The present invention relates to compositions and methods for the prevention and treatment of disorders characterized and/or caused by high levels of fibroblast growth factor 23 (FGF23) , especially chronic kidney disease (CKD) .

Chronic kidney disease (CKD) , or chronic renal disease, is a chronic condition characterized by progressive loss of kidney function over a period of months to years and describes all stages (1-5) of decreased renal function ranging from mild and moderate to severe chronic kidney failure (end-stage kidney dis ^¬ ease requiring dialysis) . CKD is classified according to the Kidney Disease Improving Global Outcomes (KDIGO) based on the glomerular filtration rate (GFR) and proteinuria (condition characterized by an excess amount of protein in the urine) . GFR describes the flow rate of filtered fluid through the kidney and is calculated based on age, sex and blood creatinine.

CKD is an important public health problem associated with a number of serious complications, including increased incidence of cardiovascular disease, anemia and metabolic bone disease. The most common causes for developing CKD are diabetes, high blood pressure and genetic predisposition, which are responsible for about two-thirds of the cases.

Early stages of CKD often remain undiagnosed due to patients being asymptomatic. Early detection and treatment of CKD however is vital to prevent progression of renal function decline and to reduce sequelae such as cardiovascular and metabolic disease. Over the last decade, substantial evidence has been accumulated regarding the role of high circulating levels of fibroblast growth factor 23 (FGF23) as a predictor of adverse outcomes in patients with CKD at all stages.

FGF23 is a phosphaturic hormone secreted from bone cells, which together with PTH and the biologically active vitamin D hormone la, 25-dihydroxyvitamin D3 [ 1 , 25 (OH) 2D3 ] , is a major sys ^¬ temic regulator of phosphate homeostasis. In the kidney, FGF23 reduces phosphate reabsorption from urine by a downregulation of sodium phosphate co-transporters (2a and 2c) in proximal renal tubular epithelium. In addition, FGF23 downregulates renal l - hydroxylase expression, thereby suppressing the production of la, 25-dihydroxyvitamin D3.

FGF23 was first described in the context of several heredi ^¬ tary and acquired human diseases (Ruppe MD. X-Linked Hypophos ^¬ phatemia. 2012 Feb 9 [Updated 2014 Oct 16] . In: Pagon RA, Adam MP, Ardinger HH, et al . , editors. GeneReviews® [Internet] . Seat ^¬ tle (WA) : University of Washington, Seattle; 1993-2017). Autoso ^¬ mal dominant hypophosphatemic rickets (ADHR) is a rare genetic renal phosphate-wasting disorder caused by a missense mutation in the FGF23 gene, which results in the stabilization of the ac ^¬ tive form of the protein leading to prolonged or enhanced FGF23 activity. In contrast, tumor-induced osteomalacia (TIO) , also known as oncogenic osteomalacia (OOM) , is an acquired parane ^¬ oplastic syndrome caused by high levels of serum FGF23 that are secreted by mesenchymal tumors. Further disorders associated with high levels of FGF23 and phosphate-wasting are the McCune- Albright syndrome and linear nevus sebaceous syndrome (or epi ^¬ dermal nevus syndrome) . The hypophosphatemic rickets observed in McCune-Albright syndrome are associated with overproduction of FGF23 by the fibrous dysplastic bone resulting in renal phos ^¬ phate wasting. Hypophosphatemia in linear nevus sebaceous syn ^¬ drome, which is characterized by multiple cutaneous nevi, is due to excessive production of FGF23 by fibrous dysplasia. Excessive FGF23 levels are also associated with acute diseases such as acute myocardial infarction and acute kidney injury (Schnedl C et al., Disease Markers, 2015. Article ID 358086).

Generally, increased serum levels of FGF23 result in hypo ^¬ phosphatemia, rickets and/or ostemalacia, mainly because defects in phosphate metabolism lead to poor bone mineralization and fractures. In a study performed in CKD patients, Pande S et al. (Nephron Physiol., 2006. 104(1): p23-p32) demonstrated that FGF23 levels rapidly decreased after kidney transplantation sug ^¬ gesting that FGF23 is cleared by the kidney. In contrast, loss of FGF23 in mice results in hyperphosphatemia and hypervitamino- sis D further highlighting the important function of FGF23 in regulating the phosphate metabolism.

In CKD patients, serum FGF23 concentrations rise in parallel with the decline in glomerular filtration rate (GFR) , and can reach levels 1,000-fold above normal in more advanced renal failure. The reason why FGF23 increases during CKD progression is not entirely clear. It has been shown that the increase in circulating intact FGF23 occurs independently of hyperphos ^¬ phatemia and increased parathyroid hormone (PTH) in early CKD stages. Therefore, other factors such as reduced renal elimina ^¬ tion of FGF23, or increased secretion of FGF23-stimulating substances such as aldosterone or proinflammatory cytokines may be involved in the upregulation of blood concentrations of intact FGF23 in CKD. Prospective studies in CKD patients have demon ^¬ strated that elevated FGF23 levels are independently associated with CKD progression, left ventricular hypertrophy (LVH) , cardiovascular risk, and all-cause mortality.

In typical FGF23 target tissues such as the kidney, the bio ^¬ logical effects of FGF23 are mediated through a receptor complex consisting of FGF receptors (FGFRs) and of the co-receptor Klotho (Klotho) . FGF23 is not only a phosphaturic, but also a calcium (Ca ²⁺ ) - and sodium (Na ⁺ ) -conserving hormone. FGF23 directly increases the apical membrane abundance of the epithelial Ca ²⁺ channel transient receptor potential vanilloid-5 (TRPV5) and of the Na ⁺ :C1 co-transporter (NCC) in distal renal tubules through a signalling cascade involving FGFRlc/Klotho receptor complex, extracellular signal-regulated kinase 1 and 2 (ERK1/2), serum/glucocorticoid-regulated kinase-1 (SGK1), and with-no ly ^¬ sine kinase-4 (WNK4) .

WNKs are key regulators of intracellular transport of mem ^¬ brane proteins, thereby acting as a complex of WNK1, 3, and 4. WNKs bind to and stimulate the kinases SPAK (STE20/SPSl-related proline/alanine-rich kinase) and OSR1 (oxidative stress- responsive kinase 1), which directly phosphorylate and stimulate Cl ^~ -importing, Na ⁺ -driven cation-chloride cotransporters (CCCs) or inhibit the Cl ^~ -extruding, K ⁺ -driven CCCs. The inventors have previously shown that FGF23 is involved in the physiological regulation of Ca ²⁺ and Na ⁺ reabsorption in renal distal tubules by Klotho-dependent activation of WNK4 (Andrukhova 0 et al . , EM- BO J., 2014. 33, 229-246; Andrukhova 0 et al . , EMBO Mol. Med., 2014. 6, 744-759) .

WNKs have a vital role in the control of the blood pressure through regulation of the NCC and NKCCl/2 co-transporters (Richardson C & Alessi D R, J. Cell Sci., 2008. 121, 3293-3304). Therefore, the link between FGF23 and volume homeostasis and blood pressure regulation may at least partially explain the as ^¬ sociation between circulating FGF23 and cardiovascular risk in CKD .

Interestingly, the inventors of the present invention found that the FGF23-induced WNK activation with subsequent Na ⁺ reten ^¬ tion, volume overload and hypertension is the key driving factor underlying the pro-hypertrophic effects of excessive circulating Fgf23 in CKD (Andrukhova 0 et al . , EMBO Mol. Med., 2014. 6, 744- 759) . The finding was supported by epidemiological studies that have linked elevated circulating FGF23 levels with hypercalcemia and hypernatremia in CKD progression (Pavik I et al., Nephrol. Dial. Transplant., 2013. 28, 352-359; Russo D & Battaglia Y, Int. J. Nephrol., 2011. 364890). Several clinical studies fur ^¬ ther confirmed the association between circulating FGF23, volume overload and adverse cardiac outcomes in CKD patients (Hung S C et al . , J. Am. Heart Assoc., 2015. 4; Unver S et al . , Ren Fail. 37, 2015. 951-956) .

Furthermore, by employing genetic loss-of-function models in Fgf23 and Klotho deficient mice together with pharmacological FGF23 inhibition models, the inventors of the present invention have recently shown that FGF23 signalling plays a pivotal role in the pathogenesis of CKD, whereby activation of WNK signalling turned out to be the disease-driving factor.

They have shown that low dose anti-FGF23 Ab treatment sub ^¬ stantially ameliorated disease progression and left ventricular dysfunction by partially preventing volume overload, hypertension, and vascular calcification in CKD mice. In contrast, Shal- houb V et al . (J. Clin. Invest, 2012. 122, 2543-2553) previously reported that high dose anti-FGF23Ab therapy increased mortality and vascular calcifications in CKD rats on a high phosphate di ^¬ et. The discrepant findings can probably be explained by the fact that complete elimination of Fgf23 function by high dose anti-FGF23 Ab leads to a Fgf23 deficiency-like phenotype charac ^¬ terized by inappropriately high production of l,25(OH)2D3, and subsequent hypercalcemia and hyperphosphatemia.

In order to exploit the strikingly beneficial effects of lowering blood Fgf23 concentrations in CKD, it seems necessary to carefully titrate the dose of anti-FGF23 Ab to prevent the toxic effects of over-suppression. Because the toxic effect of anti-FGF23 Ab therapy is mainly based on reduction of the sup ^¬ pressive effect of FGF23 on phosphate reabsorption and l,25(OH)2D3 production in proximal renal tubules, the inventors found a safer therapeutic strategy with a wider therapeutic win ^¬ dow by selectively interfering with the detrimental effects of excessive FGF23 signalling in distal renal tubules by inhibiting WNK signalling.

Therefore, to overcome the adverse effects of FGF23 target ^¬ ing therapies, new therapeutic strategies for patients diagnosed with disorders associated with elevated serum FGF23 levels, es ^¬ pecially CKD patients, are needed.

It is an object of the present invention to provide means and methods for use in preventing and treating disorders caused by elevated levels of serum FGF23, especially chronic kidney disease (CKD) .

The present invention therefore relates to an agent used to lower blood FGF23 concentrations in subjects with a disorder as ^¬ sociated with or caused by elevated serum FGF23 levels, in par ^¬ ticular subjects with CKD. According to the present invention, it was surprisingly found that WNK signalling inhibitors, in particular closantel, are potent agents to reduce the adverse FGF23-mediated WNK signaling activation in the kidney in vivo. The inventors have further shown for the first time that admin ^¬ istration of a WNK signalling inhibitor prevented CKD progression in mice. Therefore, it is an important feature of the pre ^¬ sent invention that its administration is not only suited for late stage CKD, but also for early stages of CKD. It therefore is an object of the present invention to also provide prevention of disease progression in a subject diagnosed with a disorder that is caused and/or characterized by excessive levels of serum FGF23.

Several possible strategies for inhibiting the WNK-SPAK/OSRI signalling cascade have been proposed (Alessi D R et al . , Sci Signal., 2014. 7, 334) . One possible approach is to directly in ^¬ hibit the kinase activity of either the WNK or the SPAK/OSRI ki ^¬ nases. For example, Yamada, K et al . (Nature Chem Biology, 2016., 12(11), 896-898) discloses an orally bioavailable inhibi ^¬ tor of WNK1 and WNK4, called WNK463, that decreases SPAK and OSR1 phosphorylation in a dose-dependent manner. Further allo- steric WNK kinase inhibitors are disclosed in Yamada et al . , ACS Chem. Biol. 11 (2016), 3338-3346; WNK1-4 inhibitors). Indirect inhibition through targeting WNK upstream regulators, e.g. KLHL3, or SPAK/OSRI scaffold proteins, M025 a or β isoforms, were also proposed, however the underlying mechanism and speci ^¬ ficity is not yet fully understood. Thus far, inhibition of the interaction between WNKs and SPAK/OSRI by targeting the CCT domain within SPAK/OSRI seems to be the most selective approach of WNK pathway inhibition. Mori T et al . (Biochem J, 2013. 455, 339-345) performed high-throughput screening of 17000 compounds and discovered two novel WNK signalling inhibitors (STOCK1S- 50699 and STOCK2S-26016) preventing SPAK phosphorylation in a dose-dependent manner by preventing WNK-SPAK binding.

