IntroductionCalcineurin inhibitors (CNIs), like tacrolimus and cyclosporine A, are main pillars of immunosuppressive therapy in patients with solid organ transplantation and autoimmune disease 1Thirty Years of Tacrolimus in Clinical Practice.. Calcineurin or protein phosphatase 3 (PP3) is a calcium-calmodulin activated serine-threonine protein phosphatase that is composed of two subunits, which both are essential for the function of this phosphatase. The catalytic (A) subunit provides the enzymatic activity and is encoded by three separate genes (Ppp3ca, Ppp3cb, and Ppp3cc). The regulatory (B) domain carries the Ca2+-binding pockets and is encoded by two separate genes (Ppp3r1, Ppp3r2) 2Calcineurin: form and function.. 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 3

Aramburu J, Heitman J, Crabtree GR. Calcineurin: A central controller of signalling in eukaryotes. Workshop on the calcium/calcineurin/NFAT pathway: Regulation and function. In: EMBO Reports. Vol 5. ; 2004:343-348. doi:10.1038/sj.embor.7400133

. 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.,.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 2Calcineurin: form and function. . The broad tissue distribution of calcineurin may explain the plethora of side effects of CNI treatment 1Thirty Years of Tacrolimus in Clinical Practice.. Arterial hypertension, hyperkalemia, hypomagnesemia, hypercalciuria and metabolic acidosis are frequently observed under CNI treatment and were attributed at least in part to a malfunction of the renal distal convoluted tubule (DCT) 6McCormick J.A. Ellison D.H. Distal convoluted tubule.. 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.. The DCT impacts on K+ and acid-base balance mainly via indirect effects. For example, a lowered NCC activity, 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 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 co-workers 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.. 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.,9Lazelle R.A. McCully B.H. Terker A.S. et al.Renal deletion of 12 kDa FK506-binding protein attenuates tacrolimus-induced hypertension.. 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., 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.,10Ishizawa K. Wang Q. Li J. et al.Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling. . 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. Tzadok-Witkon M. Pauzner R. Farfel Z. Pseudohypoaldosteronism type II: Marked sensitivity to thiazides, hypercalciuria, normomagnesemia, and low bone mineral density.. 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. possibly mediated via a reduced EGF-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.. However, the underlying molecular mechanism and a possible contribution of other Mg2+ transporters like SLC41A3 14 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. 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.. 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 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.,18Pfluger P.T. Kabra D.G. Aichler M. et al.Calcineurin Links Mitochondrial Elongation with Energy Metabolism..Materials and methods AnimalsAll 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 shown schematically in Figure 1A,B. Activation of cre was induced by oral gavage of tamoxifen (Sigma-Aldrich, St. Louis, Montana, USA, 2 mg/animal/day in ethanol – sunflower oil mixture with a ratio of 1:9) for five consecutive days. Pilot studies showed that CnB1 deletion for 10 weeks, but not for 3 weeks reduces the fractional volume of the DCT (Suppl. Figure 1). 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.

Figure 1DCT-specific targeting of CnB1. A: Breeding scheme of CnB1flox/flox:NCCwt (Control) and CnB1flox/flox:NCCCre/wt (CnB1-KO) mice. B: Breeding scheme of CnB1wt/wt:NCCCre/wt:Tomatoflox/wt (Control-Tomato) and CnB1flox/flox:NCCCre/wt:Tomatoflox/wt (CnB1-KO-Tomato) mice. C: Immunoblotting of whole kidney homogenates to reveal the abundance of DCT-specific proteins in CnB1wt/wt:NCCwt and CnB1wt/wt:NCCCre/wt mice. D: Densitometric analysis of immunoblot in C. Band intensities are normalized to β-actin and average abundance in Control group (n = 3 mice /group). Values are mean ± SEM. E: Immunofluorescent detection of NKCC2 (green), NCC (green), AQP2 (green), and Tomato (red) in kidneys of Control-Tomato mice. Tomato-related fluorescence starts directly at the transition from the NKCC2-positive TAL (T) to the NKCC2-negative early DCT (D1), and extends along the entire NCC-positive DCT (D1) just until the transition from the AQP2-negative late DCT (D2) to the AQP2-positive CNT (CN). F: Representative selection window (left panel) used at the Biosorter to isolate red fluorescent DCTs as shown as a pellet in an Eppendorf tube (middle panel) or as single tubules under the microscope (right panel). G: Detection of NKCC2 and AQP2 mRNA in Biosorter-isolated DCTs from Control and CnB1-KO mice 3 weeks after induction of CnB1 deletion. For the analysis of NKCC2 and AQP2 in DCT, mRNA from total kidney served as a positive control. Shown are data points with mean ± SEM. (n = 4-5 mice per group; ****p≤0.0001, unpaired Student’s t-test). H: Detection of CnB1 mRNA and calcineurin activity in Biosorter-isolated DCTs from Control and CnB1-KO mice 3 weeks after induction of CnB1 deletion. Calcineurin activity was measured with a calorimetric assay that is based on the detection of free-phosphate generated by calcineurin. Hence, although DCT samples were washed with phosphate-free TBS after DCT-sorting, the measured values in CnB1-KO could reflect both a residual calcineurin activity and remnants of PBS in the analyzed samples. Shown are points with mean ± SEM (n = 3 mice / group; **p≤0. 01, unpaired Student’s t-test)..

