Efficacy and safety of therapeutic alpha-1-microglobulin RMC-035 in reducing kidney injury after cardiac surgery: a multicentre, randomised, double-blind, parallel group, phase 2a trial.

Background: Cardiac surgery invariably triggers acute kidney stress causing adverse renal outcomes. The AKITA study evaluated the efficacy and safety of RMC-035, a novel analogue of alpha-1-microglobulin, for reducing cardiac surgery-associated kidney injury. Methods: In this randomised double-blind placebo-controlled phase 2a study, we randomly assigned (1:1) adult hospitalised patients undergoing open-chest cardiac surgery at high risk for acute kidney injury (AKI) at 21 sites in North America and Europe to receive either RMC-035 (1.3 or 0.65 mg/kg) or placebo (1:1) for 2 days (5 intravenous infusions), stratified by region and renal function. Eligible patients had at least one pre-defined AKI risk factor. Patients with severe renal impairment (estimated glomerular filtration rate [eGFR] <30 mL/min/1.73 m2) were excluded. The co-primary efficacy and safety endpoints were AKI (Kidney Disease: Improving Global Outcomes definition) within 72 h after surgery and nature, frequency, and severity of treatment-emergent adverse events (TEAEs). Secondary endpoints included eGFR and Major Adverse Kidney Events (MAKE) up to Day 90. Randomised patients who had received at least one dose of study drug were analysed for primary and safety analyses. Participants, investigators and sponsor were masked to treatment allocation. This study is registered at ClinicalTrials.gov (NCT05126303) and EudraCT (2021-004040-19). Findings: Patient enrolment was stopped at interim analysis due to futility. Between March 31, 2022 and July 12, 2023, 177 patients (RMC-035: 89, placebo: 88) were randomised and treated. AKI rate for RMC-035 vs placebo was 50.6% (n = 45) and 39.8% (n = 35) (relative risk [RR]: 1.30, 90% confidence interval [90% CI]: 0.99, 1.71; p = 0.12). A short-lived creatinine increase was observed with the higher RMC-035 dose. Treatment with RMC-035 was associated with improved secondary renal outcomes at Day 90: placebo-adjusted eGFR change from baseline 4.3 mL/min/1.73 m2, 90% CI 0.51-8.12, p = 0.06; and MAKE 6.7% (n = 6) vs 15.9% (n = 14); RR: 0.41, 90% CI: 0.19, 0.88, p = 0.05. The most frequently reported TEAEs for RMC-035 were chills (30.3%), nausea (21.3%), anaemia (20.2%); and atrial fibrillation (29.5%), anaemia (20.5%), hypervolemia (14.8%) for placebo. The majority of TEAEs in both treatment groups were mild or moderate in severity. In the RMC-035 group, 26 (29.2%) patients experienced at least one severe or life-threatening TEAE and in the placebo group 16 (18.2%) patients. There were 4 deaths per treatment arm (one treatment-related, in placebo group). Interpretation: In this proof-of-concept study, RMC-035 did not reduce AKI 72 h after cardiac surgery. Evaluations may have been confounded by a drug-induced transient creatinine increase in a subgroup of patients. RMC-035 was associated with improved secondary renal outcomes. These results merit further investigation and should be interpreted with caution, as the study was not powered for these outcomes. Funding: Guard Therapeutics.

Therapeutic α-1-microglobulin ameliorates kidney ischemia-reperfusion injury.

