Calcineurin inhibitors (CNIs), including tacrolimus and cyclosporin A, are potent immunosuppressive drugs that exert their effects by inhibiting protein phosphatase 3, also known as calcineurin. CNIs are widely used clinically, particularly for preventing organ rejection following transplantation. However, their use is frequently associated with significant adverse effects. In particular, CNIs commonly affect the kidney and can lead to acute and chronic kidney injury. In addition, they interfere with renal electrolyte reabsorption resulting in electrolyte disturbances (e.g., hyperkalemia, hypomagnesemia) and hypertension. Notably, calcineurin subunits are abundantly expressed throughout the nephron. Consequently, calcineurin inhibition affects electrolyte transport in virtually all nephron segments. In this review, we summarize the current knowledge on the effects of CNIs on renal tubular ion transport processes and highlight the underlying mechanism by which these effects contribute to CNI-induced electrolyte disturbances.

Introduction Klotho acts as a coreceptor for the phosphaturic hormone fibroblast growth factor-23 (FGF-23) and exists in both a membrane-bound and a soluble form (sKlotho) found in blood and urine. Klotho protein is moderately expressed in kidney proximal tubule and more abundant in the distal convolution (DC), which includes the distal convoluted tubule (DCT) and connecting tubule (CNT). However, the function of Klotho in the DC, particularly its role in sKlotho release and regulation of mineral metabolism, remains unclear. Methods scRNA-seq was performed on isolated mouse DC cells. Four novel gene-modified mouse models were generated with Klotho deleted in the entire DC, the late DCT/CNT, the DCT only, and pan-tubular. Results Using scRNA-seq on isolated mouse DC tubules, we showed that Klotho is more abundant in the late-DCT/CNT than in the early DCT. The composite data from three DC specific Klotho knockout mice support DC to be the primary source of urinary sKlotho, with 80% coming from the late-DCT/CNT and only 20% from the DCT. Notably, mice lacking Klotho in the entire DC (Kl-KODC) maintained normal serum sKlotho, FGF-23, and phosphate homeostasis. Bulk RNA-seq of isolated fluorescent DC segments from Kl-KODC_Tomato mice revealed suppressed signaling by mitogen activated protein kinase and downregulation of several genes involved in kidney calcium ion handling (Trpv5, Vdr, Pth1r, Klk1). Consistently, the Kl-KODC mice exhibited profound hypercalciuria and reduced bone density. On the other hand, pan-tubular Klotho deficiency in mice led to severe phosphate imbalance and loss of both serum/urine sKlotho. Conclusions DC-derived Klotho regulates urinary sKlotho levels and controls calcium ion reabsorption, while Klotho in proximal tubule maintains phosphate homeostasis and likely regulates circulating sKlotho levels.

The renal thick ascending limb (TAL) plays a key role in water and ion homeostasis. Apical potassium secretion via the renal outer medullary potassium channel (ROMK) is essential for transepithelial sodium reabsorption via the furosemide-sensitive Na-K-2Cl-cotransporter and creates the electrochemical gradient for paracellular ion transport through Claudin tight junction proteins. Interestingly, the TAL exhibits transcriptomic heterogeneity and variable apical ROMK abundance. Single-cell RNA sequencing suggests that the cortical TAL consists of at least three distinct cell types, but whether ROMK distribution aligns with these types remains unclear. We analyzed perfusion-fixed mouse kidneys using RNAscope in situ hybridization (ISH), iterative indirect immunofluorescence imaging (4i multiplexing), and machine learning. ROMK mRNA expression was seen in all TAL cells. In contrast, apical ROMK protein abundance was found on almost all macula densa (MD) cells but was heterogeneous along the rest of the TAL. In the remaining TAL, only about 60% of the TAL cells had strong apical ROMK staining, while 40% lacked apical ROMK but showed weak perinuclear signals. ISH revealed that apical ROMK-positive cells express Ptger3 mRNA, whereas apical ROMK-negative cells express Foxq1 mRNA. Multiplexing analysis showed that ROMK-positive cells form Claudin-10b-positive tight junctions, while ROMK-negative cells form Claudin-16/19-positive junctions and express basolateral Kir4.1. Despite universal ROMK mRNA expression, apical ROMK distribution aligns with molecularly distinct TAL cell types. This unique ROMK expression pattern suggests functional heterogeneity for ROMK along the TAL.

