute Mouse Genomes database, with their wild-type littermates, whereas urine volume of Acer2+/ 2 2 we found that none of these variations resulted in an amino mice was in between that of Acer2+/+ and Acer2 / mice. Urine acid change predicted to be deleterious for protein structure or osmolality of control diet mice was similar between the differ- function. Thus, rather than a protein-inactivating mutation, ent genotypes, whereas urine osmolality of lithium-treated 2 2 it suggests the expression of Acer2 might be associated with Acer2 / mice was significantly lower as compared with 2 the susceptibility to Li-NDI development. wild-type controls, and urine osmolality of Acer2+/ mice 2 2 was again between that of the Acer2+/+ and Acer2 / mice Acer2 Expression Determines Susceptibility to the (Figure 6B). Importantly, serum and urine lithium levels Development of Li-NDI were not different among the different groups (Supplemental To investigate the role of Acer2 in the development Figure 13, A and B). To determine whether the effect of Acer2 of Li-NDI, we obtained an Acer2 mutant mouse strain28 and on urine output and osmolality was due to changes in AQP2 2 2 2 treated Acer2 / animals and Acer2+/ and Acer2+/+ littermates abundance, AQP2 localization, or principal cell–intercalated for 10 days with a control or lithium diet. In the control groups, cell ratio, we performed AQP2 Western blotting and IHC. Al- 24-hour urine volume was not different between the different though AQP2 abundance in the whole kidney did not signifi- 2 2 genotypes (Figure 6A). With lithium treatment, however, urine cantly differ among lithium-treated Acer2+/+ and Acer2 / mice 2 2 volume was 1.8-times higher in Acer2 / mice as compared (data not shown), we found that AQP2 abundance was reduced

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A B 8 6000 * * 6 4500

3000 4 1200

2 600 Urine osmolality (mOsm/kg) Urine output (mL/20g output bw/24h) Urine 0 0 Ctr Li Ctr Li Ctr Li Ctr Li Ctr Li Ctr Li Acer2 +/+ Acer2 +/- Acer2 -/- Acer2 +/+ Acer2 +/- Acer2 -/- C D Acer2 +/+ Acer2 -/- Acer2 +/+ Acer2 -/- 125 * Ctr Ctr Ctr Li Li kDa 100 * 45 75

50 31 25 AQP2 abundance (%) abundance AQP2 0 Cm Ctr Li Ctr Li AQP2 Acer2 +/+ Acer2 -/- E

'

2 2 2 Figure 6. The development of Li-NDI is aggravated in Acer2 knockout mice. Acer2+/+,Acer2+/ , and Acer2 / mice were bred and, at an age of 10 weeks, treated with lithium for 10 days. The development of Li-NDI was studied by housing the mice during the last 48 hours in metabolic cages and analyzing (A) urine output and (B) urine osmolality of the urine from the last 24 hours. (C) Furthermore, kidneys were isolated and subjected to Western blotting to determine AQP2 abundance. Molecular masses of proteins are indicated