α-1-Microglobulin (A1M) is a circulating glycoprotein with antioxidant, heme-binding, and mitochondrial protection properties. The investigational drug RMC-035, a modified therapeutic A1M protein, was assessed for biodistribution and pharmacological activity in a broad set of in vitro and in vivo experiments, supporting its clinical development. Efficacy and treatment posology were assessed in various models of kidney ischemia and reperfusion injury (IRI). Real-time glomerular filtration rate (GFR), functional renal biomarkers, tubular injury biomarkers (NGAL and KIM-1), and histopathology were evaluated. Fluorescently labeled RMC-035 was used to assess biodistribution. RMC-035 demonstrated consistent and reproducible kidney protection in rat IRI models as well as in a model of IRI imposed on renal impairment and in a mouse IRI model, where it reduced mortality. Its pharmacological activity was most pronounced with combined dosing pre- and post-ischemia and weaker with either pre- or post-ischemia dosing alone. RMC-035 rapidly distributed to the kidneys via glomerular filtration and selective luminal uptake by proximal tubular cells. IRI-induced expression of kidney heme oxygenase-1 was inhibited by RMC-035, consistent with its antioxidative properties. RMC-035 also dampened IRI-associated inflammation and improved mitochondrial function, as shown by tubular autofluorescence. Taken together, the efficacy of RMC-035 is congruent with its targeted mechanism(s) and biodistribution profile, supporting its further clinical evaluation as a novel kidney-protective therapy.NEW & NOTEWORTHY A therapeutic A1M protein variant (RMC-035) is currently in phase 2 clinical development for renal protection in patients undergoing open-chest cardiac surgery. It targets several key pathways underlying kidney injury in this patient group, including oxidative stress, heme toxicity, and mitochondrial dysfunction. RMC-035 is rapidly eliminated from plasma, distributing to kidney proximal tubules, and demonstrates dose-dependent efficacy in numerous models of ischemia-reperfusion injury, particularly when administered before ischemia. These results support its continued clinical evaluation.

Study protocol of a phase 2, randomised, placebo-controlled, double-blind, adaptive, parallel group clinical study to evaluate the efficacy and safety of recombinant alpha-1-microglobulin in subjects at high risk for acute kidney injury following open-chest cardiac surgery (AKITA trial).

INTRODUCTION: Acute kidney injury (AKI) is a common complication after cardiac surgery (CS) and is associated with adverse short-term and long-term outcomes. Alpha-1-microglobulin (A1M) is a circulating glycoprotein with antioxidant, heme binding and mitochondrial-protective mechanisms. RMC-035 is a modified, more soluble, variant of A1M and has been proposed as a novel targeted therapeutic protein to prevent CS-associated AKI (CS-AKI). RMC-035 was considered safe and generally well tolerated when evaluated in four clinical phase 1 studies. METHODS AND ANALYSIS: This is a phase 2, randomised, double-blind, adaptive design, parallel group clinical study that evaluates RMC-035 compared with placebo in approximately 268 cardiac surgical patients at high risk for CS-AKI. RMC-035 is administered as an intravenous infusion. In total, five doses will be given. Dosing is based on presurgery estimated glomerular filtration rate (eGFR), and will be either 1.3 or 0.65 mg/kg.The primary study objective is to evaluate whether RMC-035 reduces the incidence of postoperative AKI, and key secondary objectives are to evaluate whether RMC-035 improves postoperative renal function compared with placebo. A blinded interim analysis with potential sample size reassessment is planned once 134 randomised subjects have completed dosing. An independent data monitoring committee will evaluate safety and efficacy data at prespecified intervals throughout the trial. The study is a global multicentre study at approximately 30 sites. ETHICS AND DISSEMINATION: The trial was approved by the joint ethics committee of the physician chamber Westfalen-Lippe and the University of Münster (code '2021-778 f-A') and subsequently approved by the responsible ethics committees/relevant institutional review boards for the participating sites. The study is conducted in accordance with Good Clinical Practice, the Declaration of Helsinki and other applicable regulations. Results of this study will be published in a peer-reviewed scientific journal. TRIAL REGISTRATION NUMBER: NCT05126303.

A Transgenic Model Reveals the Role of Klotho in Pancreatic Cancer Development and Paves the Way for New Klotho-Based Therapy.

