Specific disruption of calcineurin-signaling in the distal convoluted tubule impacts the transcriptome and proteome, and causes hypomagnesemia and metabolic acidosis
Authors: Eszter Banki, Viktoria Fisi, Sandra Moser, Agnieszka Wengi, Monique Carrel, Dominique Loffing-Cueni, David Penton, Denise V. Kratschmar, Ludovica Rizzo, Soeren Lienkamp, Alex Odermatt, Markus M. Rinschen, Johannes Loffing
Journal: Kidney International (2021)

Adverse effects of calcineurin inhibitors (CNI), such as hypertension, hyperkalemia, acidosis, hypomagnesemia and hypercalciuria, have been linked to dysfunction of the distal convoluted tubule (DCT). To test this, we generated a mouse model with an inducible DCT-specific deletion of the calcineurin regulatory subunit B alpha (CnB1-KO). Three weeks after CnB1 deletion, these mice exhibited hypomagnesemia and acidosis, but no hypertension, hyperkalemia or hypercalciuria. Consistent with the hypomagnesemia, CnB1-KO mice showed a downregulation of proteins implicated in DCT magnesium transport, including TRPM6, CNNM2, SLC41A3 and parvalbumin but expression of calcium channel TRPV5 in the kidney was unchanged. The abundance of the chloride/bicarbonate exchanger pendrin was increased, likely explaining the acidosis. Plasma aldosterone levels, kidney renin expression, abundance of phosphorylated sodium chloride-cotransporter and abundance of the epithelial sodium channel were similar in control and CnB1-KO mice, consistent with a normal sodium balance. Long-term potassium homeostasis was maintained in CnB1-KO mice, but in-vivo and ex-vivo experiments indicated that CnB1 contributes to acute regulation of potassium balance and sodium chloride-cotransporter. Tacrolimus treatment of control and CnB1-KO mice demonstrated that CNI-related hypomagnesemia is linked to impaired calcineurin-signaling in DCT, while hypocalciuria and hyponatremia occur independently of CnB1 in DCT. Transcriptome and proteome analyses of isolated DCTs demonstrated that CnB1 deletion impacts the expression of several DCT-specific proteins and signaling pathways. Thus, our data support a critical role of calcineurin for DCT function and provide novel insights into the pathophysiology of CNI side effects and involved molecular players in the DCT. Adverse effects of calcineurin inhibitors (CNI), such as hypertension, hyperkalemia, acidosis, hypomagnesemia and hypercalciuria, have been linked to dysfunction of the distal convoluted tubule (DCT). To test this, we generated a mouse model with an inducible DCT-specific deletion of the calcineurin regulatory subunit B alpha (CnB1-KO). Three weeks after CnB1 deletion, these mice exhibited hypomagnesemia and acidosis, but no hypertension, hyperkalemia or hypercalciuria. Consistent with the hypomagnesemia, CnB1-KO mice showed a downregulation of proteins implicated in DCT magnesium transport, including TRPM6, CNNM2, SLC41A3 and parvalbumin but expression of calcium channel TRPV5 in the kidney was unchanged. The abundance of the chloride/bicarbonate exchanger pendrin was increased, likely explaining the acidosis. Plasma aldosterone levels, kidney renin expression, abundance of phosphorylated sodium chloride-cotransporter and abundance of the epithelial sodium channel were similar in control and CnB1-KO mice, consistent with a normal sodium balance. Long-term potassium homeostasis was maintained in CnB1-KO mice, but in-vivo and ex-vivo experiments indicated that CnB1 contributes to acute regulation of potassium balance and sodium chloride-cotransporter. Tacrolimus treatment of control and CnB1-KO mice demonstrated that CNI-related hypomagnesemia is linked to impaired calcineurin-signaling in DCT, while hypocalciuria