In a compound library screening approach, Kikuchi E et al . (J Am Soc Nephrol, 2014. 1046-6673/2608) further identified STOCKlS-14279 (N- [4- ( 1 -bromonaphthalen-2 -yl ) oxy-3-chlorophenyl ] - 3, 5-dichloro-2-hydroxybenzamide) and closantel (N- [5-chloro-4- [ ( 4-chlorophenyl ) -cyanomethyl ] -2-methylphenyl ] -2-hydroxy-3, 5- diiodobenzamide) as potent inhibitors of the WNK-regulated ki ^¬ nase SPAK (Ste20-like proline/alanine-rich kinase).

Targeting the WNK signalling pathway in a therapeutic manner was described before. For example, WO 03/007793 A2 discloses mammalian WNK inhibitors, e.g. anti-WNK antibodies or isolated nucleic acids complementary to isolated nucleic acids encoding a human WNK, for use in a method of treating human patients with hypertension and pseudohypoaldosteronism type II. A similar approach of targeting WNK is described in JP 2007/074951 A, where ^¬ in the interaction of WNK1 and WNK4 with SPAK is disclosed and the inhibition of their interactions for treating hypertension proposed. Furthermore, US 2009/0203012 Al discloses methods com ^¬ prising identifying genetic predisposition for developing a metabolic syndrome, screening compounds that inhibit the kinase ac ^¬ tivity of STK39/SPAK and using said inhibitory compounds for treating a metabolic syndrome, hypertension, cardiovascular disease and T2D. According to the prior art, WNK signalling inhibition is obviously beneficial in the treatment of metabolic dis ^¬ eases. However, nothing in the prior art so far provided a link between WNK signalling inhibition as therapy for disorders caused and/or characterized by excessive FGF23 levels, especial ^¬ ly CKD.

WO 2007/066130 A2 relates to compositions comprising closan ^¬ tel to "control endoparasites in animals". Meena et al . , CMLS Cell. Mol. Life Sci., 74 (2016) 1261-1280, disclose the specific WNK signalling inhibitors STOCKlS-50699 and STOCK2S-26016, WNK 463, 1S-14279 and closantel.

The present invention cannot be construed from the prior art in an obvious manner: In Shaloub et al . , 2012, FGF23 inhibition aggravated vascular calcification so that mortality was higher in FGF23 antibody-treated rats because FGF23 inhibition in ^¬ creased vitamin D hormone production and worsened hyperphos ^¬ phatemia. This program was stopped after this finding. In this document, WNK is not mentioned at all. In fact, at that time (2012), it was completely unknown that FGF23 also targets WNK in distal renal tubules. Based on Shaloub et al . , 2012, a person with average skills in the art could never think of WNK signal ^¬ ing as a target in CKD. Conversely, in Yamada et al . , 2016, CKD and especially FGF23 is not mentioned. Although hypertension of ^¬ ten accompanies CKD, it is a different disease entity, and one would not automatically extend the findings to CKD. If such link would be obvious, it would have been mentioned in the prior art (e.g. in WO 03/007793 A2, Yamada et al . , 2016, Meena et al . , 2016, or Kikuchi et al . , 2014. This is not the cause, because the link is not obvious. Therefore, it is clear that the present invention discloses WNK signaling inhibition as a drug target for CKD therapy and prevention for the first time.

According to preferred embodiments of the present invention, the WNK signalling inhibitor used to reduce the adverse effects of increased renal FGF23 signalling in subjects with a disorder associated with or caused by elevated serum FGF23 levels, is closantel (N- [5-chloro-4- [ (4-chlorophenyl) -cyanomethyl ] -2- methylphenyl] -2-hydroxy-3, 5-diiodobenzamide) , STOCKlS-14279 (N- [4- (l-bromonaphthalen-2-yl) oxy-3-chlorophenyl ] -3, 5-dichloro-2- hydroxybenzamide) , STOCKlS-50699 ( (2Z) -3-ethyl-2- [[(3Z)-3-[(E)- 3- (3-ethyl-l, 3-benzothiazol-3-ium-2-yl) prop-2-enylidene] -5- methylcyclohexen-l-yl ] methylidene] -1, 3-benzothiazole) , STOCK2S- 26016 (7-Ethoxy-N3- (2-furanylmethyl) -3, 9-acridinediamine) ,

WNK463 (N-tert-butyl-3- [1- [5- [5- (trifluoromethyl) -1, 3, 4- oxadiazol-2-yl ] pyridin-2-yl ] piperidin-4-yl ] imidazole-4- carboxamide) , or any combination thereof.

Closantel (N- [5-chloro-4- [ (4-chlorophenyl) -cyanomethyl] -2- methylphenyl] -2-hydroxy-3, 5-diiodobenzamide) is known as an an ^¬ thelmintic from the group of salicylanilide . Salicylanilides are important anti-parasitics in the control of Haemonchus spp . and Fasciola spp. infestation in sheep and cattle, and Oestrus ovis in sheep. Macielag M J et al . (J. Med. Chem., 1998. 41 (16),

2939-45) tested for antibacterial activity of closantel and re ^¬ lated derivatives against the drug-resistant organisms, methi- cillin-resistant Staphylococcus aureus (MRSA) and vancomycin- resistant Enterococcus faecium (VREF) . Furthermore, WO 2016/193136 Al discloses halogenated salicylanilides , e.g. closantel, or pharmaceutically acceptable salts or esters there ^¬ of, for use in treating infections caused by Clostridium bacte ^¬ ria, especially diarrhoea and colitis in humans and warm-blooded animals. Its suitability for topical treatment or prevention of Staphylococcus and Streptococcus infections is disclosed in WO 2016/038035 Al .

Preferred WNK signalling inhibitors for use in the present invention are also disclosed in Yamada et al . (J. Med. Chem. 60

(2017), 7099-7107) as "Compounds 1 to 12", namely (2- ( (4- Chlorobenzyl ) oxy) phenyl) (5- (2- (methylamino) thiazol-4-yl) indolin- 1-yl) methanone, (4-Benzylpiperazin-l-yl) (2- (3-methoxyphenyl) - quinolin-4-yl) methanone, 1- (1- ( [1, 1 '-Biphenyl] -3-yl) -lH-indol-3- yl) -N- (cyclohexylmethyl) methanamine, 1-Cyclohexyl-N- ( (1- (3 ' - methoxybiphenyl-3-yl ) -1H- indol-3-yl) methyl) methanamine, 1- Cyclohexyl-N- ( (1- (2- (3-methoxyphenyl) pyridine-4-yl) -lH-indol-3- yl) methyl) methanamine, 1-Cyclohexyl-N- ( (6-fluoro-l- (2- (3- methoxyphenyl ) -pyridin-4-yl ) -lH-indol-3-yl) methyl) methanamine, N- ( (1- (2- (3-Methoxyphenyl) -5-methylpyridin-4-yl) -lH-indol-3- yl) methyl) -2-methylpropan-l-amine, (R) -N- (1- (4-

Chlorobenzyl ) pyrrolidin-3-yl ) -2- (3-methoxyphenyl) -N- methylquinoline-4-carboxamide, (4- (4-Chlorobenzyl) piperazin-1- yl) (2- (3-methoxyphenyl) pyridin-4-yl) methanone, (4- (4-

Chlorobenzyl ) piperazin-l-yl) (2- (2- (methylamino) thiazol-4- yl) pyridin-4-yl) methanone, (5-Chloro-2- (2- (methylamino) thiazol- 4-yl) - pyridin-4 -yl ) (4- (4-chlorobenzyl) piperazin-l-yl) methanone, and (5-Chloro-2- (2- ( (methyl-d3) amino) thiazol-4-yl) -pyridin-4- yl) (4- (4-chlorobenzyl) piperazin-l-yl) methanone .

Preferred WNK signalling inhibitors are selected from the group of WNK1 inhibitors, WNK4 inhibitors and SPAK/OSRI kinase inhibitors. Preferably, inhibition potential of the compounds used according to the present invention is defined by an IC ₅ o of 10 μΜ or lower, preferably 5 μΜ or lower, more preferred 1 μΜ or lower, especially 0.1 μΜ or lower. Such inhibition potential

(IC ₅₀ value) may be determined by any suitable and specific inhi- bition test. According to the present invention, it is specifi ^¬ cally preferred to determine such inhibition potential of the WNK signalling inhibitor by the potency test disclosed in Yamada et al . , 2017, Yamada et al . , 2016 (2x) , or Kikuchi et al . , 2015. These tests are easily reproducible, specific and can, if neces ^¬ sary, be easily adapted to specific members of the WNK-SPAK/OSRI signalling cascade.

As an example for such a test, the "in Vitro Radiometric As ^¬ says for WNK Family Selectivity Assessment" as disclosed by Yamada et al . (ACS Chem. Biol. 11 (2016), 3338-3346) can be cit ^¬ ed: To address the selectivity of the inhibitors among WNK fami ^¬ ly kinases, a radiometric kinase assay using WNK1,WNK2, WNK3, and WNK4 proteins (CarnaBioscience) was developed: "The assay utilized 5 to 10 nM of WNK1-4 protein compared to 25 nM used for mobility shift assay, enabling a more accurate comparison of se ^¬ lectivity among potent inhibitors. Assays measured the incorpo ^¬ ration of 33 P from [γ- ³³ Ρ]ΑΤΡ into myelin basic protein (MBP) coated in the wells of 96-well ScintiPlates ( PerkinElmer ) . Each well of the MBP-coated ScintiPlates held 100 ]i of a solution containing 20 mM HEPES at pH 7.3, 5 mM MnCl ₂ (WNK1 and WNK4) or 3 mM MnCl 2 (WNK2 and WNK3) , 0.01% Tween-20 (WNK1, WNK3, and WNK4) or 0.02% Tween-20 (WNK2), 1 mM TCEP, 2% DMSO, 1 μΜ ATP (WNK1, WNK3, and WNK4) or 2 μΜ ATP (WNK2), 1 μθί [γ- ³³ Ρ]ΑΤΡ (WNK1, WNK2, WNK4) or 0.25 μθί [γ-33Ρ]ΑΤΡ (WNK3), WNK kinase enzyme (5 nM WNK1, 10 nM WNK2 , 5 nM WNK3, or 10 nM WNK4), and the compound at the desired concentration. The plate was sealed and mixed for 20 s at 800 rpm on a benchtop plate shaker. The plate was then placed in a 25 °C shaking incubator at 175 rpm. Reactions were run within the linear range of the assay for each en ^¬ zyme and stopped by the addition of 50 ]i of quench buffer (45 mM EDTA, 0.01% Tween-20, and 20 mM HEPES, pH 7.3) . The content of each well was then aspirated, and the well was washed three times with 300 ]i of assay wash buffer (150 mM NaCl, 0.02% Tween-20, and 50 mM Tris/HCl at pH 7.4) . Incorporation of ³³ P in ^¬ to the bound MBP substrate was measured using a MicroBeta TriLux LSC and Luminescence plate counter. Nonspecific background was measured in the absence of WNK enzyme.

Usually, IC ₅₀ values do not significantly differ with dif ^¬ ferent ATP concentrations in such inhibition assays (see e.g. Yamada et al . , 2017, Yamada et al . , 2016 (2x) , or Kikuchi et al . , 2015), however, in case of doubt, the IC ⁵⁰ values referred to herein should relate to an ATP concentration of 10 mM ATP.

The present invention therefore provides a new unexpected therapeutic area for WNK signalling inhibitors, especially closantel. In particular, the present invention discloses WNK signalling inhibitors and/or pharmaceutic compositions thereof for the use in preventing and/or treating disorder caused and/or characterized by elevated serum FGF23 levels, especially CKD.