Excessive dietary salt intake is a global health concern, affecting cardiovascular, renal, and bone health. While the renin-angiotensin-aldosterone system (RAAS) is a known regulator of dietary salt-induced hormonal responses, the impact of adrenal cortisol remains unclear. Here, we performed a retrospective analysis in individuals (n=292) consuming a random diet. Dietary salt intake positively correlated with urinary cortisol and inversely correlated with plasma fibroblast growth factor 23 (FGF23), a bone-derived hormone regulating phosphate and vitamin D homeostasis. Controlled salt diets in healthy individuals confirmed a dose-dependent increase in urinary cortisol and suppression of plasma FGF23. In mice, oral corticosterone, a cortisol analogue, reduced circulating FGF23 levels. RNA-seq analysis of corticosterone-treated MC3T3 osteoblasts identified suppression of FGF23 via glucocorticoid receptor activation, anti-inflammatory pathways, and reduced osteoblast activity. Our findings reveal a novel endocrine cascade where high salt intake elevates cortisol and suppresses FGF23, with potential implications for bone, kidney, and cardiovascular health. ### Competing Interest Statement The authors have declared no competing interest. This work was supported by the Swiss National Science Foundation through the National Center of Competence in Research NCCR Kidney. C.H. [grant number N-403-03-55 (to G. P.)], funds from the Clinical Research Priority Program HYRENE of the University of Zurich (UZH) and intramural funding of the UZH (to J.L.). M.S.S. received funding from the Medical Research Council (United Kingdom) as part of a Clinical Research Training Fellowship (MR/T008172/1).

Macrophages play crucial roles in organ-specific functions and homeostasis. In the adrenal gland, macrophages closely associate with sinusoidal capillaries in the aldosterone-producing zona glomerulosa. We demonstrate that macrophages preserve capillary specialization and modulate aldosterone secretion. Using macrophage-specific deletion of VEGF-A, single-cell transcriptomics, and functional phenotyping, we found that the loss of VEGF-A depletes PLVAP+ fenestrated endothelial cells in the zona glomerulosa, leading to increased basement membrane collagen IV deposition and subendothelial fibrosis. This results in increased aldosterone secretion, called "haptosecretagogue" signaling. Human aldosterone-producing adenomas also show capillary rarefaction and basement membrane thickening. Mice with myeloid cell-specific VEGF-A deletion exhibit elevated serum aldosterone, hypokalemia, and hypertension, mimicking primary aldosteronism. These findings underscore macrophage-to-endothelial cell signaling as essential for endothelial cell specialization, adrenal gland function, and blood pressure regulation, with broader implications for other endocrine organs.

Fibroblast growth factor-23 (FGF23) is crucial for phosphate and vitamin D homeostasis. Moreover, FGF23 levels are very high in patients with chronic kidney disease (CKD) with unclear functions. Binding of FGF23 to its coreceptor Klotho is considered essential for its actions. However, recent data suggested that CKD-related high FGF23 levels may have Klotho-independent cardiotoxic and inflammatory effects but the underlying signaling mechanisms are unclear. Here, we performed a comprehensive and unbiased transcriptomic profiling in HEK293 cells, comparing the effects of 0.5 nM FGF23 for 1 hour (low-transitory: ∼physiological) and 10 nM FGF23 for 24 hours (high-prolonged: ∼pathological), in the presence and absence of Klotho. We found that, at physiological concentration, FGF23 action requires Klotho and follows the canonical MAPK signaling. Conversely, at pathological high levels, FGF23 acts both in the presence and in the absence of Klotho. In the presence of Klotho, high FGF23 activates a plethora of transcripts including the inflammatory genes (e.g. TGFB1, GDF15, ANXA1 and TNFRSF9/12 ) known to be elevated in patients with CKD. Interestingly, in the absence of Klotho, high FGF23 levels does also regulate a small and unique set of genes related to post-transcriptional modifications and translation initiation. To conclude, Klotho is essential for FGF23 signaling at physiological FGF23 concentrations. However, with pathologically high FGF23 levels, Klotho acts as a molecular switch determining the type of FGF23 response.### Competing Interest StatementThe authors have declared no competing interest.