and hyponatremia occur independently of CnB1 in DCT. Transcriptome and proteome analyses of isolated DCTs demonstrated that CnB1 deletion impacts the expression of several DCT-specific proteins and signaling pathways. Thus, our data support a critical role of calcineurin for DCT function and provide novel insights into the pathophysiology of CNI side effects and involved molecular players in the DCT. Translational StatementCalcineurin inhibitors (CNIs), such as tacrolimus and cyclosporine A, have several adverse effects, including arterial hypertension, hyperkalemia, and hypomagnesemia. The present study employed a conditional knockout mouse model to define the specific role of disrupted calcineurin signaling in the renal distal convoluted tubule. Using targeted and unbiased transcriptomic and proteomic approaches, potential underlying molecular mechanism are revealed. Thus, the study improves our current understanding of the pathogenesis of CNI adverse effects, and it may set the foundation for developing future preventive strategies. Calcineurin inhibitors (CNIs), such as tacrolimus and cyclosporine A, have several adverse effects, including arterial hypertension, hyperkalemia, and hypomagnesemia. The present study employed a conditional knockout mouse model to define the specific role of disrupted calcineurin signaling in the renal distal convoluted tubule. Using targeted and unbiased transcriptomic and proteomic approaches, potential underlying molecular mechanism are revealed. Thus, the study improves our current understanding of the pathogenesis of CNI adverse effects, and it may set the foundation for developing future preventive strategies. Calcineurin inhibitors (CNIs), like tacrolimus and cyclosporine A, are main pillars of immunosuppressive therapy in patients with solid organ transplantation and autoimmune disease.1Ong S.C. Gaston R.S. Thirty years of tacrolimus in clinical practice.Transplantation. 2021; 105: 484-495Crossref PubMed Scopus (5) Google Scholar Calcineurin or protein phosphatase 3 (PP3) is a calcium-calmodulin-activated serine–threonine protein phosphatase that is composed of 2 subunits, which are both essential for the function of this phosphatase. The catalytic (A) subunit provides the enzymatic activity and is encoded by 3 separate genes (Ppp3ca, Ppp3cb, and Ppp3cc). The regulatory (B) domain carries the Ca2+-binding pockets and is encoded by 2 separate genes (Ppp3r1, Ppp3r2).2Rusnak F. Mertz P. Calcineurin: form and function.Physiol Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1074) Google Scholar Calcineurin has strong effects on gene transcription, as it dephosphorylates and hence activates the transcription factor NFAT (nuclear factor of activated T-lymphocytes). Activated NFAT translocates to the cell nucleus where it up-regulates, among others, genes that are critical for interleukin-2 (IL-2) signaling and hence T-lymphocyte-mediated immune responses.3Aramburu J. Heitman J. Crabtree G.R. Calcineurin: a central controller of signalling in eukaryotes. Workshop on the calcium/calcineurin/NFAT pathway: regulation and function.EMBO Rep. 2004; 5: 343-348Crossref PubMed Scopus (130) Google Scholar Moreover, calcineurin or its inhibitors are thought to impact several additional signal transduction pathways including other transcription factors (e.g., forkhead transcription factors, myocyte-specific enhancer factor 2, transcription factor EB) and the mitogen-activated protein kinase (MAPK).4Bendickova K. Tidu F. Fric J. Calcineurin-NFAT signalling in myeloid leucocytes: new prospects and pitfalls in immunosuppressive therapy.EMBO Mol Med. 2017; 9: 990-999Crossref PubMed Scopus (40) Google Scholar,5Creamer T.P. Calcineurin.Cell Commun Signal. 