In the context of the present invention, disorders caused and/or characterized by elevated serum FGF23 levels are prefera ^¬ bly selected from the group consisting of tumor-induced osteoma ^¬ lacia (TIO) , X-linked hypophosphatemia, autosomal dominant hypo- phosphatemic rickets (ADHR) , McCune-Albright syndrome, linear nevus sebaceous syndrome, acute myocardial infarction, and CKD, especially CKD.

According to a preferred embodiment, the present invention is used for preventing and/or treating subjects with CKD. Importantly, the present invention provides treatment for CKD sub ^¬ jects in any stage of the disease process, wherein CKD stages are classified according to the KDIGO GFR system (KDIGO 2012 Clinical Practice Guideline for Evaluation and Management of Chronic Kidney Disease, Volume 3, Issue 1, January 2013) .

According to the present invention, the subject having a disorder caused and/or characterized by elevated levels of serum FGF23 may be human and/or a non-human animal, whereby the non- human animal is preferably a domestic companion animal, e.g. a dog or a cat.

According to the present invention, the WNK signalling inhibitor used for prevention and/or treatment of disorders caused and/or characterized by elevated serum FGF23 levels, may be for ^¬ mulated in a pharmaceutical composition comprising at least one, preferably more than one bioactive WNK signalling inhibitor se ^¬ lected from the group consisting of closantel (N- [5-chloro-4-

[ ( 4-chlorophenyl ) -cyanomethyl ] -2-methylphenyl ] -2-hydroxy-3, 5- diiodobenzamide) , STOCKlS-14279 (N- [4- ( l-bromonaphthalen-2- yl) oxy-3-chlorophenyl ] -3, 5-dichloro-2-hydroxybenzamide) ,

STOCKlS-50699 ( (2Z) -3-ethyl-2- [[(3Z)-3-[(E)-3- ( 3-ethyl- 1 , 3- benzothiazol-3-ium-2-yl ) prop-2-enylidene ] -5-methylcyclohexen-l- yl]methylidene] -1, 3-benzothiazole) , STOCK2S-26016 (7-Ethoxy-N3-

(2-furanylmethyl) -3, 9-acridinediamine) or WNK463 (N-tert-butyl- 3- [1- [5- [5- (trifluoromethyl ) -1,3, 4-oxadiazol-2-yl ] pyridin-2- yl ] piperidin-4-yl ] imidazole-4-carboxamide) , or pharmaceutically acceptable salts thereof, preferably closantel, more preferred a pharmaceutically acceptable salt of closantel.

The WNK signalling inhibitor, a pharmaceutically acceptable salt thereof, or the composition comprising a WNK signalling inhibitor or its salt, wherein the WNK signalling inhibitor is preferably closantel or a pharmaceutically acceptable salt thereof, may be administered in an amount, frequency, and dura ^¬ tion that measurably decreases levels of serum FGF23. Prefera ^¬ bly, closantel or a pharmaceutically acceptable salt thereof is administered in an amount between 0.01 and 3000 mg/day, more preferably, in an amount between 0.1 and 2000 mg/day (WO 2004/006906 A) .

In some embodiments of the present invention, the composi ^¬ tion further comprises a pharmaceutically acceptable excipient and/or solvent. The pharmaceutically acceptable excipient may be selected from the group consisting of, but not limited to, sur ^¬ factants, preferably oleic acid, thickening agents, salts, buff ^¬ ers, or any other inert excipient commonly used in pharmaceuti ^¬ cal industry.

Pharmaceutically acceptable solvents according to the in ^¬ ventive composition comprising WNK signalling inhibitors are alcohols from the group consisting of ethanol, benzyl alcohol, isopropanol, butanol, or a mixture thereof, with ethanol, benzyl alcohol or a mixture thereof being preferred. In another embodi ^¬ ment, the pharmaceutical composition comprising the WNK signal ^¬ ling inhibitor may be dissolved in propylene glycol, diethylene glycol monoethyl ether (transcutol) , ethylene glycol, butylene glycol, polyvinylpyrrolidone (PVP) , polyoxypropyl- ene/polyoxyethylene, polyethylene glycol, or the like, prefera ^¬ bly propylene glycol, diethylene glycol monoethyl ether or a mixture thereof.

In another embodiment of the present invention the pharma ^¬ ceutical composition comprising one or more WNK inhibitors, or pharmaceutically acceptable salts thereof, and one or more phar ^¬ maceutically acceptable excipient and/or solvent, is adminis ^¬ tered orally or parenterally for preventing and/or treating conditions caused/or characterized by elevated serum FGF23 levels, especially CKD. The pharmaceutical composition according to the present in ^¬ vention may be in form of tablets, lozenges, hard or soft cap ^¬ sules, aqueous or oily suspensions, emulsions, dispersible pow ^¬ ders or granules, syrups or elixirs suitable for oral admin ^¬ istration. In another embodiment, the inventive composition is in form of a sterile aqueous or oily solution suitable for par ^¬ enteral administration, e.g. intravenous, subcutaneous, intra ^¬ muscular or intraperitoneal injection.

The present invention also features a method for treating a subject having a disorder caused and/or characterized by elevat ^¬ ed serum FGF23 levels, especially CKD. This method includes the step of administering to the subject a WNK signalling inhibitor, or a salt thereof, in an amount effective to decrease FGF23- induced WNK signalling in the kidney. Accordingly, the WNK signalling inhibitor may be administered as single agent or pharma ^¬ ceutical composition, preferably as pharmaceutical composition. Desirable routes of administration of the single agent or phar ^¬ maceutical composition according to the present invention include enteral and parenteral administration, preferably oral or intravenous administration. The subject treated by the inventive method may be a human and/or a non-human animal, in particular a domesticated companion animal. Non-limiting examples of domesti ^¬ cated companion animals are dogs and cats.

In another embodiment, the inventive pharmaceutical composi ^¬ tion is for use in a method for treating a subject that suffers from a disorder associated with high levels of serum FGF23. Examples are subjects diagnosed with tumor-induced osteomalacia (TIO) , X-linked hypophosphatemia, autosomal dominant hypophos- phatemic rickets (ADHR) , McCune-Albright syndrome, linear nevus sebaceous syndrome, and acute myocardial infarction.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to de ^¬ scribe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims . As used herein, the term "WNK signalling inhibitor" refers to a compound preventing phosphorylation of SPAK and/or 0SR1 kinases, either directly or indirectly. For example, WO 2007/010210 A2 discloses a compound-screening assay for identi ^¬ fying WNK signalling modulators that prevent phosphorylation of WNK substrates, SPAK or OSRI .

The term "disorder" refers to a medical health condition associated with adverse effects on the well-being of a subject and resulting in illness.

As used herein, the term "subject" refers to any living or ^¬ ganism unless otherwise specified. In specific examples, sub ^¬ jects include: humans and non-human animals; domestic mammals such as dogs and cats. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

As used herein, the term "enteral administration" refers to any route of administration involving the esophagus, stomach, and small and large intestines (i.e., the gastrointestinal tract) . Methods of enteral administration thus include oral, sublingual (dissolving the drug under the tongue), and rectal.

The term "parenteral administration" refers to any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composi ^¬ tion through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a phar ^¬ maceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating nonsurgical wound, and the like. In particular, parenteral admin ^¬ istration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal in ^¬ jection, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient com ^¬ bined with a pharmaceutically acceptable carrier, such as ster ^¬ ile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus admin ^¬ istration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a pre- servative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained- release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. For parenteral administration, the active ingredient may also be provided in dry (i.e. powder or granular) form for re- constitution with a suitable vehicle (e. g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition .

The term "pharmaceutical composition" refers to a formula ^¬ tion comprising a biologically active ingredient and suitable pharmaceutically acceptable inert substances, such as carriers, excipients and/or solvents.

The term "pharmaceutically acceptable", such as pharmaceuti ^¬ cally acceptable carrier, excipient, etc. refers to pharmacolog ^¬ ically acceptable and substantially non-toxic to the subject to which the particular compound is administered.

The term "pharmaceutically acceptable salt" refers to those salts that are conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention, are formed from suitable non-toxic organic or inorganic acids or organic or inorganic ba ^¬ ses and are suitable for use in, on or with the tissues of hu ^¬ mans and lower animals without undue toxicity, irritation, al ^¬ lergic response and the like and are commensurate with a reason ^¬ able benefit/risk ratio.

According to a specifically preferred embodiment, the pre ^¬ sent invention relates to a pharmaceutical composition compris ^¬ ing WNK463 or Closantel for use in a method for preventing and/or treating CKD. According to another specifically preferred embodiment, the present invention relates to a pharmaceutical composition comprising a WNK signalling inhibitor for use in a method for preventing and/or treating CKD.

According to a specifically preferred embodiment, the pre ^¬ sent invention relates to a pharmaceutical composition compris ^¬ ing a WNK signalling inhibitor for use in a method for preventing and/or treating CKD, wherein the WNK signalling inhibitor is selected from the group consisting of WNK1 inhibitors, WNK4 in- hibitors and SPAK/OSRI kinase inhibitors.

According to a specifically preferred embodiment, the pre ^¬ sent invention relates to a pharmaceutical composition compris ^¬ ing a WNK signalling inhibitor for use in a method for preventing and/or treating CKD, wherein the WNK signalling inhibitor has an inhibition potential defined by an IC ₅₀ of 10 μΜ or lower, preferably 5 μΜ or lower, more preferred 1 μΜ or lower, espe ^¬ cially 0.1 μΜ or lower.

According to a specifically preferred embodiment, the pre ^¬ sent invention relates to a pharmaceutical composition compris ^¬ ing a WNK signalling inhibitor for use in a method for preventing and/or treating CKD, wherein the WNK signalling inhibitor is selected from the group consisting of Closantel (N- [5-chloro-4- [ ( 4-chlorophenyl ) -cyanomethyl ] -2-methylphenyl ] -2-hydroxy-3, 5- diiodobenzamide) , STOCKlS-14279 (N- [4- ( l-bromonaphthalen-2- yl) oxy-3-chlorophenyl ] -3, 5-dichloro-2-hydroxybenzamide) ,

STOCKlS-50699 ( (2Z) -3-ethyl-2- [[(3Z)-3-[(E)-3- ( 3-ethyl- 1 , 3- benzothiazol-3-ium-2-yl ) prop-2-enylidene ] -5-methylcyclohexen-l- yl]methylidene] -1, 3-benzothiazole) , STOCK2S-26016 (7-Ethoxy-N3- (2-furanylmethyl) -3, 9-acridinediamine) , WNK463 (N-tert-butyl-3- [ 1- [ 5- [ 5- ( trifluoromethyl ) -1 , 3 , 4-oxadiazol-2-yl ] pyridin-2- yl ] piperidin-4-yl ] imidazole-4-carboxamide) , (2- ( (4-

Chlorobenzyl ) oxy) phenyl) (5- (2- (methylamino) thiazol-4-yl) indolin- 1-yl) methanone, (4-Benzylpiperazin-l-yl) (2- (3-methoxyphenyl) - quinolin-4-yl) methanone, 1- (1- ( [1, 1 '-Biphenyl] -3-yl) -lH-indol-3- yl) -N- (cyclohexylmethyl) methanamine, 1-Cyclohexyl-N- ( (1- (3 ' - methoxybiphenyl-3-yl ) -1H- indol-3-yl) methyl) methanamine, 1- Cyclohexyl-N- ( (1- (2- (3-methoxyphenyl) pyridine-4-yl) -lH-indol-3- yl) methyl) methanamine, 1-Cyclohexyl-N- ( (6-fluoro-l- (2- (3- methoxyphenyl ) -pyridin-4-yl ) -lH-indol-3-yl) methyl) methanamine, N- ( (1- (2- (3-Methoxyphenyl) -5-methylpyridin-4-yl) -lH-indol-3- yl) methyl) -2-methylpropan-l-amine, (R) -N- (1- (4-

Chlorobenzyl ) pyrrolidin-3-yl ) -2- (3-methoxyphenyl) -N- methylquinoline-4-carboxamide, (4- (4-Chlorobenzyl) piperazin-l- yl) (2- (3-methoxyphenyl) pyridin-4-yl) methanone, (4- (4-

Chlorobenzyl ) piperazin-l-yl) (2- (2- (methylamino) thiazol-4- yl) pyridin-4-yl) methanone, (5-Chloro-2- (2- (methylamino) thiazol- 4-yl) - pyridin-4 -yl ) (4- (4-chlorobenzyl) piperazin-l-yl) methanone, (5-Chloro-2- (2- ( (methyl-d3) amino) thiazol-4-yl) -pyridin-4-yl) (4- (4-chlorobenzyl) piperazin-l-yl) methanone, or any combination thereof, preferably Closantel, WNK463 or a pharmaceutically ac ^¬ ceptable salt thereof.