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2 2 in the medulla of control and lithium-treated Acer2 / mice as treatment. The decreased blood pH in BUB mice coincided compared with their wild-type control mice (Figure 6, C and D). with a significant decrease in blood bicarbonate, whereas blood IHC for AQP2 in the inner stripe of the outer medulla pCO2 was not changed, demonstrating that the small decrease (Figure 6E) and the inner medulla (Supplemental Figure 13C) in blood pH was indeed from metabolic origin. In contrast, in did not demonstrate any difference in AQP2 localization be- P mice blood bicarbonate at 28 days was not affected, whereas +/+ 2/2 tween the Acer2 and Acer2 mice. In addition, there were blood pCO2 levels seemed to increase. This effect might result no significant changes in the fraction of H+-ATPase–positive from the observed sensitivity of this strain to the repetitive cells (marker of intercalated cells) in the inner medulla between blood draws (maximum 150 ml per draw), because these 2 2 the four groups (Acer2+/+ control, 0.2960.02 [n=5]; Acer2 / con- mice needed more time to recover than all other strains. More- 2 2 trol, 0.2860.02 [n=5]; Acer2+/+ Li, 0.3260.04 (n=5); Acer2 / Li, over,themiceonlithiumtreatmentseemedmoreaffected 0.2160.04 [n=4]) (Supplemental Figure 14). To reveal the than the control mice. The relatively mild effects on blood mechanism by which an altered expression of Acer2, an alkaline pH might be due to the rather low serum lithium levels ceramidase, would affect the susceptibility for Li-NDI, (0.34–0.71 mM) in our study, although most other studies we tested whether Acer2 mutant mice demonstrated altered with blood lithium levels around 1 mM also do not find a ceramide levels in medulla tissue, however we did not find lithium-induced metabolic acidosis.15–20 Finally, we found significant differences (Supplemental Figure 15). that lithium treatment for 28 days increased the total free urinary H+ levels in eight strains. Because we did not determine urinary net acid excretion, we cannot determine whether the elevated H+ DISCUSSION levels coincided with a net overall increase in acid production. This is likely because our recent study using C57BL/6J mice The Genetic Background of a Mouse Strain Determines treated with lithium in identical conditions revealed increased Its Susceptibility to Develop Li-NDI, Metabolic urine ammonium levels,48 a phenomenon also found in recent Acidosis, or Hypercalcemia studies with rats.18,49 Altogether, it is likely that lithium impairs A substantial number of patients with bipolar disorder receiv- acid excretion, followed by a compensatory increase in produc- ing lithium treatment develop side effects such as NDI, hyper- tion of titratable acids and acid excretion. calcemia, and possibly metabolic acidosis. In this study, we Finally, elevated blood calcium ion levels were found in treated 29 different mouse inbred strains with a control or SWR, A, C3H, and CBA mice after 10, but not after 28, days lithium diet and analyzed the development of these side effects of lithium treatment. It must be noted that the development of after 10 and 28 days. Although lithium did not cause side effects hypercalcemia is likely dependent on blood lithium levels, in some strains, other strains developed side effects. Because which were rather low in our study. Altogether we identified treatment and housing conditions were identical and blood different mouse strains with an increased susceptibility to lithium concentrations were similar, the different susceptibility develop lithium-induced hypercalcemia, which will facilitate of these strainsto develop side effects must be due to differences follow-up studies on this topic. in their genetic background. NDIwas found in many strains after 10 or 28 days of lithium Identification of Acer2 as a First Susceptibility Gene for treatment, although there were also strains in which lithium Li-NDI Using Haplotype Association Mapping and did not affect urine concentration. In most strains, Li-NDI Acer2 Knockout Mice development after 10 days did not differ much from 28 days. Genome-wide analysis on data of Li-NDI resulted in the iden- During treatment all mice had access to a salt lick. We observed tification of eight gene-containing loci whichwere significantly that strains with severe Li-NDI used more of the salt lick than associated with the susceptibility of strains to develop NDI. strains without Li-NDI. Increased salt use as a consequence of Having selected Acer2 as a most promising candidate to volume lossdue to Li-NDIdevelopment is likely the underlying be involved in Li-NDI, we investigated its potential role in reason. Furthermore, as we did not measure the actual salt Li-NDI. At similar blood lithium levels, Acer2 knockout intake, we cannot exclude the possibility that differences in mice were more susceptible to develop Li-NDI than their salt craving between strains might have had a confounding wild-type littermates because the homozygous knockout effect on Li-NDI development, as very low salt intake attenu- mice demonstrated a significantly higher urine output and ates the development of Li-NDI.47 lower urine osmolality after lithium treatment. These effects Asmallbutsignificant decrease in blood pH was identified coincided with a further reduction of AQP2 abundance in in BUB and P mice after 10 and 28 days, respectively, of lithium both control as lithium-treated conditions. The fact that

on the left (in kDa). (D) The signals for nonglycosylated (29 kDa) and complex-glycosylated (40–45 kDa) AQP2, as indicated, were den- sitometrically semiquantified. Coomassie staining of the blots confirmed loading of protein equivalents. (E) Furthermore, IHC was per- formed to determine AQP2 abundance and localization. Control, Ctr; Hom, homozygous knockout mice; ISOM, inner stripe of the outer medulla; Li, lithium treated; mOsm, milliosmole; WT, wild type.