Klotho plays a critical role in the regulation of ion and fluid homeostasis. A previous study reported that haplo-insufficiency of Klotho in mice results in increased aldosterone synthase (CYP11B2) expression, elevated plasma aldosterone, and high blood pressure. This phenotype was presumed to be the result of diminished Klotho expression in zona glomerulosa (zG) cells of the adrenal cortex; however, systemic effects on adrenal aldosterone production could not be ruled out. To examine whether Klotho expressed in the zG is indeed a critical regulator of aldosterone synthesis, we generated a tamoxifen-inducible, zG-specific mouse model of Klotho deficiency by crossing Klotho-flox mice with Cyp11b2-CreERT mice (zG-Kl-KO). Tamoxifen-treated Cyp11b2-CreERT animals (zG-Cre) served as controls. Rosa26-mTmG reporter mice were used for Cre-dependent lineage-marking. Two weeks after tamoxifen induction, the specificity of the zG-Cre line was verified using immunofluorescence analysis to show that GFP expression was restricted to the zG. RNA in situ hybridization revealed a 65% downregulation of Klotho messenger RNA expression in the zG of zG-Kl-KO female mice at age 12 weeks compared to control mice. Despite this significant decrease, zG-Kl-KO mice exhibited no difference in plasma aldosterone levels. However, adrenal CYP11B2 expression and the CYP11B2 promotor regulatory transcription factors, NGFIB and Nurr1, were enhanced. Together with in vitro experiments, these results suggest that zG-derived Klotho modulates Cyp11b2 but does not evoke a systemic phenotype in young adult mice on a normal diet. Further studies are required to investigate the role of adrenal Klotho on aldosterone synthesis in aged animals.

ABSTRACT Fibroblast growth factor-23 (FGF23) is a bone-derived hormone that has recently received much attention due to its association with the progression of chronic kidney disease, cardiovascular disease, and associated mortality. Extracellular sodium concentration ([Na + ]) plays a significant role in bone metabolism. Hyponatremia (low serum [Na + ]) has recently been shown to be independently associated with FGF23 levels in patients with chronic systolic heart failure. However, nothing is known about the direct impact of [Na + ] on FGF23 production. Here, we show that an elevated [Na + ] (+20 mM) suppressed FGF23 formation, whereas low [Na + ] (−20 mM) increased FGF23 synthesis in the osteoblast-like cell line UMR-106. Similar bidirectional changes in FGF23 abundance were observed when osmolality was altered by mannitol but not by urea, suggesting a role of tonicity in FGF23 formation. Moreover, these changes in FGF23 were inversely proportional to the expression of NFAT5 (nuclear factor of activated T cells-5), a transcription factor responsible for tonicity-mediated cellular adaptations. On the other hand, arginine vasopressin (AVP), which is often responsible for hyponatremia, did not affect FGF23 production. Next, comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low vs. high [Na + ] revealed several novel genes involved in cellular adaptation to altered tonicity. Additional analysis of cells with Crisp-Cas9 mediated NFAT5 deletion indicated that NFAT5 controls numerous genes associated with FGF23 synthesis, thereby confirming its role in [Na + ]-mediated FGF23 regulation. In line with these in vitro observations, we found that human hyponatremia patients have higher FGF23 levels. Our results suggest that [Na + ] is a critical regulator of FGF23 synthesis. SIGNIFICANCE STATEMENT Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that controls phosphate and vitamin D metabolism. Excess FGF23 is postulated to cause left ventricular hypertrophy, while FGF23 deficiency reduces life span and mimics age-related diseases in mice. FGF23 is also a potential biomarker for chronic kidney disease and cardiovascular disorders, but its role in disease progression is unclear. Therefore, it is important to explore the regulation of FGF23 production, which is incompletely understood. Our paper identifies extracellular-sodium-NFAT5 signaling as a key regulator of FGF23 formation.

With-no-lysine kinases regulate the phosphorylation and activity of the Na + -Cl – and Na + -K + -2Cl – cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

Significance Statement Rapid renal responses to ingested potassium are essential to prevent hyperkalemia and also play a central role in blood pressure regulation. Although local extracellular K + concentration in kidney tissue is increasingly recognized as an important regulator of K + secretion, the underlying mechanisms that are relevant in vivo remain controversial. To assess the role of the signaling kinase mTOR complex-2 (mTORC2), the authors compared the effects of K + administered by gavage in wild-type mice and knockout mice with kidney tubule-specific inactivation of mTORC2. They found that mTORC2 is rapidly activated to trigger K + secretion and maintain electrolyte homeostasis. Downstream targets of mTORC2 implicated in epithelial sodium channel regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. These findings offer insight into electrolyte physiologic and regulatory mechanisms. Background Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K + ]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. Methods We used Cre-Lox–mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins after a K + load by gavage. Results A K + load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type, but not in knockout, mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K + ] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). Conclusions The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K + ] in vivo . The effects of K + on this signaling module are specific, in that other downstream mTORC2 targets, such as PKC and Akt, are not acutely affected, and ROMK and Large-conductance K + (BK) channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K + in vivo .