Klotho is an anti-aging transmembrane protein, which can be shed and can function as a hormone. Accumulating data indicate that klotho is a tumor suppressor in a wide array of malignancies, and designate the subdomain KL1 as the active region of the protein towards this activity. We aimed to study the role of klotho as a tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Bioinformatics analyses of The Cancer Genome Atlas (TCGA) datasets revealed a correlation between the survival of PDAC patients, levels of klotho expression, and DNA methylation, and demonstrated a unique hypermethylation pattern of klotho in pancreatic tumors. The in vivo effects of klotho and KL1 were examined using three mouse models. Employing a novel genetic model, combining pancreatic klotho knockdown with a mutation in Kras, the lack of klotho contributed to PDAC generation and decreased mousece survival. In a xenograft model, administration of viral particles carrying sKL, a spliced klotho isoform containing the KL1 domain, inhibited pancreatic tumors. Lastly, treatment with soluble sKL prolonged survival of Pdx1-Cre; KrasG12D/+;Trp53R172H/+ (KPC) mice, a model known to recapitulate human PDAC. In conclusion, this study provides evidence that klotho is a tumor suppressor in PDAC. Furthermore, these data suggest that the levels of klotho expression and DNA methylation could have prognostic value in PDAC patients, and that administration of exogenous sKL may serve as a novel therapeutic strategy to treat PDAC.

Longitudinal change in c-terminal fibroblast growth factor 23 and outcomes in patients with advanced chronic kidney disease.

BACKGROUND: Fibroblast growth factor23 (FGF23) is elevated in CKD and has been associated with outcomes such as death, cardiovascular (CV) events and progression to Renal Replacement therapy (RRT). The majority of studies have been unable to account for change in FGF23 over time and those which have demonstrate conflicting results. We performed a survival analysis looking at change in c-terminal FGF23 (cFGF23) over time to assess the relative contribution of cFGF23 to these outcomes. METHODS: We measured cFGF23 on plasma samples from 388 patients with CKD 3-5 who had serial measurements of cFGF23, with a mean of 4.2 samples per individual. We used linear regression analysis to assess the annual rate of change in cFGF23 and assessed the relationship between time-varying cFGF23 and the outcomes in a cox-regression analysis. RESULTS: Across our population, median baseline eGFR was 32.3mls/min/1.73m2, median baseline cFGF23 was 162 relative units/ml (RU/ml) (IQR 101-244 RU/mL). Over 70 months (IQR 53-97) median follow-up, 76 (19.6%) patients progressed to RRT, 86 (22.2%) died, and 52 (13.4%) suffered a major non-fatal CV event. On multivariate analysis, longitudinal change in cFGF23 was significantly associated with risk for death and progression to RRT but not non-fatal cardiovascular events. CONCLUSION: In our study, increasing cFGF23 was significantly associated with risk for death and RRT.

Mechanism-based modeling of the effect of a novel inhibitor of vascular adhesion protein-1 on albuminuria and renal function markers in patients with diabetic kidney disease.

The vascular adhesion protein-1 (VAP-1) inhibitor ASP8232 reduces albuminuria in patients with type 2 diabetes and chronic kidney disease. A mechanism-based model was developed to quantify the effects of ASP8232 on renal markers from a placebo-controlled Phase 2 study in diabetic kidney disease with 12 weeks of ASP8232 treatment. The model incorporated the available pharmacokinetic, pharmacodynamic (plasma VAP-1 concentration and activity), serum and urine creatinine, serum cystatin C, albumin excretion rate, urinary albumin-to-creatinine ratio, and urine volume information in an integrated manner. Drug-independent time-varying changes and different drug effects could be quantified for these markers using the model. Through simulations, this model provided the opportunity to dissect the relationship and longitudinal association between the estimated glomerular filtration rate and albuminuria and to quantify the pharmacological effects of ASP8232. The developed drug-independent model may be useful as a starting point for other compounds affecting the same biomarkers in a similar time scale.

Population pharmacokinetics and pharmacodynamics of a novel vascular adhesion protein-1 inhibitor using a multiple-target mediated drug disposition model.