According to a preferred embodiment, the WNK signalling in ^¬ hibitor is an allosteric inhibitor, preferably an allosteric WNK1 inhibitor, an allosteric WNK4 inhibitor, or an allosteric SPAK/OSRI kinase inhibitor.

The present invention is further explained by way of the following figures and examples, yet without being limited there ^¬ to .

Figure 1 shows that Angiotensin II infusion does not significantly aggravate 5/6 nephrectomy-induced CKD progression in C57BL/6 mice on rescue diet. (A) Scheme of the 5/6 nephrectomy

(5/6 Nx) protocol. Animals were included only if the ratio of resected left kidney/ removed right kidney was 0.55-0.72. (B) Body weight, glomerular filtration rate (GFR) , urinary albumin, serum urea, serum creatinine, and mean arterial pressure in sham-operated (sham) and 5/6 Nx C57BL/6 mice on rescue diet (2% calcium, 1.25% phosphorus, 20% lactose), receiving additional subcutaneous infusions of angiotensin II (Ang II) or saline (ve ^¬ hicle, Veh) via osmotic mini-pumps for 4 and 8 weeks postsur- gery. Each data point represents the mean ± SEM of 5 - 8 mice each. * denotes P < 0.05 vs. Sham, 1-way ANOVA followed by Stu- dent-Newman-Keuls test.

Figure 2 shows the effect of FGF23- or Klotho-deficiency on CKD-induced pathology in mice. (A) Survival curve and body weight, (B) glomerular filtration rate (GFR) and urinary albumin, (C) blood volume, left ventricular end-diastolic pressure

(EDP) , mean arterial pressure (MAP) , heart-to-body weight ratio

(HW/BW) , cardiac contractility (dP/dT), and linear plot of HW/BW ratio on blood volume, (D) von Kossa staining of aortic sec ^¬ tions, quantification of aortic calcification, representative images of kidney sections stained with periodic acid-Schiff

(PAS) stain, and semi-quantitative assessment of glomerular in ^¬ jury as evidenced by mesangial expansion in PAS-stained sections in Sham-operated (Sham) and 5/6 Nx (CKD) WT, VDR, Klotho/VDR and Fgf23/VDR compound mutant mice, 8 weeks postsurgery. Each data point in bar graphs represents the mean ± SEM of 8 - 12 mice each. Original magnification x40 in D. * denotes P < 0.05 vs. Sham animals of the same genotype, † P < 0.05 vs. Sham VDR ^{A A} mu ^¬ tants, # P < 0.05 vs. CKD WT and VDR ^A/A mice, II P < 0.05 vs. CKD Klotho/VDR compound mutants, 1-way ANOVA followed by Student- Newman-Keuls test.

Figure 3 shows that lack of Fgf23 but not of Klotho amelio ^¬ rates CKD-induced pathology. (A) Body weight, (B) serum creati ^¬ nine, (C) aortic calcium and phosphate content assessed by bio ^¬ chemical analysis, cardiac fractional shortening (FS) and tau measured by echocardiography, (D) renal Klotho protein expres ^¬ sion, (E) Fgf23 protein expression in total femur homogenates, and (F) quantification of Fgf23 mRNA expression in osteoblasts

(OB) and osteocytes (OC) harvested by laser capture microdissec ^¬ tion from femur cryo-sections in sham-operated (sham) and 5/6 Nx

(CKD) WT, VDR, Klotho/VDR and Fgf23 /VDR compound mutant mice, 8 weeks postsurgery. Each data point in A-B represents the mean ± SEM of 7 - 9 mice each. Each data point in D-E represents the mean ± SEM of 3 - 5 mice each. * denotes P < 0.05 vs. Sham ani ^¬ mals of the same genotype, † P < 0.05 vs. Sham VDR ^{A A} mutants, # P < 0.05 vs. CKD WT and VDR ^A/A mice, II P < 0.05 vs. CKD Klotho/VDR compound mutants, 1-way ANOVA followed by Student- Newman-Keuls test.

Figure 4 shows the effect of FGF23- or Klotho-deficiency on renal calcium and sodium retention in mice with CKD. (A) Serum phosphate, calcium and sodium, urinary calcium/creatinine (Ur- Ca/Crea) excretion, urinary phosphate/creatinine (UrP/Crea) ex ^¬ cretion, urinary sodium/creatinine (UrNa/Crea) excretion, serum PTH, aldosterone (ALD) and Fgf23 levels; (B) Relative TRPV5, NCC and ENaC protein expression in renal total membrane fractions or total kidney homogenates in Sham-operated (Sham) and 5/6 Nx

(CKD) WT, VDR, Klotho/VDR and Fgf23 /VDR compound mutant mice, 8 weeks postsurgery. Each data point in A represents the mean ± SEM of 8 - 12 mice each. Each data point in B represents the mean ± SEM of 4 - 5 mice each. * denotes P < 0.05 vs. Sham ani ^¬ mals of the same genotype, † P < 0.05 vs. Sham VDR ^{A A} mutants, # P < 0.05 vs. CKD WT and VDR ^A/A mutants, II P < 0.05 vs. CKD Klotho/VDR compound mutants, 1-way ANOVA followed by Student- Newman-Keuls test.

Figure 5 shows that pharmacological inhibition of FGF23 largely protects against the CKD-induced renal and cardiovascu ^¬ lar pathology. (A) Serum Fgf23 levels, (B) survival curve, (C) GFR, (D) von Kossa staining of aortic sections and quantifica ^¬ tion of aortic calcification, (E) left ventricular end-diastolic pressure (EDP) , (F) Percent tubular reabsorption of calcium (%TRCa) and of sodium (%TRNa) in Sham-operated (Sham) and 5/6 Nx (CKD) WT, VDR, and Klotho/VDR compound mutant mice treated for 8 weeks with control antibody (CoAB) or anti-FGF23 antibody (anti- FGF23 AB, 50 yg per mouse, two times per week) , 8 weeks post- surgery. Each data point represents the mean ± SEM of 7 - 9 mice each. * denotes P < 0.05 vs. Sham animals of the same genotype treated with CoAB, † P < 0.05 vs. Sham VDR ^A/A mutants treated with CoAB, # P < 0.05 vs. CKD mice of the same genotype treated with CoAB, 1-way ANOVA followed by Student-Newman-Keuls test.

Figure 6 shows that anti-FGF23 antibody treatment largely protects against the CKD-induced renal and cardiovascular pa ^¬ thology. (A) Body weight and serum creatinine, (B) urinary albu ^¬ min, tau, dP/dT, heart-to-body weight ratio, ejection fraction (EF) and fractioning shortening (FS) , (C) aortic calcium and phosphate content and serum calcium-phosphate product, (D) serum phosphate, calcium and sodium, (E) urinary phosphate/creatinine (UrP/Crea) excretion, urinary calcium/creatinine (UrCa/Crea) ex ^¬ cretion, and urinary sodium/creatinine (UrNa/Crea) excretion, (F) serum PTH and aldosterone (ALD) , (G) Representative alizarin red-stained aortic sections (n = 3 - 5 mice each) in Sham- operated (Sham) and 5/6 Nx (CKD) WT, VDR, and Klotho/VDR compound mutant mice treated for 8 weeks with control antibody (Co ^¬ AB) or anti-FGF23 antibody (anti-FGF23 AB, 50 yg per mouse, two times per week) , 8 weeks post-surgery. Each data point in A-F represents the mean ± SEM of 7 - 12 mice each. * denotes P < 0.05 vs. Sham animals of the same genotype treated with CoAB, † P < 0.05 vs. Sham VDRA/Δ mutants treated with CoAB, # P < 0.05 vs. CKD mice of the same genotype treated with CoAB, 1-way ANOVA followed by Student-Newman-Keuls test.

Figure 7 shows that anti-FGF23 antibody treatment suppresses WNK-mediated Klotho-dependent and -independent activation of re ^¬ nal calcium- and sodium-transporting molecules in CKD mice. Quantification and original Western blotting images of ENaC, βΕΝ3θ, yENaC, phospho-NKCCl (pNKCCl T203, 207, 212), phospho- WNK4 (pWNK4), phospho-WNKl (pWNKl), TRPV5, TRPV6, NCC and phos- pho-NCC (pNCC T45, 50, 55) protein expression in renal total ho- mogenates or in renal total membrane fractions (TRPV5 and TRPV6) in Sham-operated (Sham) and 5/6 Nx (CKD) WT, VDR, and Klotho/VDR compound mutant mice treated for 8 weeks with control antibody (CoAB) or anti-FGF23 antibody (anti-FGF23 AB, 50 yg per mouse, two times per week) , 8 weeks post-surgery. Each data point rep ^¬ resents the mean ± SEM of 4 -5 mice each. * denotes P < 0.05 vs. Sham animals of the same genotype treated with CoAB, † P < 0.05 vs. Sham VDRA/Δ mutants treated with CoAB, # P < 0.05 vs. CKD mice of the same genotype treated with CoAB, 1-way ANOVA fol ^¬ lowed by Student-Newman-Keuls test.

Figure 8 shows that treatment with anti-FGF23 antibody de ^¬ creases distal renal tubular TRPV6 and pNKCCl protein expression in Klotho/VDR CKD mice. (A) Representative images of TRPV6, (B) pNKCCl, and (C) NKCC1 immunohistochemistry in Sham-operated

(Sham) and 5/6 Nx (CKD) WT, VDR, and Klotho/VDR compound mutant mice treated for 8 weeks with control antibody (CoAB) or anti- FGF23 antibody (anti-FGF23 AB, 50 yg per mouse, two times per week), 8 weeks post-surgery. Original magnification ^χ 40. Scale bar, 50 ym.

Figure 9 shows that serum intact FGF23 levels are associated with WNK activation in the kidney of human CKD patients. Representative immunohistochemical images of Patient 2 (**) and Pa ^¬ tient 9 (*), and association between serum intact FGF23 with protein expression of phospho-NKCCl T212, 217 (pNKCCl) and phos- pho-WNKl S382 (pWNKl) quantified by immunohistochemistry in renal biopsies of 10 patients with CKD stages 2 - 5, and expressed as average staining intensity (a.u., arbitrary units) . Insets show Spearman rank correlation coefficients. Bar = 50 ym.

Figure 10 shows that very high concentrations of FGF23 acti ^¬ vate WNK signalling in a Klotho-independent manner in kidney slices ex vivo. Quantification and original Western blotting images of phospho-WNKl (pWNKl), phospho-WNK4 (pWNK4), phospho- NKCCl (pNKCCl T203, 207, 212), TRPV5, and TRPV6 protein expres ^¬ sion in homogenates of 200-ym-thick live kidney slices from 3- month-old WT and Klotho/VDR mutant mice treated ex vivo for 2 h with vehicle or rFGF23 (100 ng/ml or 200 ng/ml) . Data represent mean ± SEM of of 3 mice each. * denotes P < 0.05 vs. vehicle- treated slices of the same genotype.