2020; 18: 137Crossref PubMed Scopus (19) Google Scholar While PPP3CC and PPP3R2 are restricted to the testis, the other calcineurin isoforms are expressed in many organs, including the kidney, lung, heart, brain, adrenal gland, skeletal muscle, and liver.2Rusnak F. Mertz P. Calcineurin: form and function.Physiol Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1074) Google Scholar The broad tissue distribution of calcineurin may explain the plethora of adverse effects of CNI treatment.1Ong S.C. Gaston R.S. Thirty years of tacrolimus in clinical practice.Transplantation. 2021; 105: 484-495Crossref PubMed Scopus (5) Google Scholar Arterial hypertension, hyperkalemia, hypomagnesemia, hypercalciuria, and metabolic acidosis are frequently observed under CNI treatment and have been attributed at least in part to a malfunction of the renal distal convoluted tubule (DCT).6McCormick J.A. Ellison D.H. Distal convoluted tubule.Compr Physiol. 2015; 5: 45-98PubMed Google Scholar Na+ transport in the DCT occurs via the Na+-Cl– cotransporter (NCC), while Ca2+ and Mg2+ reabsorption is mediated through transient receptor potential vanilloid 5 (TRPV5) and transient receptor potential ion channel 6 (TRPM6), respectively.6McCormick J.A. Ellison D.H. Distal convoluted tubule.Compr Physiol. 2015; 5: 45-98PubMed Google Scholar The DCT impacts on K+ and acid–base balance mainly via indirect effects. For example, a lowered NCC activity level, as it occurs with thiazide treatment, increases plasma aldosterone levels and Na+ delivery to the downstream localized renal collecting system (i.e., the connecting tubule [CNT] and the collecting duct [CD]). The high plasma aldosterone levels and the increased intratubular Na+ concentration stimulate Na+ reabsorption in the collecting system via the epithelial Na+ channel (ENaC), which enhances the electrochemical driving force for the secretion of K+ and protons, via apical K+ channels (e.g., renal outer medulla potassium channel [ROMK]) in principal cells and the proton adenosine triphosphatase (H+-ATPase) in intercalated cells, respectively. Consistent with the idea that many of the CNI-induced adverse effects are related to a malfunction of the DCT, Hoorn and coworkers reported that the salt-sensitive hypertension under tacrolimus is associated with a strong upregulation of the expression, phosphorylation, and functional activity of the DCT-specific NCC.7Hoorn E.J. Walsh S.B. McCormick J.A. et al.The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension.Nat Med. 2011; 17: 1304-1309Crossref PubMed Scopus (229) Google Scholar Mice that lack the CNI binding protein FKBP12 in the kidney are protected from CNI-induced hypertension, hypomagnesemia, and hypercalciuria.8Gratreak B.D.K. Swanson E.A. Lazelle R.A. et al.Tacrolimus-induced hypomagnesemia and hypercalciuria requires FKBP12 suggesting a role for calcineurin.Physiol Rep. 2020; 8: 1-8Crossref Scopus (10) Google Scholar,9Lazelle R.A. McCully B.H. Terker A.S. et al.Renal deletion of 12 kDa FK506-binding protein attenuates tacrolimus-induced hypertension.J Am Soc Nephrol. 2016; 27: 1456-1464Crossref PubMed Scopus (34) Google Scholar The effect of CNIs is thought to be related to a reduced dephosphorylation of the ubiquitin-ligase complex protein Kelch-like 3 (KLHL3),10Ishizawa K. Wang Q. Li J. et al.Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling.Proc Natl Acad Sci U S A. 2019; 116: 3155-3160Crossref PubMed Scopus (25) Google Scholar which activates the with-no-lysine (K) kinase 4 (WNK4) and Stk39 serine/threonine kinase 39/oxidative stress–responsive kinase 1 (SPAK/OSR1) pathway and finally leads to an enhanced phosphorylation and activation of NCC.7Hoorn E.J. Walsh S.B. McCormick J.A. et al.The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension.Nat Med. 2011; 17: 1304-1309Crossref PubMed Scopus (229) Google Scholar,10Ishizawa K. Wang Q. Li J. et al.Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling.Proc Natl Acad Sci U S A. 2019; 116: 3155-3160Crossref PubMed Scopus (25) Google Scholar However, the activation of the KLHL3-WNK4-SPAK/OSR1-NCC axis cannot explain CNI-related hypomagnesemia, because hypomagnesemia is not seen in patients with familial hyperkalemic hypertension, who have an activation of this cascade.11Mayan H. Vered I. Mouallem M. et al.Pseudohypoaldosteronism type II: marked sensitivity to thiazides, hypercalciuria, normomagnesemia, and low bone mineral density.J Clin Endocrinol Metab. 2002; 87: 3248-3254Crossref PubMed Scopus (0) Google Scholar Instead, CNI-related renal Mg2+ wasting was linked to a significant downregulation of TRPM6,12Nijenhuis T. Hoenderop J.G.J. Bindels R.J.M. Downregulation of Ca2+ and Mg2+ transport proteins in the kidney explains tacrolimus (FK506)-induced hypercalciuria and hypomagnesemia.J Am Soc Nephrol. 2004; 15: 549-557Crossref PubMed Scopus (155) Google Scholar possibly mediated via reduced epidermal growth factor signaling.13Ledeganck K.J. De Winter B.Y. Van den Driessche A. et al.Magnesium loss in cyclosporine-treated patients is related to renal epidermal growth factor downregulation.Nephrol Dial Transplant. 2014; 29: 1097-1102Crossref PubMed Scopus (29) Google Scholar However, the underlying molecular mechanism and a possible contribution of other Mg2+ transporters such as the solute carrier family 41 member 3 (SLC41A3)14De Baaij J.H.F. Arjona F.J. Van Den Brand M. et al.Identification of SLC41A3 as a novel player in magnesium homeostasis.Sci Rep. 2016; 6: 28565Crossref PubMed Scopus (31) Google Scholar and cyclin M2 (CNNM2)15Stuiver M. Lainez S. Will C. et al.CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia.Am J Hum Genet. 2011; 88: 333-343Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar remained obscure. Another enigma are the strong DCT effects, despite the very low expression levels of calcineurin subunits in the DCT compared with other nephron portions.16Lee J.W. Chou C.L. Knepper M.A. Deep sequencing in microdissected renal tubules identifies nephron segment-specific transcriptomes.J Am Soc Nephrol. 2015; 26: 2669-2677Crossref PubMed Scopus (312) Google Scholar Hence, it is unclear whether the observed strong effects of CNIs on the kidney are directly related to an impaired calcineurin signaling in the DCT or rather if the DCT is indirectly affected due to an altered function of other nephron segments or organs. To address the precise role of calcineurin in the DCT, we generated a novel mouse model with an inducible DCT-specific deletion of the calcineurin subunit B alpha (CnB1). CnB1 is known to be critical for calcineurin activity in brain and muscle.17Cottrell J.R. Levenson J.M. Kim S.H. et al.Working memory impairment in calcineurin knock-out mice is associated with alterations in synaptic vesicle cycling and disruption of high-frequency synaptic and network activity in prefrontal cortex.J Neurosci. 2013; 33: 10938-10949Crossref PubMed Scopus (31) Google Scholar,18Pfluger P.T. Kabra D.G. Aichler M. et al.Calcineurin links mitochondrial elongation with energy metabolism.Cell Metab. 