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AQP2 expression was already lowered in the control situation Dr. de Groot, Ms. Ebert, Dr. Christensen, Dr. Deen, and Dr. Korstanje but did not have strong effects on urine output or osmolality drafted and revised the paper; all authors approved the final version of in these conditions suggests that Acer2 might affect water bal- the manuscript. ance, which can be compensated for in the control situation, but becomes problematic during lithium conditions. ACER2 belongs to the family of which remove DISCLOSURES fatty acids from the lipid molecule ceramide, thereby produc- ing , which can be phosphorylated to form sphin- None. gosine-1-phosphate (S1P).50 Ceramide, sphingosine, and S1P have important signaling functions which affect cellular process- es like proliferation, apoptosis, and differentiation.50,51 In this FUNDING study, we did not find altered ceramide levels in medulla tissue of Acer2 knockout mice. The absence of any difference in ceram- This project received support from a grant from the Society of Experimental ide levels might be due to other ceramidases regulating renal Laboratory Medicine to Dr. Deen and a Marie Curie fellowship PIOF-GA- 2012-332395 and a Niels Stensen Fellowship to Dr. de Groot. ceramide levels. Despite the specific expression of Acer2 in the collecting duct, and the absence or noncollecting duct–specific expression of Acer1 and Acer3, respectively (data not shown), we cannot be sure that collecting duct ACER2 regulates Li- SUPPLEMENTAL MATERIAL NDI. Rather, a recent publication demonstrated that mice with Acer2 knockout in whole-body or specifically in hemato- This article contains the following supplemental material online at poietic cells exhibit decreased S1P blood levels.28 Importantly, http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018050549/-/ S1P also increases sodium excretion,52,53 likely by inhibition of DCSupplemental. ENaC.52 The reduced S1P blood levels in the Acer2 knockout Supplemental Figure 1. The effect of lithium on urine osmolality in mice might lead to ENaC activation which, during lithium 29 mouse strains. treatment, might lead to an enhanced uptake of lithium and Supplemental Figure 2. The effect of lithium on water intake in thus an increased susceptibility to Li-NDI. Follow-up studies 29 mouse strains. should investigate whether S1P indeed plays an essential role in Supplemental Figure 3. Relationship between serum lithium levels the development of Li-NDI and whether its application might and development of Li-NDI. attenuate Li-NDI. Supplemental Figure 4. Relationship Li-NDIdevelopment between day 10 and 28. From GWAS in Mice to Determining Susceptibility for Supplemental Figure 5. The effect of a 10-day lithium treatment on Lithium-Induced Side Effects in Patients urinary PGEM levels in various mouse strains. In this study, we identified that Acer2 plays a role in Li-NDI, Supplemental Figure 6. Relationship between blood pH and Li- however many other genes remain to be investigated. This NDI development. might lead to the identification of novel pathways in the de- Supplemental Figure 7. The effect of lithium on blood pCO2 in velopment of Li-NDI. Having established a more complete set various mouse strains. 2 of pathways involved in Li-NDI, the next step would be Supplemental Figure 8. The effect of lithium on blood HCO3 in to perform GWAS in the human population and find the var- various mouse strains. iations that significantly correlate with the development of Supplemental Figure 9. The effect of lithium on urine pH in lithium-induced side effects. Using the insight of important 29 mouse strains. proteins and pathways in Li-NDI, as established by previous, Supplemental Figure 10. The effect of lithium on free urinary H+ current, and future animal studies, this would enhance the content in 29 mouse strains. chances of successfully performing an expensive GWAS on Supplemental Figure 11. Relationship between effect of lithium on patients treated with lithium. blood Ca2+ and development of Li-NDI. Supplemental Figure 12. Genomic inflation. Supplemental Figure 13. Serum and urine lithium content and inner medullary AQP2 localization in ACER2 knockout mice. ACKNOWLEDGMENTS Supplemental Figure 14. The density of intercalated cells is not different for wildtype and Acer2 knockout mice. We would like to thank Ms. Holly Savage, Mr. Thomas O’Rourke, and Supplemental Figure 15. Ceramide levels in medullary tissue of Ms. Rita O’Rourke (from the Jackson Laboratory) for their expert help. male and female Acer2 Ko mice. Dr.deGroot,Dr.Deen,andDr.Korstanjedesignedthestudy; Supplemental Table 1. Names of the 29 inbred mouse strains, their Dr.deGroot,Ms.Ebert,Dr.Christensen,Dr.Andralojc,Ms.Cheval, abbreviations and number of mice used per analysis. and Dr. Sandhoff carried out experiments and analyzed the data; Supplemental Table 2. Blood Na+ and Hct levels in 29 different Dr.deGroot,Ms.Ebert,andDr.Christensenmadethefigures; mouse strains treated for 10 and 28 days with lithium.

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14 JASN JASN 30: ccc–ccc,2019 www.jasn.org BASIC RESEARCH

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AFFILIATIONS

1The Jackson Laboratory, Bar Harbor, Maine; Departments of 2Physiology, 6Molecular Biology, 7Biochemistry, and 8Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands; 3Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands; 4Department II of Internal Medicine, Center for Molecular Medicine Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; 5Department of Biomedicine, Aarhus University, Aarhus, Denmark; 9Cordeliers Research Center, Sorbonne University, Pierre and Marie Curie University Paris 06, INSERM (Institut National de la Santé et de la Recherche Médicale), Paris Descartes University, Sorbonne Paris Cité, UMR_S (Unité Mixte de Recherche en Sciences) 1138, Paris, France; 10Physiology of Renal and Tubulopathies, CNRS (Centre National de la Recherche Scientifique) ERL 8228, Cordeliers Research Center, INSERM, Sorbonne University, Sorbonne Paris Cité University, Paris Descartes University, Paris Diderot University, Paris, France; 11Department of Medicine, Stony Brook University, Stony Brook, New York; 12Stony Brook Cancer Center, Stony Brook, New York; 13Reinier- Haga Medical Diagnostic Center, Delft, The Netherlands; 14Lipid Pathobiochemistry Group, Department of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Heidelberg, Germany; and 15Centre for Applied Sciences at Technical Universities (ZAFH)–Applied Biomedical Mass Spectrometry (ABIMAS), Mannheim, Germany

JASN 30: ccc–ccc,2019 GWAS in Mouse Lithium-Induced NDI 15 Table of Contents PAGE Figure S1. The effect of lithium on urine osmolality in 29 mouse strains. 2 Figure S2. The effect of lithium on water intake in 29 mouse strains. 3 Figure S3. Relationship between serum lithium levels and development of Li-NDI. 4 Figure S4. Relationship Li-NDI development between day 10 and day 28. 5 Figure S5. The effect of a 10-day lithium treatment on urinary PGEM levels in various mouse strains. 6 Figure S6. Relationship between blood pH and Li-NDI development. 7 Figure S7. The effect of lithium on blood pCO 2 in various mouse strains. 8 Figure S8. The effect of lithium on blood HCO 3- in various mouse strains. 9 Figure S9. The effect of lithium on urine pH in 29 mouse strains. 10 Figure S10. The effect of lithium on free urinary H+ content in 29 mouse strains. 11 Figure S11. Relationship between effect of lithium on blood Ca 2+ and development of Li-NDI. 12 Figure S12. Genomic inflation. 13 Figure S13. Serum and urine lithium content and inner medullary AQP2 localization in ACER2 knockout mice. 14 Figure S14. The density of intercalated cells is not different for wildtype and Acer2 knockout mice. 15 Figure S15. Ceramide levels in medullary tissue of male and female Acer2 Ko mice. 16 Supplementary Table S1. Names of the 29 inbred mouse strains, their abbreviations and number of mice used per analysis. 17 Supplementary Table S2. Blood Na + and Hct levels in 29 different mouse strains treated for 10 and 28 days with lithium. 18

