Lithium Chloride and GSK3 Inhibition Reduce Aquaporin-2 Expression in Primary Cultured Inner Medullary Collecting Duct Cells Due to Independent Mechanisms

cells Article Lithium Chloride and GSK3 Inhibition Reduce Aquaporin-2 Expression in Primary Cultured Inner Medullary Collecting Duct Cells Due to Independent Mechanisms Marc Kaiser 1 and Bayram Edemir 2,* 1 Medizinische Klinik D, Experimentelle Nephrologie, Universitätsklinikum Münster, 48143 Münster, Germany; [email protected] 2 Department of Medicine, Hematology and Oncology, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany * Correspondence: [email protected]; Tel.: +49-345-557-4890; Fax: +49-345-557-2950 Received: 20 March 2020; Accepted: 19 April 2020; Published: 23 April 2020 Abstract: Lithium chloride (LiCl) is a widely used drug for the treatment of bipolar disorders, but as a side effect, 40% of the patients develop diabetes insipidus. LiCl affects the activity of the glycogen synthase kinase 3 (GSK3), and mice deficient for GSK3β showed a reduction in the urine concentration capability. The cellular and molecular mechanisms are not fully understood. We used primary cultured inner medullary collecting duct cells to analyze the underlying mechanisms. LiCl and the inhibitor of GSK3 (SB216763) induced a decrease in the aquaporin-2 (Aqp2) protein level. LiCl induced downregulation of Aqp2 mRNA expression while SB216763 had no effect and TWS119 led to increase in expression. The inhibition of the lysosomal activity with bafilomycin or chloroquine prevented both LiCl- and SB216763-mediated downregulation of Aqp2 protein expression. Bafilomycin and chloroquine induced the accumulation of Aqp2 in lysosomal structures, which was prevented in cells treated with dibutyryl cyclic adenosine monophosphate (dbcAMP), which led to phosphorylation and membrane localization of Aqp2. Downregulation of Aqp2 was also evident when LiCl was applied together with dbcAMP, and dbcAMP prevented the SB216763-induced downregulation. We showed that LiCl and SB216763 induce downregulation of Aqp2 via different mechanisms. While LiCl also affected the mRNA level, SB216763 induced lysosmal degradation. Specific GSK3β inhibition had an opposite effect, indicating a more complex regulatory mechanism. Keywords: aquaporin; diabetes insipidus; lithium chloride 1. Introduction In the renal collecting duct, the final urine concentration is predominantly regulated by the action of the antidiuretic hormone vasopressin (AVP), which is secreted by the neuronal lobule of the pituitary gland [1]. The binding of AVP to the vasopressin type 2 receptor (V2R) at the basolateral membrane of the collecting duct’s principal cells leads to, via an activation of a Gs-protein, the stimulation of the adenylyl cyclase and an increase of the cyclic adenosine monophosphate (cAMP) level [2]. This activates the protein kinase A (PKA), which in turn phosphorylates the water channel aquaporin-2 (Aqp2) at serine 256 [3]. This phosphorylation induces the translocation and fusion of Aqp2-containing vesicles with the luminal plasma membrane. Thus, water can enter the cell along an osmotic gradient and leaves the cell on the basolateral site facilitated by aquaporin-3 (Aqp3) and aquaporin-4 (Aqp4) [4]. Defects in this pathway often lead to a urine concentration deficiency, a disorder called diabetes insipidus (DI) [5]. This disorder can be inherited, either when specific mutations lead to a defect in Cells 2020, 9, 1060; doi:10.3390/cells9041060 www.mdpi.com/journal/cells Cells 2020, 9, 1060 2 of 15 the secretion of AVP from the pituitary gland (central DI) or to nephrogenic diabetes insipidus (NDI) when the V2R or Aqp2 genes are mutated [5]. Alternatively, DI can be acquired, e.g., due to therapy with the drug lithium chloride (LiCl) or due to diseases affecting the pituitary gland [6,7]. LiCl is widely used for the treatment of bipolar disorders and causes NDI in up to 40% of these patients as severe side effect [8]. Various mechanisms have been described of how LiCl could induce NDI. One postulated mechanism is the stimulation of the Gi protein by LiCl, which would inhibit the activity of the adenylate cyclase [8]. As a consequence, the trafficking of Aqp2-containing vesicles to the apical membrane and Aqp2 expression, which is also cAMP and protein kinase A (PKA) dependent, would be reduced [9]. Another postulated mechanism is that LiCl reduces the density of the V2R at the plasma membrane, which would have the same effect as described above. Moreover, LiCl might cause a defect independent of the classical V2R pathway [10,11]. LiCl, for example, inhibits the activity of the glycogen synthase kinase 3 alpha/beta (GSK3α/β), but it is still not well understood how the inhibition of GSK3α/β by LiCl causes NDI [12]. A global GSK3β-deficient mouse was embryonic lethal [13], and in contrast, a