 Metabolic cage studies, analysis of urine, feces, and blood

Mice were maintained on a standard rodent diet (0.3% Na+ and 0.8% K+) unless stated otherwise. Urine and feces were collected for 24h or 48h in metabolic cages. At the end of 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 (Radiometer ABL 505, Copenhagen, Denmark). Plasma and urine ion and creatinine concentrations as well as fecal Mg2+ content were determined as described in supplement.

 Biochemical analysis

Plasma aldosterone levels, alpha-ketoglutarate concentrations in urine, and calcineurin activity in isolated DCTs were measured as described in supplement.

 Quantitative RT-PCR

Total RNA was isolated from kidney, distal colon mucosa and renal tubules with the SV Total RNA Isolation System (Promega, Fitchburg, Wisconsin, USA) and the Absolutely RNA Nanoprep Kit (Agilent, Santa Clara, CA), respectively. Real-time PCR was performed using a LightCycler 480 Instrument II (Roche, Switzerland). Primers are given in Suppl. Table 1.

 Immunoblotting

Kidneys were processed for immunoblotting and probed with antibodies listed in Suppl. Table 2. Moreover, a new rat monoclonal anti-pT58 NCC antibody was developed and characterized (Suppl. Figure 2). More details are given in the supplement.

 Immunohistochemistry and electron microscopy

Perfusion-fixed (3% paraformaldehyde) kidneys were processed for immunohistochemistry, morphometric analysis and electron microscopy as described in supplement.

 Thiazide test

Mice received a single i.p. injection of hydrochlorothiazide (HCTZ 50 μg/g BW) as described in the supplement.

 Tacrolimus treatment of induced renal tubular epithelial cells (iRECs)Direct reprogramming of fibroblasts to induced renal tubular epithelial cells (iRECs) 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.. iRECs were then treated for 24h either with vehicle (DMSO) or 20 nM tacrolimus (see supplement for more details). Tacrolimus treatment of mice

Mice received for 16 days daily s.c. injections of Tacrolimus (3 μg/g BW/day) as described in supplement.

 Telemetric monitoring of blood pressure

Blood pressure measurements were conducted in the Zürich Integrative Rodent Physiology (ZIRP) core-facility as described in supplement.

 Acute K+ challenge via gastric gavageMice received by oral gavage (15μl/g body weight) either a vehicle solution (2% sucrose) or 2% K+ in 2% sucrose as described earlier 20Sorensen M.V. Grossmann S. Roesinger M. et al.Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice. and in supplement. Kidney slices

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, United Kingdom, 20 nM) as described in the supplement.

 Automated isolation of Tomato-positive DCTs and deep RNA sequencing

Control-Tomato and CnB1-KO-Tomato mice were used for automated isolation of red fluorescent DCTs using a biosorter (Union Biometrica, Somerville, MA) as described in the supplement and were processed for RNA sequencing done by the Functional Genomics Centre Zurich (see supplement). Data are deposited at the Gene Expression Omnibus (GEO) database with the accession number GSE169721.