1

Figure S1. The effect of lithium on urine osmolality in 29 mouse strains. 29 different inbred mouse strains were treated with a control (n=8) or lithium (n=10) diet for 10 and 28 days and were housed in metabolic cages from day 8-10 and day 26-28. At day 10 ( A) and 28 (B), 24-hour urine production was collected and urine osmolality was determined. Order strains on x-axis ( A and B) is based on Figure 1B . *P<0.0017, significantly different from control.

A

12000 Ctr Li +

9000

6000

* 3000 * * * * * * * * * * * * * * * * * 0 Urine osmolality from day 9-10 (mOsmo/kg) from osmolality Urine A P LP B6 PL D2 KK SM SJL 129 B10 C3H FVB MRL BUB CBA NZO NOD NON NZW PWD SWR RIIIS WSB C57L CAST BLKS BTBR BALB B 8000 Ctr Li +

6000

4000 * * * * * 2000 * * * * * * * * * * * 0 Urine osmolality from day from 27-28 osmolality (mOsmo/kg) Urine A P B6 PL LP D2 KK SM SJL 129 B10 C3H FVB BUB MRL CBA NZO NOD NON NZW SWR RIIIS WSB PWD C57L BLKS BTBR BALB CAST

2 Figure S2. The effect of lithium on water intake in 29 mouse strains. 29 different inbred mouse strains were treated with a control (n=8) or lithium (n=10) diet for 10 and 28 days and were housed in metabolic cages from day 8-10 and day 26-28. Water intake was assessed at day 10 ( A) and 28 ( B). Order strains on x-axis ( A and B) is based on Figure 1B . *P<0.0017, significantly different from control.

A 20 Ctr * Li +

15 * * * * 10 * * * * 5 * (mL/20 g (mL/20 bodyweight/24h) Water intake from day intake9-10 from Water

0 A P B6 LP D2 PL KK SM SJL 129 B10 C3H FVB BUB CBA NZO MRL NZW NOD NON PWD SWR RIIIS WSB C57L CAST BALB BLKS BTBR B 20 * Ctr Li +

15 * * * 10 * * * * *

5 *

(mL/20 g(mL/20 bodyweight/24h) * Water intake from day intake27-28 from Water * *

0 A P B6 LP PL D2 KK SM SJL 129 B10 C3H FVB BUB MRL CBA NZO NZW NOD NON PWD SWR RIIIS WSB C57L CAST BLKS BTBR BALB C D 6 6 r=0.82 r=0.80 p<0.0001 p<0.0001

4 4

2 2 intake per strain from day 9-10 9-10 day from strain per intake intake per strain from day 27-28 27-28 day from strain per intake Lithium-induced fold increase in water water in increase fold Lithium-induced Lithium-induced fold increase in water water in increase fold Lithium-induced 0 0 0 4 8 12 16 0 4 8 12 16 Lithium-induced fold increase in urine Lithium-induced fold increase in urine output per strain from day 9-10 output per strain from day 27-28

3 Figure S3. Relationship between serum lithium levels and development of Li-NDI. Serum lithium levels, determined after 18 days of lithium treatment were compared with urine output after 10 (A) and 28 days (C) and with urine osmolality after 10 (B) and 28 days (D) of lithium treatment. No significant correlations were found in these four comparisons.

A 0.8 C 0.8 r=0.0091 p=0.96 0.6 0.6

0.4 0.4

0.2 r=0.17 0.2 p=0.41

Serum lithium per strain at day 18 (mM) 18 day at strain per lithium Serum 0.0 (mM) 18 day at strain per lithium Serum 0.0 0 4 8 12 16 0 4 8 12 16 Lithium-induced fold increase in urine output Lithium-induced fold increase in urine output per strain from day 9-10 per strain from day 27-28

B D 0.8 0.8 r=0.19 p=0.34 r=0.0049 p=0.91 0.6 0.6

0.4 0.4

0.2 0.2 Serum lithium per strain at day 18 (mM) 18 day at strain per lithium Serum Serum lithium per strain at day 18 (mM) 18 day at strain per lithium Serum 0.0 0.0 0 4 8 12 16 0 4 8 12 16 Lithium-induced fold decrease in urine Lithium-induced fold decrease in urine osmolality per strain from day 9-10 osmolality per strain from day 27-28

4 Figure S4. Relationship Li-NDI development between day 10 and day 28. Various inbred mouse strains were treated with a control (n=8) or lithium (n=10) diet. Urine output (A) or urine osmolality ( B) of each strain from day 9-10 was compared with the outcome from day 27-28.