Figure 11 shows that very high concentrations of FGF23 stim ^¬ ulate distal tubular calcium and sodium transport in kidney slices by Klotho- and FGFRl-independent activation of NKCC1 and TRPV6 channels. (A) Original images and quantification of intra ^¬ cellular Ca2+ levels in renal distal tubules in Fluo-4-loaded, 200-μπι-thick, live kidney slices from 3-month-old WT and Klotho/VDR mutant mice treated for 2 h with vehicle, rFGF23 (100 ng/ml or 200 ng/ml) alone or combined with a FGFR1 inhibitor

(iFGFRl) or a pan FGFR inhibitor (iFGFR pan) . Ruthenium red (RR) was used as TRPV inhibitor, 1 μΜ RR was used for TRPV5 inhibi ^¬ tion and 10 μΜ RR was used for TRPV6 inhibition. (B) Original images and quantification of intracellular Na+ levels in renal distal tubules in SBFI-loaded, 200^m-thick, live kidney slices from 3-month-old WT and Klotho/VDR mutant mice treated for 2 h with vehicle, rFGF23 (100 ng/ml or 200 ng/ml), alone or in com ^¬ bination with a FGFR1 inhibitor (iFGFRl) or a pan FGFR inhibitor

(iFGFR pan) . Chlorothiazide (CTZ, 10 μΜ) was used as NCC inhibi ^¬ tor and bumetanide (BF, 15 μΜ) was used as NKCC1 inhibitor. Flu ^¬ orescence intensity in A and B was quantified in 4-6 regions of interest per image, sample, and time point from 2-3 independent experiments. Data represent mean ± SEM. * denotes P < 0.05 vs. vehicle-treated, # P < 0.05 vs. slices treated with 200 ng/ml rFGF23 alone.

Figure 12 shows renal FGFR mRNA expression and effects of FGFR1/3 and/or WNK signalling inhibition on distal tubular FGF23-mediated calcium and sodium uptake in kidney slices of Klotho/VDR mutants. (A) FGFRlc, FGFR2, FGFR3 and FGFR4 mRNA expression levels in distal (DT) and proximal renal tubules (PT) of WT mice harvested by laser capture microdissection. Data rep ^¬ resent mean ± SEM of 4 animals. * denotes P < 0.05 vs. FGFRlc mRNA expression, # P < 0.05 vs. FGFR2 mRNA expression, 1-way ANOVA followed by Student-Newman-Keuls test. (B) Original images and quantification of intracellular Ca ²⁺ levels in renal distal tubules in Fluo-4-loaded, 200^m-thick, live kidney slices of 3- month-old Klotho/VDR mutant mice treated for 2 h with vehicle

(PBS), rFGF23 (200 ng/ml) alone or in combination with the WNK signalling inhibitor closantel or a FGFR1/3 inhibitor (iFGFR3) .

(C) Original images and quantification of intracellular Na+ levels in renal distal tubules in SBFI-loaded, 200^m-thick, live kidney slices of 3-month-old Klotho/VDR mutant mice treated for 2 h with vehicle, rFGF23 (200 ng/ml) alone or combined with the WNK signalling inhibitor closantel or a FGFR1/3 inhibitor

(iFGFR3) . Fluorescence intensity was quantified in 4-6 regions of interest per image, sample, and time point from 2-3 independ ^¬ ent experiments. Data represent mean ± SEM.* denotes P < 0.05 vs. vehicle-treated, # P < 0.05 vs. slices treated with 200 ng/ml rFGF23 alone, † P < 0.05 vs. slices treated with iFGFR3 alone, 1-way ANOVA followed by Student-Newman-Keuls test.

Figure 13 shows that the WNK signalling inhibitor closantel largely protects against disease progression in CKD mice. (A) Body weight, HW/BW ratio, GFR, blood volume, mean arterial pres ^¬ sure (MAP), urinary sodium/creatinine (UrNa/Crea) excretion, and

(B) quantification and original Western blotting images of phos- pho-NCC (pNCC) , phospho-NKCCl (pNKCCl), and phospho-WNKl (pWNKl) protein expression in renal total homogenates of Sham-operated

(Sham) and 5/6 Nx (CKD) WT mice on rescue diet treated over 8 weeks once a week with closantel orally via the diet, 8 weeks postsurgery. Each data point represents the mean ± SEM of 7 - 9 mice each. * denotes P < 0.05 vs. Sham + vehicle (Veh) , # P < 0.05 vs. CKD + Veh, 1-way ANOVA followed by Student-Newman-Keuls test .

Figure 14 shows the calculated plasma levels (arbitrary units) after oral administration of closantel at varying doses once a week via the diet. The plasma level after the loading dose (5 mg/kg body weight, corresponding to 30 mg/kg diet) given on Day 1 was set to 100. Closantel-containing food at the concentrations (mg/kg diet) shown in the graph were administered on Days 1, 8, 15, 22, 29, 36, 43, and 50 (arrows) after Sham sur ^¬ gery or 5/ 6-Nx .

Figure 15 shows the WNK signaling inhibitor WNK463 partial ^¬ ly protects against disease progression in CKD mice. Body weight, heart weight/body weight (HW/BW) ratio, mean arterial pressure (MAP) , urinary excretion of creatinine, urinary sodium/creatinine excretion, urinary calcium/creatinine excretion, serum creatinine, serum urea, and GFR in Sham-operated (Sham) and 5/6 Nx wild-type mice on rescue diet treated over 8 weeks once a day with WNK463 orally via gavage, 8 weeks postsurgery. Each data point represents the mean ± SEM of 5 - 8 mice each. * denotes P < 0.05 vs. Nx + vehicle (Veh), 1-way ANOVA followed by Holm-Sidak' s multiple comparison test.

EXAMPLES

Materials and methods : Human study

The human study was approved by the Ethical Committee of the Medical University of Vienna on Nov 17, 2016 (#2006/2016), and each patient provided written consent for the use of the biopsy and plasma for this specific study. We prospectively collected kidney biopsies and serum from ten patients with various stages of chronic kidney disease (CKD stage 2 to 5) . Serum was aliquot- ed and biopsy specimen split in half for paraffin and cryo- embedding. Five-ym-thick paraffin sections were cut on a microtome and further processed for anti-pWNKl, anti-pNCC, and anti- pNKCCl immunohistochemistry . Each patient received a full set of routine lab work up few days before the biopsy including elec ^¬ trolytes, creatinine, BUN, iPTH, 1,25 (OH) 2D, and intact FGF23 (ELISA, Kainos) measurements.

Animals

All animal studies were approved by the Ethical Committee of the University of Veterinary Medicine, Vienna and by the Austri ^¬ an Federal Ministry of Science and Research and were undertaken in strict accordance with prevailing guidelines for animal care. All efforts were made to minimize animal suffering. Heterozygous VDR ^+/A mutant mice (Erben R G et al . , Mol. Endocrinol., 2002. 16, 1524-1537) and heterozygous Fgf23 ^+/" (Sitara D et al., Matrix Bi ^¬ ol., 2004. 23, 421-432) and Klotho ^+/" (Lexicon Genetics, Mutant Mouse Regional Resource Centers, University of California, Da ^¬ vis) mice were mated to generate double heterozygous animals. The VDR, Fgf23 and Klotho mutant mice used for the matings had been backcrossed to C57BL/6 genetic background for 10 genera ^¬ tions. The double heterozygous offspring from these matings were intercrossed to generate wild-type (WT) , Fgf23 ^_/" , VDR ^A/A , Fgf23 ^_/" /VDR ^A/A and Klotho ^"/_ /VDR ^A/A mice. Genotyping of the mice was performed by multiplex PCR using genomic DNA extracted from the tail as described in Hesse M et al . (Matrix Biol., 2007. 26, 75- 84) and Anour R et al . (PLoS. One., 2012. 7, e31376). The mice were kept at 24°C with a 12 h/12 h light/dark cycle, and were allowed free access to a normal rodent chow or the rescue diet and tap water. The rescue diet (Ssniff, Soest, Germany) containing 2.0% calcium, 1.25% phosphorus, 20% lactose and 600 IU vita ^¬ min D/kg was fed starting from 16 days of age. This diet has been shown to normalize mineral homeostasis in VDR-ablated mice (Erben R G et al . , Mol. Endocrinol., 2002. 16, 1524-1537). All experiments were performed on 3-month-old male offspring of dou ^¬ ble heterozygous x double heterozygous matings. Urine was col ^¬ lected in metabolic cages before necropsy. At necropsy, mice were exsanguinated from the abdominal V. cava under general an ^¬ esthesia (ketamine/xylazine, 100/6 mg/kg i.p.) for serum collec ^¬ tion.

Serum and urine biochemistry

Serum and urinary calcium, phosphorus, albumin and creatinine were analyzed on a Cobas clll analyzer (Roche) . Renal tubu ^¬ lar reabsorption of calcium (TRCa) and sodium (TRNa) were calculated according to the formula %TRCa = [1- (UrCa*SeCrea) / ( SeCa*UrCrea) ] *100 and %TRNa = [1-

(UrNa*SeCrea) / ( SeNa*UrCrea) ] *100 (Ur, urinary; Se, serum; Crea, creatinine; Ca, calcium; Na, sodium) . Serum intact PTH, serum aldosterone (ALD) , and serum intact FGF23 were determined by ELISA (Immutopics, NovaTec, and Kainos, respectively).

5/6 nephrectomy (5/6 Nx) model

The 5/6 nephrectomy (5/6 Nx) was performed in two stages (Leelahavanichkul A et al . , Kidney Int. 78, 2010. 1136-1153) un ^¬ der isoflurane anesthesia (Fig. 1A) . Pain was managed by s.c. injections of buprenorphine, metamizol and meloxicam. Addition ^¬ ally, the animals were treated for 3 days postsurgery with the analgesic piritramid via the drinking water, starting 6 h postsurgery. At the first stage (wk -1), the left kidney was de- capsulated to avoid ureter and adrenal damage, and the upper and lower poles were partially resected via a left flank incision. Bleeding was controlled by microfibrillar collagen hemostasis (Avitene, Davol, Cranston, RI) . The upper and lower poles were weighed. One week later, the entire right kidney was removed via a right flank incision and weighed. Animals with insufficient kidney resection as determined by a ratio of < 0.55 for the re ^¬ moved left kidney weight to the removed right kidney weight were euthanized. In the Sham surgery control mice, a left flank inci ^¬ sion was performed at step 1, both poles of kidney were identi ^¬ fied, then the flank incision was closed; a subsequent right flank incision was performed at step 2; the right renal artery was identified, then the flank incision was closed. After each flank incision, muscles and skin were repositioned and sewed. Mortality was tracked throughout the study. Necropsy was not performed on animals that died early.

Angiotensin II administration

Angiotensin II (Ang II) 0.75 μg/kg/min (Sigma) in physiological saline or vehicle was infused by subcutaneous osmotic mini- pumps (Alzet model 1004, Cupertino, CA) (Leelahavanichkul A et al . , Kidney Int. 78, 2010. 1136-1153). The osmotic minipumps were inserted subcutaneously at the back under isoflurane anes ^¬ thesia at 5 days after right NX or Sham surgery (Fig. 1A) .

Neutralizing anti-FGF23 antibody administration

Neutralizing anti-FGF23 antibody (anti-FGF23Ab, 50 yg per mouse) or an isotype control Ab (Co Ab, 50 yg per mouse) were administered 2 times per week for 8 weeks beginning on day 0 (baseline) via intravenous administration into a tail vein.

Closantel administration

For this experiment, WT Sham and 5/6-Nx mice were fed pow ^¬ dered rescue diet throughout the study. Closantel (Abeam) was dissolved in 100% ethanol (vehicle) , and mixed into the mois ^¬ tened diet. To achieve a loading dose of 5 mg/kg body weight, the drug was mixed into the food at an initial concentration of 30 mg/kg diet. The concentration in the food was calculated based on a mean food consumption of 8.5 g per day per mouse, and an approximate bioavailability of 50%. To maintain constant plasma levels, closantel was administered once a week using the dosing regimen shown in Fig. 14. The model used to calculate the theoretical plasma levels was based on the assumption of a plas ^¬ ma half-life of 14 days (WHO Food Additives Series 27, http : //www . inchem . org/documents/j ecfa/j ecmono/v27j e02. htm) . All mice were killed 8 weeks postsurgery.