 Manual microdissection of renal tubules and proteome analysisManual 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. (for qRT-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. (for protein analysis). Isolated DCTs were processed for proteome analysis as described in supplement. Summed protein expression (LFQ) 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 initially 2364. Remaining missing values were imputed. A two-tailed t-test was performed. Proteins with fold change and p-value cutoff as indicated were further analysed. Data are available through ProteomExchange/PRIDE 23Perez-Riverol Y. Csordas A. Bai J. et al.The PRIDE database and related tools and resources in 2019: Improving support for quantification data.,24Vizcaíno J.A. Deutsch E.W. Wang R. et al.ProteomeXchange provides globally coordinated proteomics data submission and dissemination.: PXD number: PXD017455, account: [email protected] , and password: otMM0mtn. Statistics

Unpaired Student’s t test was performed to compare two groups, while one-way ANOVA with Sidak’s multiple comparison post hoc test or two-way ANOVA for multiple comparisons. All statistical analyses were performed using Graphpad Prism 8. Data are expressed as mean +/- SEM.

DiscussionWe developed and characterized a novel gene-modified mouse-model to study the DCT-specific role of calcineurin signaling in adult mice. In these mice, the regulatory subunit of calcineurin (CnB1) is efficiently deleted along the entire DCT upon tamoxifen-dependent cre activation. Despite the rather low expression levels of calcineurin in normal DCT cells 16Lee J.W. Chou C.L. Knepper M.A. Deep sequencing in microdissected renal tubules identifies nephron segment-specific transcriptomes., we observed a striking effect of the induced DCT-specific disruption of CnB1 on renal function and the expression of numerous mRNAs and proteins involved in DCT ion transport. Although the immunofluorescent studies indicated much higher CnB1 abundances in late DCT (DCT2) than in early DCT (DCT1), the observed effects are unlikely related to effects on the DCT2 only. In the adult mouse kidney, the DCT2 is rather short compared to the DCT1 34Schnoz C. Carrel M. Loffing J. Loss of sodium chloride co-transporter impairs the outgrowth of the renal distal convoluted tubule during renal development.. Moreover, the DCT1-specific marker protein parvalbumin 35Loffing J. Loffing-Cueni D. Valderrabano V. et al.Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron. was one of the genes, which are most drastically affected by the CnB1 deletion.The prevalence of arterial hypertension in patients treated with CNIs is high (23% and 60%) 36Foy M.C. Vaishnav J. Sperati C.J. Drug-Induced Hypertension.. With the exception of a few studies reporting a down-regulation of NCC and KS-WNK1 37Sun W.L. Kyung O.A. Mee R.S. et al.Downregulation of renal sodium transporters and tonicity-responsive enhancer binding protein by long-term treatment with cyclosporin A.,38Cui Y. Huang Q. Auman J.T. et al.Genomic-derived markers for early detection of calcineurin inhibitor immunosuppressant-mediated nephrotoxicity., the hypertension was linked to an altered abundance and phosphorylation of NCC and its regulating kinases WNK3, WNK4, SPAK or the ubiquitin ligase protein Kelch-like 3 7Hoorn E.J. Walsh S.B. McCormick J.A. et al.The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension.,10Ishizawa K. Wang Q. Li J. et al.Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling.,30Blankenstein K.I. Borschewski A. Labes R. et al.Calcineurin inhibitor cyclosporine A activates renal NA-K-CL cotransporters via local and systemic mechanisms.,31Shoda W. Nomura N. Ando F. et al.Calcineurin inhibitors block sodium-chloride cotransporter dephosphorylation in response to high potassium intake.,39Tutakhel O.A.Z. Moes A.D. Valdez-Flores M.A. et al.NaCl cotransporter abundance in urinary vesicles is increased by calcineurin inhibitors and predicts thiazide sensitivity.,40Melnikov S. Mayan H. Uchida S. Holtzman E.J. Farfel Z. Cyclosporine metabolic side effects: association with the WNK4 system.. However, we did not see an upregulation of NCC activity in the CnB1-KO mice. Three weeks after CnB1 deletion, the levels of NCC phosphorylation, thiazide-induced natriuresis, phospho-SPAK abundance in DCT, renal renin expression, plasma aldosterone and blood pressure were similar for Control and CnB1-KO mice. Ten weeks after CnB1 disruption, the abundance and phosphorylation of NCC were even lower in CnB1-KO than Control mice (Suppl. Figure 1). Several reasons may explain these unexpected results. First of all, we used a genetic inactivation