A 12 B 12

9 9

6 6

3 3 r=0.79 r=0.74

output per strain from day 27-28 27-28 day from strain per output p<0.0001 p<0.0001 osmolality per strain from day 27-28 27-28 day from strain per osmolality Lithium-induced fold increase in urine urine in increase fold Lithium-induced 0 urine in decrease fold Lithium-induced 0 0 4 8 12 16 0 4 8 12 16 Lithium-induced fold increase in urine Lithium-induced fold decrease in urine output per strain from day 9-10 osmolality per strain from day 9-10

5 Figure S5. The effect of a 10-day lithium treatment on urinary PGEM levels in various mouse strains. Inbred mouse strains were treated with a control (n=8) or lithium (n=10) diet for 10 and 28 days and were housed in metabolic cages from day 8-10 and day 26-28. At day 10 24-hour urine production was collected and urinary PGEM content was determined. Order strains on x-axis is based on Figure 1B .

1.5 Ctr Li +

1.0

0.5 (ng/gbodyweight/24h)

Urinary PGEM contentPGEM day 9-10from Urinary 0.0 A P B6 LP PL D2 KK SM SJL 129 B10 C3H FVB MRL BUB CBA NZO NOD NON NZW SWR PWD RIIIS WSB C57L BLKS BTBR BALB

6 Figure S6. Relationship between blood pH and Li-NDI development. (A) The effect of lithium on blood pH per individual strain at day 10 was compared with its effect after 28 days. The effect on blood pH was also compared with the effect of lithium on urine output after 10 (B) and after 28 days (C).

7 Figure S7. The effect of lithium on blood pCO 2 in various mouse strains. Different inbred mouse strains were treated with a control (n=8) or lithium (n=10) diet and after 10 ( A) and 28 days ( B) blood was isolated to determine pCO 2 levels. Order strains on x-axis ( A and B) is based on Figure 2A .

A 8 Ctr Li + 6

4 at day 10(kPa) 2

2 Blood pCO

0 P A LP PL D2 B6 KK SM SJL 129 B10 FVB C3H BUB MRL CBA NOD NZW NON RIIIS SWR C57L BTBR B BLKS BALB 8 Ctr Li +

6

4 at day 28 (kPa) 2

2 Blood pCO

0 P A PL D2 B6 LP KK SM 129 SJL B10 C3H FVB CBA MRL BUB NON NZW NOD SWR RIIIS C57L BLKS BALB BTBR

8 Figure S8. The effect of lithium on blood HCO 3- in various mouse strains. Different inbred mouse strains were treated with a control (n=8) or lithium (n=10) diet and after 10 ( A) and 28 days ( B) blood was isolated to determine HCO 3- levels. *P<0.0020, significantly different from control. Order strains on x-axis ( A and B) is based on Figure 2A .

A 30 Ctr Li +

* 20 at day 10 (mM) - 3

10 Blood HCO

0 P A B6 PL D2 LP KK SM 129 SJL B10 C3H FVB CBA MRL BUB NON NZW NOD SWR RIIIS C57L BLKS BALB BTBR

B 30 Ctr Li +

20 at day28 (mM) - 3

10 BloodHCO

0 P A B6 LP PL D2 KK SM SJL 129 B10 C3H FVB CBA BUB MRL NOD NON NZW SWR RIIIS C57L BLKS BALB BTBR

9 Figure S9. The effect of lithium on urine pH in 29 mouse strains. 29 different inbred mouse strains were treated with a control (n=8) or lithium (n=10) diet and urine pH was determined from day 9-10 ( A) and from day 27-28 ( B). Order strains on x-axis ( A and B) is based on Figure 2A . The effect of lithium on urine pH was then compared to urine output from day 9-10 ( C) and day 27-28 ( D). *P<0.0017, significantly different from control.

A 7.0 * * Ctr Li +

6.5 * * * 6.0 * * *

5.5 Urine pH from day 9-10 from pH Urine

5.0 A P LP PL D2 B6 KK SM SJL 129 B10 FVB C3H NZO MRL BUB CBA NZW NOD NON RIIIS PWD WSB SWR C57L BTBR B BLKS BALB 7.0 Ctr Li + * 6.5 * * * * * * 6.0 * * * * * * *

5.5 * Urine pH from day 27-28 from pH Urine

5.0 P A B6 LP PL D2 KK SM 129 SJL B10 C3H FVB CBA BUB NZO MRL NOD NON NZW SWR RIIIS PWD WSB C57L BLKS BALB CAST C BTBR D 1.3 1.3 R2=0.6994 R2=0.5414 p<0.0001 p<0.0001 1.2 1.2

1.1 1.1

1.0 1.0 from day 9-10 per individual strain individual per 9-10 day from from day 27-28 per individual strain individual per 27-28 day from

Lithium-induced fold increase in urine pH pH urine in increase fold Lithium-induced 0.9

Lithium-induced fold increase in urine pH pH urine in increase fold Lithium-induced 0.9 0 4 8 12 16 0 5 10 15 20 Lithium-induced fold increase in urine production Lithium-induced fold increase in urine production fr om d ay 9 -10 p er i ndividual s tr ain from day 27-28 per individual strain

10 Figure S10. The effect of lithium on free urinary H+ content in 29 mouse strains. Various inbred mouse strains were treated with a control (n=8) or lithium (n=10) diet and free urinary H + content was calculated from urine pH and urine output from day 9-10 ( A) and from day 27-28 ( B). Order strains on x-axis ( A and B) is based on Figure 2A . The effect of lithium on free urinary H + content was then compared to urine output from day 9-10 ( C) and day 27-28 ( D). *P<0.0017, significantly different from control.