The mineralocorticoid aldosterone, secreted by the adrenal zona glomerulosa (ZG), is critical for life, maintaining ion homeostasis and blood pressure. Therapeutic inhibition of protein phosphatase 3 (calcineurin, Cn) results in inappropriately low plasma aldosterone levels despite concomitant hyperkalemia and hyperreninemia. We tested the hypothesis that Cn participates in the signal transduction pathway regulating aldosterone synthesis. Inhibition of Cn with tacrolimus abolished the potassium-stimulated (K+-stimulated) expression of aldosterone synthase, encoded by CYP11B2, in the NCI-H295R human adrenocortical cell line as well as ex vivo in mouse and human adrenal tissue. ZG-specific deletion of the regulatory Cn subunit CnB1 diminished Cyp11b2 expression in vivo and disrupted K+-mediated aldosterone synthesis. Phosphoproteomics analysis identified nuclear factor of activated T cells, cytoplasmic 4 (NFATC4), as a target for Cn-mediated dephosphorylation. Deletion of NFATC4 impaired K+-dependent stimulation of CYP11B2 expression and aldosterone production while expression of a constitutively active form of NFATC4 increased expression of CYP11B2 in NCI-H295R cells. Chromatin immunoprecipitation revealed NFATC4 directly regulated CYP11B2 expression. Thus, Cn controls aldosterone production via the Cn/NFATC4 pathway. Inhibition of Cn/NFATC4 signaling may explain low plasma aldosterone levels and hyperkalemia in patients treated with tacrolimus, and the Cn/NFATC4 pathway may provide novel molecular targets to treat primary aldosteronism.

Membrane voltage controls the function of excitable cells and is mainly a consequence of the ratio between the extra-and intracellular potassium concentration. Potassium homeostasis is safeguarded by balancing the extra -/intracellular distribution and systemic elimination of potassium to the dietary potassium intake. These pro-cesses adjust the plasma potassium concentration between 3.5 and 4.5 mmol/L. Several genetic and acquired dis-eases but also pharmacological interventions cause dyskalemias that are associated with increased morbidity and mortality. The thresholds at which serum K+ not only associates but also causes increased mortality are hotly de-bated. We discuss physiologic, pathophysiologic, and pharmacologic aspects of potassium regulation and provide informative case vignettes. Our aim is to help clinicians, epidemiologists, and pharmacologists to understand the complexity of the potassium homeostasis in health and disease and to initiate appropriate treatment strategies in dyskalemic patients.& COPY; 2023 Published by Elsevier Inc.

The thick ascending limb (TAL) is critical for the renal control of fluid and ion homeostasis. The function of the TAL depends on the activity of the bumetanide-sensitive Na-K-2Cl co-transporter NKCC2, which is highly abundant in the luminal membrane of TAL cells. The TAL function is regulated by various hormonal and non-hormonal factors. However, many of the underlying signal transduction pathways remain elusive. Here, we describe the characterization of a novel gene-modified mouse model for an inducible and specific Cre/Lox-mediated gene modification in the TAL. In these mice, a tamoxifen-dependent Cre (CreERT2) was inserted into the 3’ UTR of the Slc12a1 gene, which encodes NKCC2 (Slc12a1-CreERT2). Although this gene-modification strategy reduced endogenous NKCC2 expression at the mRNA and protein level, the lowered NKCC2 abundance was not associated with an altered urinary fluid and ion excretion. Likewise, the renal response to loop-diuretics or water restriction was similar in wildtype (wt) and in Slc12a1-CreERT2wt/tg mice. Immunohistochemistry on kidneys from Slc12a1-CreERT2 mice revealed strong Cre expression exclusively in TAL cells but not in any other nephron portion. Cross-breeding of these mice with the mT/mG reporter mouse line showed a very low recombination rate (0.22%) at baseline, but a complete recombination (100%) after repeated tamoxifen administration. The achieved recombination encompassed the entire TAL and included also the macula densa. Thus, the new Slc12a1-CreERT2wt/tg mouse line allows an inducible and very efficient gene-targeting in the TAL and hence promises to be a powerful tool to advance our understanding of the regulation of TAL function. The research of Johannes Loffing is supported by the Swiss National Science Foundation (310030_143929/1) and the Swiss National Centre for Competence in Research “Kidney.CH” This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The intercalated cell Cl − /HCO 3 − exchanger, pendrin, modulates ENaC subunit abundance and function. Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl − absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle’s syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle’s variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle’s mutation increased total Cl − absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl − absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.