of calcineurin-activity specifically in the DCT, while the previous studies used a pharmacological approach, which may have blocked calcineurin signaling less efficient and/or that had impacted on other phosphatases and/or calcineurin in other tissues as well. Our findings in tacrolimus-treated Control and CnB1-KO mice suggest that the stimulatory effect of tacrolimus on NCC phosphorylation directly depends on an inhibition of calcineurin in the DCT. However, CnB1-KO mice do not show more NCC phosphorylation at the whole kidney level, because the stimulatory effect on NCC phosphorylation is completely compensated by a reduction of NCC mRNA and protein. The reason for the lowered NCC abundance is unclear, but may include a feed-back response to the enhanced NCC phosphorylation or might be simply part of the observed broad transcriptional effect of calcineurin disruption. Independently from this, it is important to note that we studied the mice 3 and 10 weeks after the induction of the CnB1 knockout, while most of the previous pharmacological studies analyzed cells and animals a few hours or within 2 weeks after initiation of CNI treatment 7Hoorn E.J. Walsh S.B. McCormick J.A. et al.The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension.,10Ishizawa K. Wang Q. Li J. et al.Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling.,30Blankenstein K.I. Borschewski A. Labes R. et al.Calcineurin inhibitor cyclosporine A activates renal NA-K-CL cotransporters via local and systemic mechanisms.,31Shoda W. Nomura N. Ando F. et al.Calcineurin inhibitors block sodium-chloride cotransporter dephosphorylation in response to high potassium intake.,40Melnikov S. Mayan H. Uchida S. Holtzman E.J. Farfel Z. Cyclosporine metabolic side effects: association with the WNK4 system.. Interestingly, the two studies that studied animals at later time points (i.e. 28 days) did also find a downregulation of NCC abundance 37Sun W.L. Kyung O.A. Mee R.S. et al.Downregulation of renal sodium transporters and tonicity-responsive enhancer binding protein by long-term treatment with cyclosporin A.,38Cui Y. Huang Q. Auman J.T. et al.Genomic-derived markers for early detection of calcineurin inhibitor immunosuppressant-mediated nephrotoxicity.. Similar, in CnB1-KO mice, the loss of NCC mRNA and protein gets more pronounced with time. Thus, the effect of calcineurin inhibition on NCC may vary depending on the type of inhibition (i.e. genetic deletion vs. pharmacological blockade), but also depending on the time-point at which it is assessed.As CnB1-KO mice show no evidence for an activation of NCC and have normal plasma aldosterone and renal ENaC levels, it is not surprising that these mice maintain K+ balance even when challenged with dietary K+ loading. Nevertheless, CnB1-KO mice have a slight defect in the early renal response to an oral K+ load similar to the one observed by Shoda et al. in tacrolimus-treated mice 31Shoda W. Nomura N. Ando F. et al.Calcineurin inhibitors block sodium-chloride cotransporter dephosphorylation in response to high potassium intake.. The authors linked the observed defect to an impaired K+-dependent dephosphorylation of NCC 31Shoda W. Nomura N. Ando F. et al.Calcineurin inhibitors block sodium-chloride cotransporter dephosphorylation in response to high potassium intake.. However, the sole loss of calcineurin signaling is not sufficient to significantly block K+-dependent regulation of NCC in the current and in our previous ex vivo studies 41Penton D. Czogalla J. Wengi A. et al.Extracellular K+ rapidly controls NaCl cotransporter phosphorylation in the native distal convoluted tubule by Cl−-dependent and independent mechanisms.. Only the disruption of both PP3 and PP1 phosphatase activities largely diminishes the K+-dependent NCC regulation. These observations are at variance to a recent study by Mukherjee and co-workers 42Mukherjee A. Yang C.L. McCormick J.A. Martz K. Sharma A. Ellison D.H. Roles of WNK4 and SPAK in K+-mediated dephosphorylation of the NaCl cotransporter., in which neither a pharmacological inhibition of PP1 nor of PP3, singly or in combination, was able to significantly block the K+-dependent NCC dephosphorylation. Turning-off the NCC activating WNK/SPAK pathway appeared to be more essential. Most likely, the maintenance of K+-homeostasis is coupled to a complex cooperation between kinases and phosphatases to safeguard a proper adaptation of NCC activity to altered plasma K+.Hypomagnesemia and hypercalciuria under CNI treatment were linked to a disruption of EGF and vitamin D receptor signaling that leads to a down-regulation of TRPM6, TRPV5, calbindin D28K and NCX1 12