WNK463 administration

WNK463 was given to WT 5/6 Nx mice on rescue diet at doses of 1 and 3 mg/kg BW via oral gavage at 5 μΐ/g BW once daily over 8 weeks post-Nx. Vehicle-treated Sham and 5/6 Nx mice served as controls. An aqueous suspension of 0.5% Pluronic F68 (Fisher Scientific), 0.5% hydroxypropylcellulose (M.W. 100,000, Sigma- Aldrich) , and 99% water (w/w/w) was used as vehicle. WNK463 was dissolved in 100% DMSO, and added to the vehicle, so that the final concentration of DMSO in all dosing solutions was 2%.

Transthoracic Doppler echocardiography (ECG)

Left ventricular (LV) function was non-invasively assessed in all mice prior to necropsy under anesthesia with isofluorane by ECG, using a 14MHz linear transducer (Siemens S2000) . Short axis m-mode recordings of LV were obtained from a left paraster ^¬ nal acoustic window. LV dimensions in systole and diastole were measured at the largest diameter of the ventricle at the level of the papillary muscles, and systolic parameters such as frac ^¬ tional shortening (FS) and ejection fraction (EF) were calculated. Diastolic LV function was evaluated using the pulsed-wave Doppler recording of trans-mitral blood flow velocities in the apical 4-chamber view. A minimum of 4 cardiac cycles were averaged for measurement of each parameter. Total duration of examination did not exceed 30 min.

Central arterial pressure measurements and cardiac catheterization

Central arterial pressure and cardiac hemodynamic parameters were assessed using a SPR-671NR pressure catheter (1.4 French; Millar Instruments). Mice were anesthetized with 1.5% isoflurane in oxygen. The catheter was inserted into the ascending aorta for measurement of central arterial pressure. Parameters of LV function (i.e., maximum rate of the pressure rise, contractility index, end-diastolic pressure, and isovolumetric relaxation time) were measured by placing the catheter into the left ven ^¬ tricle. All pressure recordings were performed at the same anes ^¬ thesia level. Hemodynamic measurements were recorded over 5 min, and traces were analyzed using LabChartPro software and a blood pressure (BP) module. After pressure measurements, all animals were euthanized.

Blood volume measurements

The blood volume was determined from plasma volume and hema ^¬ tocrit as described in Lee H B & Blaufox M D (J. Nucl . Med., 1985. 26, 72-76) . Plasma volume was determined by Evans blue dye dilution (Barron W M et al . , J. Clin. Invest, 1984. 73, 923- 932) . Briefly, 20 μΐ of a 0.4% (wt/vol) solution of Evans blue (Sigma) in sterile physiological saline was injected into a tail vein. Blood samples (10 μΐ) were collected at 10 min and 30 min after injection to measure disappearance kinetics. Tubes were centrifuged and the hematocrit was recorded. Evans blue concen ^¬ tration in the plasma was measured in duplicate as optical den ^¬ sity using the 2-wavelength method.

Total cell membrane isolation

Mouse kidney cortex was homogenized in a homogenizing buffer [20 mM Tris (pH 7.4/HCl), 5 mM MgCl ₂ , 5 mM NaH ₂ P0 ₄ , 1 mM eth- ylenediaminetetraacetic acid (pH 8.0/NaOH), 80 mM sucrose, 1 mM phenyl-methylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml pepstatin] and subsequently centrifuged for 15 min at 4,000 g. Supernatants were transferred to a new tube and centrifuged for an additional 30 min at 16,000 g.

Histology and morphologic evaluation of aorta and kidney

For histological analysis, kidney and thoracic aortas were fixed in 4 "6 paraformaldehyde (PFA) overnight, processed for par ^¬ affin histology, sectioned at 5 μπι thickness, and stained by routine methods, using hematoxylin/eosin (HE), von Kossa, Alizarin red, or periodic acid-Schiff (PAS) staining ( Sigma-Aldrich) . Quantification of von Kossa-stained aortas was performed using Image J software. The data were expressed as percentage of cal ^¬ cified regions relative to the total aortic area. Semi ^¬ quantitative pathohistologic scoring of glomerular lesions in the kidney was performed in a blind fashion. The total number of kidney glomeruli was counted, and the degree of glomerular dam ^¬ age was estimated at 400 ^χ magnification from the degree of mesangial expansion in PAS-stained sections as follows: <25%, 25-50%, 50-80%, >80% (MacKay K et al . , Kidney Int., 1987. 32, 827-837) .

Laser capture microdissection (LCM)

Cryosections from femurs or kidney were quickly stained with HistoStain (Arcturus) . Osteoblasts and osteocytes from cortical bone of the femur sections, and distal and proximal tubules from kidney sections were harvested using a Veritas (Arcturus) LCM system as described in Andrukhova 0 et al . (Bone, 2012. 51, 621- 628), and stored at -80°C until used.

RNA isolation and quantitative RT-PCR

Shock-frozen hearts were homogenized using TRI Reagent (Mo ^¬ lecular Research Center) . Total RNA was extracted with phenol/chloroform, precipitated using isopropanol, and then treated with RQ1 RNase-free DNase (Promega) . RNA purity and quality was determined spectrophotometrically (BioPhotometer; Eppendorf) . In samples harvested with LCM, RNA was extracted using the PicoPure RNA isolation kit (Qiagen) , and RNA purity and quality was de ^¬ termined spectrophotometrically (BioPhotometer, Eppendorf) and via the 2100 Bioanalyzer (Agilent Technologies) . After first- strand cDNA synthesis (iScript cDNA Synthesis Kit, BioRad) , quantitative RT-PCR was performed on a Rotor-Gene 6000 (Corbett Life Science) using SsoFastTM EvaGreen PCR kit (Bio-Rad) . A melting curve analysis was done for all assays to make sure that only a single PCR product was amplified. Primer sequences are available on request. Efficiencies were examined by standard curve. Gene expression data were corrected for efficiency and normalized to ornithine decarboxylase antizyme-1 (Oazl) as house-keeping gene.

Western blotting

Total kidney homogenates, kidney cortex homogenates or total cell membrane preparations were solubilized in Laemmli sample buffer, fractionated on SDS- PAGE (30 yg/well) and transferred to a nitrocellulose membrane (Thermo Scientific) . Immunoblots were incubated overnight at 4°C with primary antibodies includ ^¬ ing polyclonal rabbit anti-TRPV5 (1:1,000, Alpha Diagnostics), polyclonal rabbit anti-NCC (1:3,000, Millipore) , polyclonal sheep anti-phospho-NKCCl Thr 203,207,212 (pNKCCl T203, 207, 212; 1:1,000), polyclonal sheep anti-NKCCl (1:1,000), antiphospho-NCC Thr 46, 50, 55 (pNCC T46, 50, 55; 1:1,000), polyclonal sheep an- ti-phospho-WNK4 Ser 47 (pWNK4 S47; 1:2,000), polyclonal sheep anti-phospho-WNKl Ser 382 (pWNKl S382; 1:2,000) (antibodies produced in house by Dario R. Alessi, University of Dundee, Dundee, UK), anti- -ENaC (Novus Biologicals, 1:1,000), anti^-ENaC and anti-y-ENaC (Antikoerperonline.com, 1:1,500), and monoclonal mouse anti^-actin (1:5, 000, Sigma) in 2% (w/v) bovine serum al- bumin (BSA, Sigma) in a TBS-T buffer [150 mM NaCl, 10 mM Tris (pH 7.4/HCl), 0.2% (v/v) Tween-20]. After washing, membranes were incubated with horseradish peroxidase-conj ugated secondary antibodies (Amersham Life Sciences) . Specific signal was visual ^¬ ized by ECL kit (Amersham Life Sciences) . The protein bands were quantified by Image Quant 5.0 software (Molecular Dynamics) . Loading of the samples was normalized to Ponceau S stain. The expression levels were normalized to β-actin expression.

Ex vivo experiments with kidney slices

The preparation of kidney slices for ex vivo experiments was performed as described in Andrukhova 0 et al . (EMBO J., 2014. 33, 229-246). Longitudinal 200-ym-thick live slices from freshly isolated kidneys of 3-month-old male WT and Klotho/VDR mice were prepared using a Leica VT1000 Vibratome (Leica Microsystems) , and were used for ex vivo treatment with various substances as described below.

Immunohistochemistry

For immunohistochemistry, 5^m-thick paraffin sections of PFA-fixed kidneys or of human renal biopsies were prepared. Be ^¬ fore immunohistochemical staining, dewaxed sections were pre- treated with blocking solution containing 5% normal goat serum in PBS with 0.1% bovine serum albumin and 0.3% Triton X-100 for 60 min. Without rinsing, mouse kidney sections were incubated with rabbit anti-TRPV6 (Millipore, 1:400), sheep anti-NKCCl, sheep anti-pNKCCl T203, 207, 212, (1:1000, antibodies produced in house by Dario R. Alessi) antibodies at 4°C overnight. Sec ^¬ tions of human biopsies were incubated with sheep anti-human pNKCCl T212, 217, anti-human pNCC T46, 50, 55, and anti-human pWNKl S382 (1:1,000, antibodies produced in house by Dario R. Alessi) . After washing, sections were incubated for 1.5 h with biotinylated goat anti-rabbit or donkey anti-sheep or anti-goat secondary antibodies (Invitrogen, 1:400), followed by incubation with horseradish peroxidase-labeled streptavidin (Vector) , and DAB staining. Controls were performed by omitting the primary antibody. The slides were analyzed on a Zeiss Axioskop 2 micro ^¬ scope. For quantification of pWNKl, pNKCCl, and pNCC expression in human renal biopsies, the average staining intensity was ana ^¬ lyzed in about 20 fields per biopsy at x400, using Image J. Intracellular Na ⁺ and Ca ²⁺ imaging

Longitudinal 200-ym-thick live slices of freshly isolated kidneys were prepared using a Leica VT1000 Vibratome (Leica Mi ^¬ crosystems) . For Na ⁺ imaging, the kidney slices of WT and Klotho/VDR mice were incubated for 60 min at 37 °C with 2 μΜ of the sodium-sensitive dye SBFI (Molecular Probes) , diluted in DMSO (Merck Millipore International) and 20% Pluronic (Merck Millipore International) . For Ca ²⁺ imaging, the kidney slices were incubated for 30 min at 37°C with 2 μΜ of the calcium- sensitive dye Fluo-4 (Molecular Probes) , diluted in DMSO and 20% Pluronic. Thereafter, the slices were washed two times for 20 min each in 0.1M PBS, and were subsequently incubated in vitro with vehicle (PBS), rFGF23 R176/179Q [(100 or 200 ng/ml) kindly provided by Amgen Inc., Thousand Oaks, CA, USA], PD173074 (Sig- ma-Aldrich) (10 nM for inhibiting FGFR1 and 25 nM for inhibiting FGFR1 and 3), pan FGFR inhibitor [(120 nM) FIIN1 hydrochloride, Tocris Bioscience, Bristol, UK) ] , WNK signalling inhibitor Closantel (30 ng/mL, generous gift of Dario R. Alessi) alone or combined at 37°C, 5% C02 / 95% air humidified atmosphere for 2 h. For the inhibition of NCC or NKCC1 activity, tissue slices were incubated with 10 μΜ of chlorothiazide (CTZ, Sigma) or 15 μΜ bumetanide (BF, Sigma) or vehicle (PBS + 1% ethanol) alone or in combination with rFGF23 R176/179Q (200 ng/ml) . For inhibition of TRPV activity, tissue slices were incubated with 1 μΜ (for TRPV5) or 10 μΜ (for TRPV6) of ruthenium red (Sigma) alone or in combination with rFGF23 (200 ng/ml) . For visualization of intracellular Na ⁺ and Ca ²⁺ content, SBFI or Fluo-4 were excited by a Ti:sapphire laser (Chameleon, Coherent Inc.) at 820 nm. Images (512 x 512 pixels) were acquired every 30 sec at a depth of 60 - 80 ym. Fluorescence images were analyzed using Image J software. The whole epithelial layer of the distal tubules was selected by manually drawing the region of interest (ROI) to quantify intra ^¬ cellular Na ⁺ and Ca ²⁺ levels. Fluorescence intensity was quanti ^¬ fied in 4-9 ROIs per image, and the ratio between the fluores ^¬ cence intensity and the ROI area was calculated for each tubule. This ratio was used for all subsequent calculations.

