Johannes Loffing's research while affiliated with University of Zurich and other places

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.

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 mechanism 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.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of the Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related Proline/Alanine rich Kinase (SPAK) and Oxidative Stress Responsive kinase 1 (OSR1), which are regulated by the With No Lysine (K) kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a Protein Phosphatase 1 (PP1) inhibitor, may also play a role. In HEK293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation was studied in desmopressin-infused WNK4 -/- mice and in tubule suspensions. In HEK293 cells, only wild-type WNK4, but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4, but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation, but not of NCC or SPAK in the absence of WNK4. WNK4 -/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4 -/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of pSPAK was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.

The bone-derived hormone fibroblast growth factor-23 (FGF23) has recently received much attention due to its association with chronic kidney disease and cardiovascular disease progression. Extracellular sodium concentration ([Na⁺]) plays a significant role in bone metabolism. Hyponatremia (lower 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 lines UMR-106 and MC3T3-E1. 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. Furthermore, arginine vasopressin, which is often responsible for hyponatremia, did not affect FGF23 production. Next, we performed a comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low versus high [Na⁺], which 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 hyponatremia patients have higher FGF23 levels. Our results suggest that [Na⁺] is a critical regulator of FGF23 synthesis.

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 processes adjust the plasma potassium concentration between 3.5 and 4.5 mmol/L. Several genetic and acquired diseases 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 debated. 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.

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.

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 (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. Phosphoproteomic 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 regulates CYP11B2 expression. Thus, calcineurin 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.

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 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.

Soluble-Klotho (sKl) is the shed ectodomain of the transmembrane protein-Klotho (mKl) that exhibits pleiotropic actions, including lifespan extension, mineral metabolism, slowing-down kidney diseases and cardioprotection. The sKl is derived from the kidneys, but what type/s of renal cells secrete it is unknown. Secondly the respective roles of mKl versus sKl in regulating mineral metabolism is unclear due to the lack of appropriate in vivo models. Here, using scRNA-seq of renal distal-convolution (DC) cells, we found an unexpected pattern revealing that Klotho transcripts ( Kl) are moderately expressed in overall distal convoluted tubule (DCT), but highly enriched in the end of DCT and in connecting tubule (CNT). Immunohistochemistry further confirmed this pattern for mKl protein as well. Next, Kl was knocked-out only in renal DC to check if it affects sKl production. Interestingly, deleting Kl in the DCT and late-DCT+CNT in mice showed ~20% and ~80% reduction in sKl levels, respectively. Expectedly, knocking-out Kl along the entire DC in mice (Kl-KO DC ) abolished sKl levels. Furthermore, we found that compared to control mice, Kl-KO DC mice exhibited reduced renal TRPV5-Ca ²⁺ channel expression, profound calciuria, and loss of bone mineral density. The RNA-seq of automated-sorted DC cells from Kl-KO DC mice revealed enhanced caveolae-mediated endocytosis of TRPV5. On the other hand, Kl-KO DC mice had normal phosphate metabolism as confirmed by unchanged serum FGF23, serum phosphate, urinary phosphate excretion, and renal NaPi-IIa expression. Our findings reveal that a small population of renal DC cells accounts for the sKl levels. The lack of sKl may leads to disturbed Ca ²⁺ homeostasis and bone loss without affecting phosphate balance in mice. 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.