renal collecting duct specific knockout mouse showed only mild polyuria. However, under water deprivation or stimulation with AVP, reduced cAMP levels and in turn a urine concentration disability was observed [14]. Therefore, it was concluded that GSK3β is involved in the urine concentration under water-deprived conditions. Still, the underlying cellular and molecular mechanisms were not fully understood. To further address this issue, we used primary cultured inner medullary collecting duct (IMCD) cells and analyzed the effects of pharmacological GSK3 inhibition and LiCl on Aqp2 expression. The treatment of IMCD cells with LiCl provoked a decrease of Aqp2 expression on both the transcriptional and protein level. Inhibition of both isoforms of the GSK3 with SB216763, however, decreased only the expression of Aqp2 protein. In contrast, a sole inhibition of GSK3β by the specific inhibitor, TWS119 [15], showed an upregulation of Aqp2 on the protein level as well as in mRNA expression. Further analysis showed that LiCl and GSK3 inhibition by SB216763 enhanced the degradation rate of Aqp2 protein in lysosomes. 2. Materials and Methods 2.1. Cell Culture Primary cultured IMCD cells were prepared as previously described [16]. Female Wistar rats at the age of 23 months were sacrificed by decapitation and the kidneys were removed. The inner medulla and the papilla were isolated, cut into small pieces, and incubated at 37 ◦C for 90 min in a digesting solution, which contained phosphate buffered saline (PBS) (Biochrom, Berlin, Germany), 0.2% hyaluronidase (Sigma, Deisenhofen, Germany), and 0.2% collagenase type CLS-II (Sigma). The isolated cells were seeded with a density of approximately 105 cells/cm2 and grown to confluency in 24-well plates (Greiner Bio-One, Frickenhausen, Germany) coated with collagen type IV (Becton-Dickinson, Heidelberg, Germany). For cultivation, Dulbecco’s modified Eagle’s medium (DMEM) containing 100 IU/mL penicillin and 100 µg/mL streptomycin, 0.2% glutamine, 1% non-essential amino acids, and 1% ultroser (BioSepra Inc., Marlborough, MA, USA) was used. The medium osmolality was increased to 600 mOsmol/kg by the addition of 100 mM NaCl and 100 mM urea. Osmolality was checked using an osmometer (Knauer, Berlin, Germany). Cells were cultured for 67 days and treated with LiCl; SB216763, a potent inhibitor of both GSK3 isoforms [17] (Sigma, Deisenhofen, Germany); TWS119, a potent inhibitor of the GSK3β isoform [15] (Millipore, Darmstadt, Germany); bafilomycin A1 and chloroquine, both inhibitors of the vacuolar ATPase [18,19] and thereby lysosomal activity inhibitors; MG132, as a proteasome inhibitor [20]; and dbcAMP (all from Sigma, Deisenhofen, Germany) in different concentrations and durations, alone or together as indicated. All experiments were conducted with at least 3 independent cell cultures from different rats. All animals were handled according to German animal protection guidelines and the experiments were approved by a governmental committee on animal welfare (A 60/1993 and A 67/09). Cells 2020, 9, 1060 3 of 15 2.2. Immunoblotting Immunoblotting was performed as previously described [21]. Briefly, the same protein amounts were separated by SDS-polyacrylamide (4–20%) electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham, Freiburg, Germany). Membranes were subsequently incubated with primary antibodies in a 1:1.000 dilution. Antibodies raised against Aqp2-4 were obtained from Alomone Labs (Jerusalem, Israel) and antibodies directed against phosphorylated Aqp2 at position S256 were a generous gift from M. Knepper (National Institutes of Health, Bethesda, MD, USA). A secondary, horseradish-peroxidase-linked anti-rabbit antibody was used. Membranes were stripped (30 min/60 ◦C) using stripping buffer (15 g/L glycine, 1 g/L sodium dodecyl sulfate, 10 mL/L Tween 20, pH to 2.2) and reincubated with an antibody directed against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to ensure equal loading. Quantification of specific signals was performed using the Lumianalyst software (Roche, Basel, Switzerland). 2.3. Real-Time PCR Total RNA and cDNA synthesis were performed as described before [16]. Real-time PCR was performed using the SYBR Green PCR Master Mix with the ABI PRISM 7900 Sequence Detection System. All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). DDCt Relative gene expression values were evaluated with the 2− method using GAPDH as the reference gene [22]. Specific primer pairs were used, and a list with the gene names, accession numbers, and gene symbols is provided in Table1. Table 1. Gene names, gene symbols, accession numbers, and primer sequences of genes used for real-time PCR. Gene Description and Acc. No. Primer Sequences Sense-TGC ATC TTT GCC TCC ACC GAC GAG Aquaporin-2 (Aqp2) Antisense-CAT

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