Total cell membrane isolation

Mouse kidney cortex was homogenized in a homogenizing buffer [20 mM Tris (pH 7.4/HCl), 5 mM MgCl ₂ , 5 mM NaH ₂ P0 ₄ , 1 mM eth- ylenediaminetetraacetic acid (pH 8.0/NaOH), 80 mM sucrose, 1 mM phenyl-methylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml pepstatin] and subsequently centrifuged for 15 min at 4,000 g. Supernatants were transferred to a new tube and centrifuged for an additional 30 min at 16,000 g. For Western blotting analysis, samples were dissolved in loading buffer and stored at -80°C un ^¬ til analyzed.

Statistical analyses

Statistics were computed using SPSS for Windows 17.0. The data were analyzed by 1-way analysis of variance (ANOVA) fol ^¬ lowed by Student-Newman-Keuls multiple comparison test. P values of less than 0.05 were considered significant. The data are pre ^¬ sented as the mean ± SEM. For correlation analysis, bivariate Spearman rank correlation coefficients were calculated. A step ^¬ wise model of multiple regression analysis was performed to find explanatory models for the changes in heart-to-body weight ratio after CKD.

Example 1: Induction of CKD in C57BL/6 mice.

In order to test susceptibility of C57BL/6 mice to 5/6 ne ^¬ phrectomy (5/6-Nx)- and angiotensin II (Angll) infusion-induced CKD, 5/6-Nx was performed and osmotic minipumps continuously re ^¬ leasing vehicle or Angll were thereafter implanted into male wild-type (WT) C57B1/6 mice, which were then monitored over 8 weeks after 5/6-Nx. All mice were maintained on rescue diet.

Results: As shown in Figure 1 no significant differences in GFR, proteinuria, azotemia, or mean arterial pressure measured by arterial catheterization between Angll- and vehicle-treated 5/6-Nx mice at 4 and 8 weeks postsurgery were found.

These data indicate that 5/6-Nx is sufficient to induce an about 60% reduction in GFR in C57BL/6 mice on rescue diet, 8 weeks post-5/6-Nx.

Example 2: Assessment of FGF23 and Klotho function during 5/6- Nx-induced CKD in mice.

Loss of Fgf23 or Klotho function leads to unleashed produc ^¬ tion of l,25(OH) ₂ D ₃ due to the lacking suppressive effect of Fgf23 on renal l -hydroxylase . Chronically elevated circulating l,25(OH) ₂ D ₃ levels result in hyperphosphatemia, hypercalcemia, soft tissue calcifications, and early mortality in Fgf23 ^{_ ~} and Klotho ^{_ ~} mice. Consequently, the severe phenotype in Fgf23 ^{_ ~} and Klotho ^{_ ~} mice can be completely rescued by genetically disrupt ^¬ ing vitamin D receptor (VDR) signalling. Fgf23 and Klotho deficient mice were therefore crossed with VDR ^{A A} mice lacking a functioning VDR to generate Fgf23/VDR and Klotho/VDR compound mutants. When kept on a so-called "rescue diet" enriched with calcium, phosphate, and lactose, which serves to normalize Ca ²⁺ and phosphorus homeostasis in VDR mutant mice, Fgf23/VDR and Klotho/VDR compound mutants are healthy, and can be analysed un ^¬ til old ages.

In order to assess whether Fgf23 and Klotho deficiency in ^¬ fluences disease progression after 5/6-Nx, CKD was induced by by 5/6-Nx in 3-month-old male WT, VDR, Fgf23 /VDR, and Klotho/VDR mice .

Results: Figure 2 shows that mortality after CKD induction was low in WT, VDR and Fgf23/VDR mice, but substantial in Klotho/VDR compound mutants. In WT and VDR mutant mice, 5/6-Nx reduced GFR by -60% vs. Sham controls, 8 weeks postsurgery (Fig. 2B) . Importantly, genetic ablation of Fgf23 in Fgf23/VDR compound mutants almost completely protected against the CKD- induced decline in renal and cardiac function, heart hypertrophy as measured by heart-to-body weight (HW/BW) ratio, volume expan ^¬ sion as measured by blood volume and end-diastolic pressure, hy ^¬ pertension, left ventricular functional impairment, aortic cal ^¬ cification, and glomerular lesions observed in WT and VDR CKD mice (Fig. 2B-D and Fig. 3A-C) . In contrast to Fgf23 /VDR CKD mice, Klotho/VDR CKD mice were not protected against disease progression, and actually showed the most severe glomerular le ^¬ sions of all genotypes (Fig. 2B-D and Fig. 3A-C) . A correlation analysis revealed that serum Fgf23, aldosterone, PTH, blood pressure, as well as serum Ca ²⁺ and Na ⁺ were correlated with HW/BW ratio (Table 1) . However, blood volume showed the strong ^¬ est association with HW/BW ratio (Fig. 2C) , and remained the on ^¬ ly significant predictor of HW/BW ratio in a stepwise model of multiple regression analysis (Table 2).

Table 1: Correlation coefficient matrix for heart-to-body weight ratio, blood volume and selected serum parameters in Sham and CKD mice (Spearman rank correlation coefficients rS) . HW/BW Fgf23 Volume ALD PTH MAP Se Na Se Ca Se

Pi

HW/BW 0.565 ^* 0.733 ^* 0.527 ^* 0.589 ^* 0.665 ^* 0.420 0.600 ^* 0.22

1

Fgf23 0.565 ^" 0.548 ^* 0.883 ^* 0.812 ^* 0.634 ^* 0.019 0.445 ^*

0.11

7

Vol0.733 ^* 0.548 ^* 0.612 ^* 0.569 ^* 0.721 ^* 0.254 0.642 ^* 0.13 ume 3

ALD 0.527 ^* 0.883 ^* 0.612 ^* 0.826 ^* 0.682 ^* 0.461 ^* 0.05

0.041 7

PTH 0.589 ^" 0.812 ^* 0.569 ^* 0.826 ^* 0.704 ^* 0.191 0.452 ^* 0.10

7

MAP 0.665 ^* 0.634 ^* 0.721 ^* 0.682 ^* 0.704 ^* 0.295 0.629 ^*

0.03

2

Se Na 0.420 ^* 0.019 0.254 -41 0.191 0.295 0.294 0.11

3

Se Ca 0.600 ^* 0.445 ^* 0.642 ^* 0.461 ^* 0.452 ^* 0.629 ^* 0.294 0.31

0

Se Pi 0.221 -0.117 0.133 0.057 0.107 -0.032 0.113 0.310

Heart weight to body weight - HW/BW; serum Fgf23 - Fgf23; blood volume - volume; serum aldosterone - ALD; serum parathyroid hor ^¬ mone - PTH; serum - Se. Mean arterial pressure - MAP. * p < 0.05, ** p < 0.01.

Table 2: Stepwise multiple regression analysis of the rela ^¬ tionship between heart-to-body weight ratio, blood volume and selected serum parameters in Sham and CKD animals.

Heart weight to body weight - HW/BW; serum Fgf23, blood volume (volume) , serum aldosterone, serum PTH, serum calcium, serum phosphate, and mean arterial pressure were used as independent variables in the model.

The data therefore suggests that CKD-induced cardiac hyper- trophy is mainly driven by increased volume overload.

Example 3: Assessment of the accelerated CKD progression in Klotho/VDR compound mutants.

In order to assess differences in mortality, disease pro ^¬ gression and vascular calcification between Fgf23/VDR and Klotho/VDR CKD mice, mineral homeostasis was examined.

Results: Figure 4 shows hyperphosphatemia, hypocalcemia, re ^¬ nal Ca ²⁺ and Na ⁺ wasting, increased PTH and aldosterone levels, together with reduced renal expression of TRPV5 and NCC and aug ^¬ mented expression of ENaC in Sham Fgf23/VDR and Klotho/VDR compound mutants relative to WT and VDR controls. In WT and VDR mu ^¬ tant mice, 5/6-Nx induced hypercalcemia, hypernatremia, reduced renal excretion of phosphate and Ca ²⁺ , as well as increased serum levels of PTH, aldosterone, and Fgf23 (Fig. 4A-C) . Notably, 5/6- Nx resulted in hyperphosphatemia in WT but not in VDR mutant mice (Fig. 4A-C) . Since disease progression was not different between VDR mutant and WT CKD mice (Fig. 2B-D) , this unexpected but interesting finding suggests that hyperphosphatemia is not a key driving factor for disease progression after 5/6-Nx. In line with the higher serum levels of Fgf23, PTH, and aldosterone, re ^¬ nal expression of fully glycosylated TRPV5, NCC, and aENaC was increased in WT and VDR CKD mice (Fig. 4D) . Significant differ ^¬ ences between Fgf23/VDR and Klotho/VDR CKD mice were found only for total serum Ca ²⁺ , urinary Ca ²⁺ excretion, and serum aldosterone concentration. Interestingly, 5/6-Nx failed to induce renal Ca ²⁺ retention in Fgf23 /VDR in contrast to Klotho/VDR mice (Fig. 4A-C) . However, TRPV5 expression in the kidney was upregulated in a similar fashion in Fgf23 /VDR and Klotho/VDR CKD mice (Fig. 4D) .

While renal Klotho protein expression remained unchanged in CKD mice of all genotypes (Fig. 3D), Klotho/VDR compound mutants showed the highest circulating intact Fgf23 levels among all CKD groups (Fig. 4C) . Similarly, Fgf23 mRNA expression as evidenced by laser capture microdissection, and Fgf23 protein expression assessed by Western blotting showed the strongest upregulation in bones of Klotho/VDR CKD animals compared to the other CKD groups (Fig. 3E-F) . Fgf23 expression was augmented in both oste ^¬ oblasts and osteocytes in Klotho/VDR CKD animals (Fig. 3E-F) . Example 4: Pharmacological inhibition of Fgf23 largely protects against the CKD-induced renal and cardiovascular pathology.

To answer the question whether the increased circulating Fgf23 was the cause of accelerated CKD progression in Klotho/VDR compound mutants, WT, VDR and Klotho/VDR CKD mice were treated with low doses of blocking anti-FGF23 antibody (anti-FGF23Ab, 50 yg per mouse, two times per week) or control antibody (Co Ab) over 8 weeks post-surgery. High-dose anti-FGF23 therapy was shown to be actually detrimental in a rat CKD model, increasing mortality and aortic calcification by inducing hyperphosphatemia together with excessive stimulation of vitamin D hormone production (Hoorn, E.J. & Ellison, D.H., Exp. Cell Res., 2012, 318, 1020-1026) . The rationale behind this experiment was that phar ^¬ macological neutralization of Fgf23 would ameliorate CKD pro ^¬ gression only in case that elevated circulating Fgf23 was driving disease progression, but not if lack of Klotho was the cause of aggravated disease progression in Klotho/VDR CKD mice.