2015; 22: 838-850Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar All animal experiments were approved by the veterinary administration of the Canton of Zurich, Switzerland. Gene-modified littermates for experiments were obtained as described in the supplement and are shown schematically in Figure 1a and b. Activation of cre was induced by oral gavage of tamoxifen (Sigma-AldrichMT; 2 mg/animal per day in ethanol—sunflower oil mixture with a ratio of 1:9) for 5 consecutive days. Pilot studies showed that CnB1 deletion for 10 weeks, but not for 3 weeks, reduces the fractional volume of the DCT (Supplementary Figure S1). To avoid confounding effects of the reduced DCT volume on the interpretation of results (e.g., immunoblots on whole kidneys), we decided to perform all subsequent experiments after 3 weeks of tamoxifen induction. Mice were maintained on a standard rodent diet (0.3% Na+ and 0.8% K+), unless stated otherwise. Urine and feces were collected for 24 hours or 48 hours, in metabolic cages. At the end of the experiments, mice were anesthetized with isoflurane, venous blood was drawn from inferior vena cava, and organs (kidney, distal colon mucosa) were harvested. Blood pH and blood gas parameters were measured immediately by a blood gas analyzer (ABL80 Flex Basic, Radiometer). Plasma and urine ion and creatinine concentrations, as well as fecal Mg2+ content, were determined as described in the Supplementary Materials and Methods. Plasma aldosterone levels, alpha-ketoglutarate concentrations in urine, and calcineurin activity in isolated DCTs were measured as described in the Supplementary Materials and Methods. Total RNA was isolated from kidney, distal colon mucosa, and renal tubules with the SV Total RNA Isolation System (Promega) and the Absolutely RNA Nanoprep Kit (Agilent), respectively. Real-time PCR was performed using a LightCycler 480 Instrument II (Roche). Primers are given in Supplementary Table S1. Kidneys were processed for immunoblotting and probed with the antibodies listed in Supplementary Table S2. Moreover, a new rat monoclonal anti-pT58 NCC antibody was developed and characterized (Supplementary Figure S2). More details are given in the Supplementary Materials and Methods. Perfusion-fixed (3% paraformaldehyde) kidneys were processed for immunohistochemistry, morphometric analysis, and electron microscopy, as described in the Supplementary Materials and Methods. Mice received a single i.p. injection of hydrochlorothiazide (HCTZ 50 μg/g BW), as described in the Supplementary Materials and Methods. Direct reprogramming of fibroblasts to induced renal tubular epithelial cells was performed as described,19Kaminski M.M. Tosic J. Kresbach C. et al.Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors.Nat Cell Biol. 2016; 18: 1269-1280Crossref PubMed Scopus (77) Google Scholar The cells were then treated for 24 hours, with either vehicle (dimethylsulfoxide) or 20 nM tacrolimus (see Supplementary Materials and Methods for more details). Mice received daily s.c. injections of tacrolimus (3 μg/g body weight per day) for 16 days, as described in the Supplementary Materials and Methods. Blood pressure measurements were conducted at the Zürich Integrative Rodent Physiology (ZIRP) core-facility, as described in the Supplementary Materials and Methods. Mice received by oral gavage (15 μl/g body weight) either a vehicle solution (2% sucrose) or 2% K+ in 2% sucrose, as described previously20Sorensen M.V. Grossmann S. Roesinger M. et al.Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice.Kidney Int. 2013; 83: 811-824Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar and in the Supplementary Materials and Methods. Kidney slices of 280 μm thickness were incubated ex vivo with different extracellular K+ concentrations with or without the protein phosphatase 1 inhibitor calyculin A (Tocris Bioscience, 20 nM), as described in the Supplementary Materials and Methods. Control-Tomato and CnB1-KO-Tomato mice were used for automated isolation of red fluorescent DCTs using a biosorter (Union Biometrica) and processed for RNA sequencing by the Functional Genomics Centre Zurich (see Supplementary Materials and Methods). Data are deposited at the Gene Expression Omnibus (GEO) database, with the accession number GSE169721. Manual microdissection of renal tubules was performed as described by Chabardès et al.21Chabardès D. Firsov D. Aarab L. et al.Localization of mRNAs encoding Ca2+-inhibitable adenylyl cyclases along the renal tubule. Functional consequences for regulation of the cAMP content.J Biol Chem. 1996; 271: 19264-19271Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar (for quantitative RT-PCR) or as described by Wagner et al.22Wagner C.A. Lükewille U. Valles P. et al.A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse.Pflugers Arch Eur J Physiol. 2003; 446: 623-632Crossref PubMed Scopus (39) Google Scholar (for protein analysis). Isolated DCTs were processed for proteome analysis as described in the Supplementary Materials and Methods. Summed protein expression (label-free quantitation) values were log-transformed and normalized by subtraction of the mean. The data were filtered for at least 3 valid values a group, decreasing the total amount of proteins to 1600 from an initial 2364. Remaining missing values were imputed. A 2-tailed t test was performed. Proteins with fold change and P value cutoff as indicated were further analyzed. Data are available through ProteomExchange/PRIDE23Perez-Riverol Y. Csordas A. Bai J. et al.The PRIDE database and related tools and resources in 2019: improving support for quantification data.Nucleic Acids Res. 2019; 47: D442-D450Crossref PubMed Scopus (3647) Google Scholar,24Vizcaíno J.A. Deutsch E.W. Wang R. et al.ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (1976) Google Scholar: PXD number PXD017455; account: [email protected] ; and password: otMM0mtn. The unpaired Student’s t test was performed to compare 2 groups, and 1-way analysis of variance with Sidak’s multiple-comparison post hoc test, or 2-way analysis of variance for multiple comparisons. All statistical analyses were performed using GraphPad Prism 8. Data are expressed as mean ± SEM. Mice with floxed alleles of the CnB1 gene25Neilson J.R. Winslow M.M. Hur E.M. Crabtree G.R. Calcineurin B1 is essential for positive but not negative selection during thymocyte development.Immunity. 2004; 20: 255-266Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar were mated with mice that express the cre-recombinase under the control of the DCT-specific NCC promoter26Schnoz C. Moser S. Kratschmar D.V. et al.Deletion of the transcription factor Prox-1 specifically in the renal distal convoluted tubule causes hypomagnesemia via reduced expression of TRPM6 and NCC.Pflugers Arch Eur J Physiol. 2021; 473: 79-93Crossref PubMed Scopus (4) Google Scholar (Figure 1a and b). Heterozygous NCC-cre mice show the same expression level for DCT-specific proteins as wild-type mice do (Figure 1c and d). When NCC-cre mice are crossed with TdTomato-red reporter mice,27Madisen L. Zwingman T.A. Sunkin S.M. et al.A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci. 2010; 13: 133-140Crossref PubMed Scopus (3487) Google Scholar tamoxifen-treatment induces a strong red fluorescence in all NCC-positive DCT cells, but not in other renal cell types (Figure 1e). To confirm the successful disruption of CnB1-dependent calcineurin signaling, we isolated red fluorescent DCTs from control and CnB1-KO TdTomato-red reporter mice using a fluorescence-activated Biosorter (Figure 1f). Quantitative RT-PCR excluded relevant contaminations of sorted DCTs with adjacent thick ascending limb (TAL) and CNT cells and showed that CnB1 expression was absent from the DCTs of CnB1-KO mice (Figure 1g and h). Likewise, calcineurin activity was largely diminished in DCTs of CnB1-KO mice (Figure 1h). Consistent with previous data showing low CnB1 (Ppp3r1) mRNA expression levels in the rat DCT,16Lee J.W. Chou C.L. Knepper M.A. Deep sequencing in microdissected renal tubules identifies nephron segment-specific transcriptomes.J Am Soc Nephrol. 