Introduction: Glucagon is raised during hypoglycemia in order to bring blood glucose back to normal levels. However, glucagon is paradoxically also raised under pathophysiological conditions where plasma glucose is high such as in diabetes mellitus (DM). DM is associated with hypertension, which contributes to diabetic kidney disease and other co-morbidities. The NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT) contributes to blood pressure control and the DCT also expresses the glucagon receptor. Therefore, we hypothesized that glucagon directly stimulates NCC via the glucagon receptor, and this may be a novel mechanism to modulate blood pressure. Methods: In vivo effects on NCC were studied in mice injected with glucagon. Ex vivo kidney tubules isolated from mouse and human kidney (obtained from tumor nephrectomized patients) were cultured and exposed to varying doses of glucagon alone, or in the presence of various inhibitors. The phosphorylation status of NCC was used as a surrogate marker for activity (increased phosphorylation = increased activity). Results: In kidneys isolated from mice 30 min after being injected with glucagon, NCC phosphorylation was increased. In ex vivo mouse kidney tubules, glucagon (1 nM to 1000 nM) exposure for 30 min increased NCC phosphorylation in a dose-dependent manner. 10 nM glucagon increased NCC phosphorylation after 5 min, an effect lasting up to 4 h. The glucagon receptor inhibitor 168,049 completely blocked the effect of 10 nM glucagon on NCC phosphorylation. The With No Lysine kinase (WNK) inhibitor StockS2, the inward-rectifier potassium channel 4.1/5.1 (Kir 4.1/5.1) inhibitor VU0134992, and the non-specific protein kinase A (PKA) inhibitor, H89, greatly reduced the effect of 10 nM glucagon on NCC phosphorylation. NCC phosphorylation was also increased in ex vivo human kidney slices exposed to 100 nM glucagon. Conclusion: We show for the first time that glucagon increases NCC phosphorylation in the mouse kidney both in vivo and ex vivo. The effect occurs via the glucagon receptor and involves WNK kinases, Kir4.1/5.1 and PKA. Glucagon effects can be observed ex vivo in human kidney slices, supporting that the findings may be relevant in humans. Whether glucagon-induced NCC phosphorylation contributes to the etiology of pathophysiological conditions manifested with raised plasma glucagon levels, such as DM, remains to be investigated. Novo Nordisk Foundation (NNF21OC0067647) and the Leducq Foundation (17CVD05). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The kidney is a structurally and functionally complex organ responsible for the control of water, ion, and other solute homeostasis. Moreover, the kidneys excrete metabolic waste products and produce hormones, such as renin and erythropoietin. The functional unit of the kidney is the nephron, which is composed by a serial arrangement of a filter unit called the renal corpuscle and several tubular segments that modulate the filtered fluid by reabsorption and secretion. Within each kidney, thousands of nephrons are closely intermingled and surrounded by an intricate network of blood vessels and various interstitial cell types, including fibroblasts and immune cells. This complex tissue architecture is essential for proper kidney function. In fact, kidney disease is often reflected or even caused by a derangement of the histologic structures. Frequently, kidney histology is studied using microscopic analysis of 2-dimensional tissue sections, which, however, misses important 3-dimensional spatial information. Reconstruction of serial sections tries to overcome this limitation, but is technically challenging, time-consuming, and often inherently linked to sectioning artifacts. In recent years, advances in tissue preparation (e.g., optical clearing) and new light- and electron-microscopic methods have provided novel avenues for 3-dimensional kidney imaging. Combined with novel machine-learning algorithms, these approaches offer unprecedented options for large-scale and automated analysis of kidney structure and function. This review provides a brief overview of these emerging imaging technologies and presents key examples of how these approaches are already used to study the normal and the diseased kidney.