A 0.4 Ctr Li +

0.3

0.2 contentday9-10 from +

0.1 (nmol//g bodyweight/24h) (nmol//g

Free urinary H urinary Free 0.0 P A B6 PL D2 LP KK SM 129 SJL B10 C3H FVB CBA NZO MRL BUB NON NZW NOD SWR PWD WSB RIIIS C57L BLKS BALB B BTBR 0.5 Ctr Li + 0.4

0.3 * *

contentday 27-28 from 0.2 + * * * 0.1 * * (nmol//g bodyweight/24h) (nmol//g *

Free urinary H urinary Free 0.0 A P PL D2 B6 LP KK SM 129 SJL B10 C3H FVB CBA NZO MRL BUB NON NZW NOD PWD WSB SWR RIIIS C57L BLKS BALB CAST BTBR

C D + +

5 R2=0.04176 5 R2=0.2395 p=0.2969 p=0.0076 4 4

3 3

2 2

1 1

0 0 content from day 9-10 per individual strain individual per 9-10 day from content content from day 27-28 per individual strain individual per 27-28 day from content Lithium-induced fold increase in free urine H urine free in increase fold Lithium-induced Lithium-induced fold increase in free urine H urine free in increase fold Lithium-induced 0 4 8 12 16 0 4 8 12 16 Lithium-induced fold increase in urine production Lithium-induced fold increase in urine production from day 9-10 per individual strain from day 27-28 per individual strain

11

Figure S11. Relationship between effect of lithium on blood Ca 2+ and development of Li- NDI. For each individual strain the effect of lithium on blood Ca 2+ levels after 10 ( A) and 28 days ( B) was also compared with serum lithium levels determined after 18 days of lithium treatment and with urine output ( C and D, respectively).

12 Figure S12. Genomic inflation. Mice were treated with lithium for 10 and 28 days and genomic inflation was calculated from the GWA analysis from urine outcome ( A and D , respectively), urine osmolality ( B and E , respectively) or a combination of both datasets (C and F , respectively, see also Table 1) and are displayed in QQ plots.

AA BB CC λ = 0.60 6 λ = 0.57 300 λ = 0.55 5 5 250 4 4 200 3 3 150 2 2 100 1 1 50 Observed -log10 P-value Observed-log10 P-value 0 Observed-log10 P-value 0 0 −4 −2 0 2 4 −4 −2 0 2 4 −4 −2 0 2 4 Expected -log10 P-value Expected -log10 P-value Expected -log10 P-value

AD BE CF λ = 0.79 λ = 0.90 6 λ = 0.56 8 4 5 6 3 4 3 4 2 2 2 1 1

Observed -log10 P-value 0 Observed -log10 P-value 0 Observed -log10 P-value 0 −4 −2 0 2 4 −4 −2 0 2 4 −4 −2 0 2 4 Expected -log10 P-value Expected -log10 P-value Expected -log10 P-value

13 Figure S13. Serum and urine lithium content and inner medullary AQP2 localization in ACER2 knockout mice. Wildtype, Acer2 +/- and Acer2 -/- mice were bred and, at an age of 10 weeks, treated with lithium for 10 days. (A) At day 10 blood was isolated just by cheek puncture to determine serum lithium concentrations, while ( B) urinary lithium content was determined by collecting urine using metabolic cages. (C) To determine AQP2 localization kidneys were isolated and subjected to immunohistochemistry for AQP2. A B 0.8 6

0.6 4

0.4

2

0.2 content Li Urine (mmol/gbw/24hr)

Serum Li concentration SerumLi (mM) 0.0 0 Ctr Li Ctr Li Ctr Li Ctr Li Ctr Li Ctr Li Acer2+/+ Acer2+/- Acer2-/- Acer2+/+ Acer2+/- Acer2-/- C

14 Figure S14. The density of intercalated cells is not different for wildtype and Acer2 knockout mice. Representative images of immunohistochemical stainings using an anti- H+ATPase antibody on kidney sections from wildtype (WT) and homozygote Acer2 knockout mice (Hom) treated with lithium for 10 days. The density of H +-ATPase positive cells is not different among the different groups of mice.

15 Figure S15. Ceramide levels in medullary tissue of male and female Acer2 Ko mice. Wildtype, Acer2 +/- and Acer2 -/- mice were bred and, at an age of 10 weeks, treated with lithium for 10 days. After 10 days kidneys were isolated, stored and ceramide levels were determined from medullary lysates. Cer, ceramide, (N/A+S/dS/Sd/P)-Cer, ceramide containing either non-hydroxfatty acyl (N) or alpha-hydroxy acyl (A) together with C18- sphingoid base, which is either sphingosine (S), dihydrosphingosine (dS), sphingadienine (Sd) or phytosphingosine (P). S(20) indicates a C20-sphingosine.

Acer2+/+ NP-Cer Acer2+/- Acer2-/-

NS(20)-Cer

ASd-Cer

NdS-Cer

NSd-Cer

AS-Cer

NS-Cer

0 2500 5000 40000 80000 Ceramide (pmol/mg protein)

16 Supplementary Table S1. Names of the 29 inbred mouse strains, their abbreviations and number of mice used per analysis.