Results: Figure 5 shows that treatment of CKD mice with low dose anti-FGF23Ab reduced circulating Fgf23 in CKD mice to Sham control levels in all genotypes, but did not completely elimi ^¬ nate circulating Fgf23. Notably, anti-Fgf23 treatment reduced the increased mortality in Klotho/VDR CKD mice to WT and VDR control levels (Fig. 5B) , suggesting that increased circulating Fgf23 is indeed the cause of increased mortality in Klotho/VDR CKD mice. In analogy to the genetic loss-of-function model, treatment of WT, VDR and Klotho/VDR CKD mice with anti-FGF23Ab partially or completely protected against the CKD-induced weight loss, reduction in GFR, albuminuria, hypercalcemia, aortic cal ^¬ cification, hypertension, heart hypertrophy, and left ventricular functional impairment (Fig. 5C-E, Fig. 6) . Importantly, an- ti-Fgf23 treatment reduced end-diastolic pressure in CKD mice of all genotypes to Sham control levels (Fig. 5E) , corroborating the notion that Fgf23-induced volume overload is a major patho ^¬ genetic factor in CKD progression. Since anti-Fgf23 therapy re ^¬ duced renal tubular Ca ²⁺ and Na ⁺ reabsorption also in Klotho/VDR CKD mice (Fig. 5F) , it seems that Fgf23 must have Klotho- independent effects on Ca ²⁺ and Na ⁺ homeostasis. Although anti- Fgf23 therapy might theoretically aggravate hyperphosphatemia in CKD mice, we did not observe anti-Fgf23 therapy-induced changes in serum phosphate levels or urinary phosphate excretion in any of the genotypes, despite the fact that anti-Fgf23 therapy low ^¬ ered circulating intact PTH in CKD mice (Fig. 6) .

The data suggests that in order to exploit the strikingly beneficial effects of lowering blood Fgf23 concentrations in CKD, it would be necessary to carefully titrate the dose of an- ti-FGF23 Ab to prevent the toxic effects of Fgf23 over- suppression .

Example 5: Protein expression analysis of the main Ca ²⁺ and Na ⁺ channels in the distal nephron as well as expression of activat ^¬ ed WNK1 and WNK4 , two of the most important regulators of intra ^¬ cellular ion channel trafficking in distal renal tubules (Nilius et al., Br. J. Pharmacol., 2001. 134, 453-462).

In order to assess how excessive intact Fgf23 can modulate renal Ca ²⁺ and Na ⁺ handling in CKD mice in a Klotho-independent manner, protein expression of ENaC, yENaC, TRPV5, NCC, phospho (p) -NCC, pNKCCl (Na+-potassium-chloride cotransporter-1 ) , pWNKl, and pWNK4 was performed.

Results: Figures 7 and 8 show that 5/6-Nx upregulated renal protein expression of aENaC, yENaC, TRPV5, NCC, phospho (p) -NCC, pNKCCl (Na ⁺ -potassium-chloride cotransporter-1), pWNKl, and pWNK4 in all genotypes. Interestingly, TRPV6 expression was profoundly upregulated in Klotho/VDR, but not in WT or VDR CKD mice (Fig. 7 and 8) . Anti-Fgf23 Ab therapy reduced aENaC, NCC, pNCC, pNKCCl, pWNKl, and pWNK4 in all genotypes (Fig. 7 and 8) . In addition, anti-Fgf23 therapy reduced the augmented TRPV6 expression in Klotho/VDR CKD mice to Sham control levels (Fig. 7) . In contrast, yENaC and TRPV5 expression remained unchanged in anti- Fgf23-treated CKD mice (Fig. 7) .

Collectively, these results indicate that excessive circu ^¬ lating Fgf23 activates WNK signalling, and upregulates the ex ^¬ pression and activation of Ca ²⁺ - and Na ⁺ -transporting molecules in a partially Klotho-independent manner in CKD mice.

Example 6: Serum intact FGF23 levels are associated with WNK ac ^¬ tivation in the kidney of human CKD patients.

In order to assess whether increased circulating intact FGF23 is also associated with WNK activation in humans, a pro ^¬ spective study in ten patients with CKD stages 2 - 5 undergoing diagnostic renal biopsies was performed. Results: pNCC, pNKCCl, and pWNKl expression by immunohisto- chemistry was quantified in renal biopsies. pNCC expression was not significantly correlated with serum intact FGF23 (r _s =0.297; P=0.407) . However, Figure 9 shows a strong association between serum intact FGF23 and distal renal tubular pWNKl and pNKCCl ex ^¬ pression .

This data suggests that FGF23-induced WNK activation in dis ^¬ tal renal tubules is a pivotal mechanism in human CKD pathophys ^¬ iology .

Example 7: Very high concentrations of FGF23 activate WNK sig ^¬ nalling in a Klotho-independent manner in kidney slices ex vivo.

In order to examine the Klotho-independent effects of exces ^¬ sive Fgf23 signalling on TRPV6 expression and phosphorylation of NCC, NKCCl and WNK4, and to rule out indirect effects of altered circulating aldosterone and PTH levels in CKD mice, 200^m-thick kidney slices isolated from WT and Klotho/VDR animals were treated with two different concentrations of recombinant FGF23 (rFGF23) , 100 ng/ml (high) and 200 ng/ml (very high) , for 2 h ex vivo, and analysed protein expression of the target molecules by Western blotting.

Results: One-hundred ng/ml rFGF23 increased pWNKl, pWNK4, and TRPV5 expression in WT, but with the exception of pWNKl not in Klotho/VDR kidney slices (Fig. lOA-C) . In contrast, Figure 10 further shows that 200 ng/ml of rFGF23 upregulated pWNKl, pWNK4, pNKCCl, and TRPV6 expression in both WT and Klotho/VDR kidney slices, indicating that very high concentrations of FGF23 are able to activate WNK signalling, NKCCl phosphorylation, and TRPV6 expression in a Klotho-independent fashion in vitro.

Example 8: Very high concentrations of FGF23 stimulate distal tubular calcium and sodium transport in kidney slices by Klotho- and FGFRl-independent activation of NKCCl and TRPV6 channels.

To gain more insight into the functional role of the rFGF23- induced increase in distal renal tubular ion channel expression and the FGF receptors involved, we performed intracellular Ca ²⁺ and Na ⁺ imaging by 2-photon microscopy in Fluo-4- or SFBI-loaded and subsequently rFGF23-treated (100 or 200 ng/ml for 2 h) , 200- μιη-thick renal slices prepared from WT and Klotho/VDR mice.

Results: Figure 11A-D shows that rFGF23 increased intracel- lular Ca ²⁺ and Na ⁺ fluorescence in WT distal tubules at both con ^¬ centrations, whereas only 200 ng/ml rFGF23 was able to increase intracellular Ca ²⁺ and Na ⁺ fluorescence in kidney slices of Klotho/VDR mice. The FGF23-induced Ca ²⁺ uptake was reversed in WT kidney slices by co-treatment with the TRPV blocker ruthenium red at 1 μΜ. Ruthenium red at 1 μΜ is known to block only TRPV5, but not TRPV6 channel function (Nilius B et al . , Br. J. Pharma ^¬ col., 2001. 134, 453-462) . In contrast, co-treatment with 10 μΜ ruthenium red was necessary to antagonize the rFGF23-induced in ^¬ crease in distal tubular Ca ²⁺ uptake in Klotho/VDR kidney slices

(Fig. 11A and C) , suggesting the involvement of TRPV6 in this process. The inhibitory effects of the specific NCC and NKCC1 blockers chlorothiazide (CTZ) or bumetanide (BF) respectively

(Monroy A et al . , Am. J. Physiol Renal Physiol, 2000. 279, F161- F169; Isenring P et al . , J. Biol. Chem., 1998. 273, 11295- 11301), was tested to analyse the molecules involved in the FGF23-induced Na ⁺ uptake. Figure 11B and D show that both, CTZ and BF, decreased SFBI fluorescence intensity in distal tubules of rFGF23-treated WT kidney slices by approximately 50%, whereas the Na ⁺ influx in Klotho/VDR kidney slices was inhibited exclu ^¬ sively by the NKCC1 inhibitor BF. To identify the FGFRs involved in the rFGF23-induced increase Ca ²⁺ and Na ⁺ uptake, kidney slices of WT and Klotho/VDR mice were co-treated with 200 ng/ml rFGF23 and an FGFR1 inhibitor (iFGFRl) or a pan FGFR inhibitor (iFGFR pan) . FGFR1 inhibition had no effect on the rFGF23-induced in ^¬ crease in Ca ²⁺ and Na ⁺ fluorescence in Klotho/VDR kidney slices, but largely blocked the effect of rFGF23 in WT kidney slices. In contrast, Figure 11A-D shows that the iFGFR pan inhibitor com ^¬ pletely blocked the rFGF23-induced increase in intracellular Ca ²⁺ and Na ⁺ fluorescence in both WT and Klotho/VDR kidney slices.

These data show that excessive concentrations of rFGF23 en ^¬ hance distal tubular Ca ²⁺ and Na ⁺ uptake in vitro by a combina ^¬ tion of Klotho-dependent FGFRl-mediated activation of TRPV5 and NCC, and of Klotho- and FGFRl-independent activation of TRPV6, NCC and NKCC1, respectively.

Example 9: Assessment of renal FGFR mRNA expression and effects of FGFR1/3 and/or WNK signalling inhibition on distal tubular FGF23-mediated calcium and sodium uptake in kidney slices of Klotho/VDR mutants. In this example it was assessed whether the Klotho- independent FGF23 signalling pathway is mediated through FGFR3 and/or 4, given that FGFR2 expression was low in distal renal tubules (Figure 12A) . To dissect between the latter receptors, Klotho/VDR kidney slices were treated with rFGF23 alone or in combination with an FGFR1/FGFR3 inhibitor and the WNK signalling inhibitor closantel.

Results: Figure 12B-C shows that closantel abrogated the FGF23-induced upregulation in distal tubular Ca ²⁺ and Na ⁺ uptake, demonstrating the essential role of WNK activation in this process. FGFR1/3 inhibition partially blocked the FGF23-induced Ca ²⁺ influx, but completely blocked the FGF23-induced increase in Na ⁺ uptake (Fig. 12B-C) .

These data suggest that the Klotho-independent activation of intracellular Ca ²⁺ uptake in distal renal tubules by excessive FGF23 in vitro is mediated through FGFR3 and 4, whereas the Klotho-independent FGF23-induced stimulation of Na ⁺ uptake is mainly mediated by FGFR3.

Example 10: Assessment of the effects of oral closantel admin ^¬ istration in CKD mice.

In order to assess whether WNK signalling inhibition is able to ameliorate CKD disease progression in vivo, WT Sham and 5/6- Nx mice on rescue diet were treated over 8 weeks with closantel orally via the diet. To maintain constant plasma levels, the mice received closantel-containing food once a week, using the dosing regimen as described in the materials and methods. The pharmacokinetic model underlying the calculation of the dosing regimen was based on the assumption of a plasma half-life of 14 days .

Results: As shown in Figure 13A-B, closantel largely pre ^¬ vented the CKD-driven decline in GFR, as well as the CKD-induced increase in HW/BW ratio, blood volume, and blood pressure by downregulation of renal pNCC, pNKCC, and pWNKl expression, which resulted in increased urinary excretion of Na ⁺ in CKD mice.

These data indicate that the small molecule WNK signalling inhibitor closantel may present a new therapeutic strategy for CKD patients.

Example 11: Assessment of the effects of oral WNK463 administra- tion in CKD mice.

Sham-operated (Sham) and 5/6 Nx wild-type mice on rescue di ^¬ et were treated over 8 weeks once a day with WNK463 orally via gavage . Eight weeks postsurgery, body weight, heart weight/body weight (HW/BW) ratio, mean arterial pressure (MAP) , urinary ex ^¬ cretion of creatinine, urinary sodium/creatinine excretion, uri ^¬ nary calcium/creatinine excretion, serum creatinine, serum urea, and GFR were measured in the mice. The results are depicted in Fig. 15. Interestingly, WNK463 had a clear diuretic effect, in ^¬ creasing urinary sodium and calcium excretion, but it did not lower arterial blood pressure. Despite the lack of an effect of WNK463 on blood pressure, it had a beneficial effect on disease progression because serum creatinine was lower, and glomerular filtration rate tended to be higher compared with vehicle- treated 5/6 Nx mice. This also shows that CKD is a complex dis ^¬ ease, high blood pressure is just one aspect. Because of the complexity of the disease the positive drug effect of closantel and WNK463 in CKD was not easily foreseeable.