2015; 26: 2669-2677Crossref PubMed Scopus (312) Google Scholar immunohistochemistry revealed a very weak CnB1 immunostaining in the early DCT (DCT1) of control mice. Only the late DCT (DCT2) and the connecting tubule and collecting duct (CNT/CD) showed a strong CnB1-immunostaining (Supplementary Figure S3). The TAL had an intermediate CnB1 protein abundance (Figure 2a). In CnB1-KO mice, CnB1 protein was gone from all NCC-positive DCT cells (DCT1 and DCT2), whereas CnB1 protein remained visible in TAL, CNT, and CD (Figure 2a). Quantitative RT-PCR of isolated tubules confirmed intact CnB1 expression in proximal tubules (PT), TAL, and CNT/CD of CnB1-KO mice (Figure 2b). Three weeks after induction, CnB1-KO mice had normal plasma Na+, K+, Ca2+, Cl–, aldosterone, and blood glucose levels. However, CnB1-KO animals showed overt hypomagnesemia and metabolic acidosis with significantly lower plasma bicarbonate levels (Table 1). Urine volume, osmolarity, and urinary excretion of α-ketoglutarate, Na+, K+, Ca2+, and Mg2+ were similar for control and CnB1-KO mice. However, the fractional excretion of Mg2+ was higher in CnB1-KO than in control mice, but the difference did not reach statistical significance (Table 1).Table 1Physiological parameters of control and CnB1-KO mice 3 weeks after CnB1 deletionParameterControlCnB1-KOP valueBlood Na+, mmol/l148.40 ± 0.58147.70 ± 0.710.440 K+, mmol/l3.88 ± 0.143.83 ± 0.100.781 Ca2+, mmol/l1.10 ± 0.041.10 ± 0.050.812 Cl–, mmol/l107.80 ± 1.20106.90 ± 0.740.559 Mg2+, mmol/l0.76 ± 0.030.63 ± 0.040.018aP < 0.05. Glucose, mmol/l8.44 ± 0.729.10 ± 0.430.453 Aldosterone, nmol/l0.31 ± 0.050.31 ± 0.020.974 pH7.41 ± 0.027.35 ± 0.020.038aP < 0.05. HCO3–, mmol/l19.66 ± 0.2717.49 ± 0.590.002bP < 0.01. cBase, mmol/l–5.64 ± 0.36–8.01 ± 0.780.006bP < 0.01. Hematocrit, %39.54 ± 1.1236.33 ± 1.100.054Urine Volume, ml/24 h1.37 ± 0.351.68 ± 0.350.535 Osmolarity, mOsm/kg3362 ± 496.23278 ± 859.40.935 α-ketoglutarate/creatinine0.47 ± 0.060.54 ± 0.060.439 Na+/creatinine28.67 ± 3.8927.64 ± 6.500.891 K+/creatinine97.59 ± 4.75113 ± 24.770.559 Ca2+/creatinine0.40 ± 0.080.56 ± 0.150.381 Mg2+/creatinine7.83 ± 0.828.11 ± 1.480.867 FE (Mg2+)8.94 ± 1.2414.14 ± 2.750.100Ca, calcium; Cl, chloride; CnB1-KO, calcineurin regulatory subunit B alpha knockout; FE, iron; K, potassium; Mg, magnesium; Na, sodium; HCO3–, hydrogencarbonate ion.Values are mean ± SEM. Blood parameters: Control (n = 11) and CnB1-KO (n = 11). Urine parameters: control (n = 6) and CnB1-KO (n = 6).a P < 0.05.b P < 0.01. Open table in a new tab Ca, calcium; Cl, chloride; CnB1-KO, calcineurin regulatory subunit B alpha knockout; FE, iron; K, potassium; Mg, magnesium; Na, sodium; HCO3–, hydrogencarbonate ion. Values are mean ± SEM. Blood parameters: Control (n = 11) and CnB1-KO (n = 11). Urine parameters: control (n = 6) and CnB1-KO (n = 6). Quantitative RT-PCR analysis (Table 2) revealed that kidneys of CnB1-KO mice have significantly lower mRNA expression levels of several DCT-enriched genes, including NCC (Slc12a3), TRPM6 (Trpm6), and cyclin M2 (Cnnm2). Interestingly, the mRNA expression of the DCT-specific Ca2+- and Mg2+-binding protein parvalbumin completely vanished, and the expression of the DCT and CNT expressed Ca2+-binding