Serum Urine output Urine osmolality Blood pH Blood calcium Li + 10d 28d 10d 28d 10d 28d 10d 28d 18d Ctr Li + Ctr Li + Ctr Li + Ctr Li + Ctr Li + Ctr Li + Ctr Li + Ctr Li + 129S1/SvImJ (129) 8 10 8 10 8 10 8 10 8 9 7 9 8 8 6 9 9 A/J (A) 8 9 8 9 8 9 8 9 6 8 6 6 6 8 6 6 8 BALB/cByJ (BALB) 8 10 8 10 8 10 8 10 8 8 6 7 8 8 6 7 10 BTBR T+ Itpr3 tf /J (BTBR) 8 10 8 9 8 9 8 9 8 9 8 8 8 9 8 8 8 BUB/BnJ (BUB) 8 10 8 9 8 10 8 9 7 7 6 7 7 7 6 7 9 C3H/HeJ (C3H) 8 10 8 9 8 10 8 9 7 9 6 7 7 9 6 7 10 C57BL/6J (B6) 8 10 8 10 8 10 7 10 5 5 5 5 5 5 5 5 10 C57BL/10J (B10) 8 10 8 10 8 10 7 10 6 8 5 6 6 8 5 6 10 C57BLKS/J (BLKS) 8 10 8 10 8 10 8 10 6 9 5 7 6 9 5 7 9 C57L/J (C57L) 7 10 8 10 7 10 8 10 6 5 6 5 6 5 6 5 10 CAST/EiJ (CAST) 4 10 8 9 4 9 7 8 4* 2* 4* 2* 6 3 4* 2* 10 CBA/J (CBA) 8 10 8 10 8 10 8 10 6 8 4 7 6 8 4 7 10 DBA/2J (D2) 7 8 7 8 7 8 7 8 3 3 3 5 3 3 3 5 5 FVB/NJ (FVB) 8 10 8 10 8 10 8 10 5 8 7 9 5 8 7 8 10 KK/HIJ (KK) 8 8 8 9 8 10 8 9 5 8 4 6 5 8 4 6 9 LP/J (LP) 8 10 8 10 8 10 8 10 8 8 7 8 8 8 7 8 10 MRL/MpJ (MRL) 8 9 10 9 8 9 9 9 7 8 4 4 7 8 4 4 6 NOD.B10Sn-H2 b/J (NOD) 8 10 8 10 8 10 8 10 6 9 7 8 6 9 7 8 10 NON/ShiLtJ (NON) 8 10 8 10 8 10 8 10 7 8 7 7 8 7 7 7 10 NZO/HILtJ (NZO) 8 9 7 6 8 9 7 6 2* 1* 0* 0* 2* 1* 0* 0* 0* NZW/LacJ (NZW) 8 10 8 9 8 10 8 9 7 8 6 9 7 8 6 9 9 P/J (P) 8 10 8 8 8 10 8 9 5 6 4 7 4 6 4 7 9 PL/J (PL) 8 10 7 7 8 10 7 10 5 7 6 5 5 7 6 5 10 PWD/PHJ (PWD) 7 9 7 7 7 9 7 7 2* 1* 0* 0* 2* 1* 0* 0* 0* RIIIS/J (RIIIS) 8 10 8 10 8 10 8 10 5 4 4 7 5 4 4 7 10 SJL/J (SJL) 8 10 8 10 8 9 8 10 5 9 5 7 5 9 5 7 10 SM/J (SM) 8 10 8 10 8 10 7 10 7 10 5 8 7 10 5 8 10 SWR/J (SWR) 8 10 8 10 8 10 8 10 5 9 4 4 5 9 4 4 10 WSB/EiJ (WSB) 8 10 8 10 7 10 7 9 0* 0* 0* 0* 0* 0* 0* 0* 0* * Groups with a sample size <3 for either control or lithium treatment were not included in the analysis.

17 Supplementary Table S2. Blood Na + and Hct levels in 29 different mouse strains treated for 10 and 28 days with lithium. *P<0.0020, significantly different from control. Grey background highlights significant differences.

Na + (mM) Hct (fraction) 10 d 28 d 10 d 28 d Ctr Li + Ctr Li + Ctr Li + Ctr Li + 1 B6 150,2 ± 0,7 150,8 ± 0,6 149,6 ± 0,5 148,4 ± 0,5 0,48 ± 0,00 0,46 ± 0,01 0,47 ± 0,01 0,46 ± 0,00 2 FVB 146,4 ± 0,6 145,5 ± 0,3 148,0 ± 0,5 145,8 ± 0,5 0,42 ± 0,01 0,43 ± 0,01 0,44 ± 0,01 0,42 ± 0,01 3 SJL 149,6 ± 0,2 150,9 ± 0,5 150,8 ± 0,5 151,1 ± 0,6 0,44 ± 0,01 0,44 ± 0,01 0,47 ± 0,01 0,48 ± 0,01 4 BTBR 149,0 ± 0,3 148,2 ± 0,8 148,8 ± 0,7 147,9 ± 0,7 0,43 ± 0,01 0,43 ± 0,00 0,42 ± 0,00 0,42 ± 0,01 5 LP 151,3 ± 0,4 152,0 ± 0,4 149,7 ± 0,2 152,3 ± 0,4* 0,47 ± 0,00 0,46 ± 0,00 0,46 ± 0,01 0,45 ± 0,01 6 MRL 151,0 ± 0,6 150,4 ± 0,7 149,0 ± 0,0 150,0 ± 1,2 0,52 ± 0,01 0,49 ± 0,01 0,52 ± 0,01 0,49 ± 0,01 7 C57L 151,8 ± 0,5 151,0 ± 0,3 154,7 ± 0,2 150,6 ± 0,5* 0,50 ± 0,01 0,49 ± 0,01 0,52 ± 0,00 0,52 ± 0,01 8 D2 151,3 ± 1,5 150,0 ± 0,6 150,3 ± 0,3 148,5 ± 0,7 0,46 ± 0,02 0,45 ± 0,01 0,45 ± 0,01 0,46 ± 0,01 9 NZW 149,4 ± 1,7 149,8 ± 0,3 149,5 ± 0,8 148,8 ± 0,6 0,41 ± 0,01 0,41 ± 0,01 0,43 ± 0,01 0,43 ± 0,01 10 CAST NA NA NA NA NA NA NA NA 11 SWR 149,6 ± 0,9 151,4 ± 0,7 150,0 ± 0,9 149,5 ± 1,0 0,49 ± 0,02 0,50 ± 0,01 NA NA 12 BLKS 150,0 ± 0,6 150,0 ± 0,6 149,8 ± 0,7 149,0 ± 0,5 0,47 ± 0,01 0,47 ± 0,01 0,45 ± 0,02 0,49 ± 0,01 13 C3H 147,4 ± 0,5 147,0 ± 0,5 147,2 ± 0,5 147,6 ± 0,4 0,42 ± 0,01 0,43 ± 0,00 0,43 ± 0,01 0,42 ± 0,00 14 CBA 147,7 ± 0,5 146,8 ± 0,5 148,5 ± 0,6 147,9 ± 0,5 0,43 ± 0,01 0,42 ± 0,01 0,46 ± 0,01 0,43 ± 0,01 15 A 149,5 ± 0,3 149,5 ± 0,4 149,7 ± 0,3 151,2 ± 0,8 0,43 ± 0,00 0,41 ± 0,01 0,43 ± 0,01 0,43 ± 0,00 16 NON 147,4 ± 0,6 146,1 ± 0,4 149,9 ± 1,1 146,7 ± 0,6 0,46 ± 0,01 0,45 ± 0,01 0,47 ± 0,02 0,45 ± 0,01 17 B10 150,7 ± 0,4 149,8 ± 0,5 149,8 ± 0,9 149,3 ± 0,3 0,46 ± 0,00 0,44 ± 0,01 0,46 ± 0,01 0,44 ± 0,01 18 BALB 148,0 ± 0,5 147,9 ± 0,4 148,0 ± 0,4 147,7 ± 0,4 0,45 ± 0,00 0,44 ± 0,00 0,45 ± 0,00 0,45 ± 0,00 19 SM 150,9 ± 0,5 151,1 ± 0,4 151,4 ± 0,4 149,4 ± 0,4 0,47 ± 0,01 0,46 ± 0,00 0,47 ± 0,00 0,47 ± 0,00 20 P 148,3 ± 0,3 149,2 ± 0,5 147,8 ± 0,3 148,3 ± 0,4 0,49 ± 0,01 0,47 ± 0,00 0,47 ± 0,00 0,47 ± 0,01 21 BUB 148,6 ± 0,2 149,6 ± 0,4 148,7 ± 0,4 147,9 ± 0,7 0,47 ± 0,01 0,45 ± 0,01 0,46 ± 0,01 0,47 ± 0,00 22 PWD NA NA NA NA NA NA NA NA 23 NZO NA NA NA NA NA NA NA NA 24 WSB NA NA NA NA NA NA NA NA 25 KK 145,2 ± 0,7 144,8 ± 0,7 148,0 ± 0,7 146,0 ± 0,6 0,44 ± 0,00 0,42 ± 0,01 0,44 ± 0,00 0,44 ± 0,01 26 NOD 151,2 ± 0,8 150,8 ± 0,5 150,9 ± 0,6 149,9 ± 0,5 0,45 ± 0,01 0,45 ± 0,01 0,44 ± 0,01 0,44 ± 0,00 27 129 149,9 ± 0,4 149,4 ± 0,4 151,1 ± 2,4 150,0 ± 0,5 0,48 ± 0,01 0,47 ± 0,01 0,48 ± 0,01 0,47 ± 0,00 28 PL 148,2 ± 0,2 147,3 ± 0,3 149,3 ± 0,4 148,0 ± 0,7 0,46 ± 0,01 0,45 ± 0,01 0,47 ± 0,01 0,44 ± 0,01 29 RIIIS 149,8 ± 0,4 149,0 ± 0,7 150,8 ± 0,5 149,0 ± 0,7 0,48 ± 0,00 0,47 ± 0,01 0,48 ± 0,00 0,48 ± 0,01 AVG 149,3 ± 0,3 149,1 ± 0,4 149,6 ± 0,3 148,8 ± 0,3 0,46 ± 0,01 0,45 ± 0,00 0,46 ± 0,01 0,45 ± 0,01

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