protein calbindin D28K was reduced by 50%. Moreover, the mRNA expression of epidermal growth factor (Egf) and vitamin D receptor (Vdr) was lower in isolated DCTs of CnB1-KO mice than in DCTs of control mice (Supplementary Table S4). Calcineurin is known to have strong transcriptional effects.3Aramburu J. Heitman J. Crabtree G.R. Calcineurin: a central controller of signalling in eukaryotes. Workshop on the calcium/calcineurin/NFAT pathway: regulation and function.EMBO Rep. 2004; 5: 343-348Crossref PubMed Scopus (130) Google Scholar Due to the lack of an appropriate native DCT cell model, we could not address this issue further. However, by using fibroblasts directly reprogrammed to renal tubular epithelial cells,19Kaminski M.M. Tosic J. Kresbach C. et al.Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors.Nat Cell Biol. 2016; 18: 1269-1280Crossref PubMed Scopus (77) Google Scholar we could demonstrate a direct effect of the calcineurin inhibitor tacrolimus on the mRNA expression of Trpm6 and Cnnm2 (Supplementary Figure S4).Table 2Expression (qRT-PCR) of genes involved in renal function detected in whole kidney homogenates from control and CnB1-KO mice 3 weeks after CnB1 deletionGeneControlCnB1-KOP valueSodium transport–related genes NKCC2 (Slc12a1)1.00 ± 0.201.58 ± 0.320.171 NCC (Slc12a3)1.00 ± 0.050.40 ± 0.05<0.0001aP < 0.0001. αENaC (Scnn1a)1.00 ± 0.180.92 ± 0.040.709 βENaC (Scnn1b)1.00 ± 0.061.10 ± 0.030.203 γENaC (Scnn1g)1.00 ± 0.071.01 ± 0.070.420Potassium transport–related genes ROMK (Kcnj1)1.00 ± 0.180.95 ± 0.100.829Magnesium transport–related genes TRPM6 (Trpm6)1.00 ± 0.070.32 ± 0.04<0.0001aP < 0.0001. TRPM7 (Trpm7)1.00 ± 0.050.97 ± 0.080.741 Cyclin M2 (Cnnm2)1.00 ± 0.050.60 ± 0.040.0003bP < 0.001. Na+/K+-transporting ATPase subunit gamma chain (Fxyd2)1.00 ± 0.170.91 ± 0.100.672 EGF (Egf)1.00 ± 0.061.17 ± 0.110.224 EGFR (Egfr)1.00 ± 0.061.00 ± 0.060.989Calcium transport–related genes TRPV5 (Trpv5)1.00 ± 0.071.04 ± 0.070.711 NCX (Slc8a1)1.00 ± 0.041.02 ± 0.030.624Intracellular Mg2+ and/or Ca2+–binder-related genes Parvalbumin (Pvalb)1.00 ± 0.090.00 ± 0.00<0.0001aP < 0.0001. Calbindin (Calb1)1.00 ± 0.140.51 ± 0.030.008cP < 0.05.Acid–base homeostasis–related genes NHE3 (Slc9a3)1.00 ± 0.111.06 ± 0.100.685 Pendrin (Slc26a4)1.00 ± 0.071.21 ± 0.100.111 H+-ATPase (Atp6v1b1)1.00 ± 0.081.39 ± 0.080.009cP < 0.05.Hormone-related genes Renin (Ren)1.00 ± 0.131.19 ± 0.150.377CnB1-KO, calcineurin regulatory subunit B alpha knockout; GAPDH, glycerinaldehyde-3-phosphate-dehydrogenase; qRT-PCR, quantitative real-time polymerase chain reaction.Gene expression was normalized to GAPDH and control values. Values are mean ± SEM. Control (n = 5) and CnB1-KO (n = 5).a P < 0.0001.b P < 0.001.c P < 0.05. Open table in a new tab CnB1-KO, calcineurin regulatory subunit B alpha knockout; GAPDH, glycerinaldehyde-3-phosphate-dehydrogenase; qRT-PCR, quantitative real-time polymerase chain reaction. Gene expression was normalized to GAPDH and control values. Values are mean ± SEM. Control (n = 5) and CnB1-KO (n = 5). The mRNA expression of other renal ion transporters and channels, including NKCC2 (Slc12a1), the 3 ENaC subunits (Scnn1a,b,g), ROMK (Kcnj1), TRPV5 (Trpv5), the sodium calcium exchanger (NCX, Slc8a1), the sodium-proton exchanger 3 (NHE3, Slc9a3), and the chloride bicarbonate exchanger (pendrin, Slc26a4), was unaltered. In contrast to the other genes, the mRNA expression of the H+-ATPase (Atp6v1b1) was upregulated. Renal renin mRNA expression was not different between genotypes (Table 2). The kidneys of CnB1-KO mice revealed significantly lower protein levels for NCC, TRPM6, parvalbumin, and calbindin than those of controls (Figure 3). Despite the