ASP8232 is a novel inhibitor of vascular adhesion protein-1 that was under evaluation for reducing residual albuminuria in patients with diabetic kidney disease. To characterize the pharmacokinetics (PK) of ASP8232 and its effect on vascular adhesion protein 1 (VAP-1) plasma activity and VAP-1 concentrations (pharmacodynamics, PD) in an integrated and quantitative manner, a target mediated drug disposition model was developed based on pooled data from four completed clinical trials with ASP8232 in healthy volunteers, and in patients with diabetic kidney disease and diabetic macular edema, respectively. In this model, the binding of ASP8232 to its soluble and membrane-bound target in the central and peripheral compartments were included. The model was able to adequately describe the non-linear PK and PD of ASP8232. The observed difference in PK between healthy volunteers and renally impaired patients could be explained by an effect of baseline estimated glomerular filtration rate on ASP8232 clearance and relative bioavailability. The relationship between ASP8232 concentration and VAP-1 inhibition was successfully established and can be applied to simulate drug exposure and degree of VAP-1 inhibition for any given dose of ASP8232 across the spectrum of renal function.

Carbazate modified dextrans as scavengers for carbonylated proteins.

A series of biocompatible and non- toxic polysaccharide molecules have been successfully fabricated and explored their potential application for scavenging the carbonyl species in vitro. These macromolecules were dextrans with different hydrazide substitution ratios determined by TNBS assay, NMR and FTIR characterization. The colorimetric assay had demonstrated that these macromolecules could effectively scavenge acrolein, oxidized bovine serum albumin (BSA) in buffer solutions as well as carbonyl proteins from serum. The scavengers could achieve twice more scavenging effects for modified dextrans with high molecular weight (Mw = 100,000) than those of low ones (Mw = 40,000) with the same substitution ratio. Protein gel electrophoresis confirmed that the formation of the complex between carbonyls and modified dextrans resulted in appearance of slower bands. It also revealed that such macromolecules could protect cultured cells against the toxicity of acrolein or its derivatives. The proposed macromolecules indicated a very promising capability as scavengers for oxidative stress plus its derivatives without side effects.

Weak acidic stable carbazate modified cellulose membranes target for scavenging carbonylated proteins in hemodialysis.

Carbazate groups were grafted on the commercial cellulose membrane (CM) to specifically scavenge the carbonylated proteins for hemodialysis. It confirmed that carbazate groups were successfully covalently attached on the CMs by XPS and EDS, and the modified CMs still saved their original morphology and crystalline structures by SEM and XRD. Furthermore, the modified CMs presented favorable physicochemical stability at wide pH range from 2.5 to 7.4. It was also found that the carbazate modified CMs could selectively remove carbonylated proteins from acrolein treated bovine serum albumin (BSA) or ESRD patient's blood serum in PBS buffer. The modified CMs showed the potential to be utilized as the substitute of dialysis membranes in hemodialysis.

Efficacy of a novel inhibitor of vascular adhesion protein-1 in reducing albuminuria in patients with diabetic kidney disease (ALBUM): a randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Many patients with diabetic kidney disease have residual albuminuria and are at risk of disease progression. The ALBUM trial investigated the efficacy of a novel, orally active inhibitor of vascular adhesion protein-1, ASP8232, compared with placebo for reducing albuminuria in individuals with type 2 diabetes and chronic kidney disease. METHODS: In this randomised, double-blind, placebo-controlled phase 2 trial, we randomly assigned individuals (aged 18-85 years) from 64 clinical sites in nine European countries to receive ASP8232 40 mg or placebo orally once daily for 12 weeks using a web-based randomisation schedule (block size 4), stratified by country. Eligible patients had a urinary albumin-to-creatinine ratio (UACR) of 200-3000 mg/g, an estimated glomerular filtration rate of at least 25 mL/min per 1·73 m2 but lower than 75 mL/min per 1·73 m2, HbA1c less than 11·0% (97 mmol/mol), and stable treatment with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers and antidiabetic medication for 3 months or more. The primary endpoint was mean change from baseline to week 12 in log-transformed first morning void UACR, which was assessed in all patients who received at least one dose of study drug and had at least one post-baseline UACR measurement (full analysis set). Safety was assessed in all patients who received at least one dose of study drug. Participants and investigators were masked to treatment allocation. This trial is registered with ClinicalTrials.gov, number NCT02358096. FINDINGS: 125 participants were randomly assigned to receive ASP8232 (n=64) or placebo (n=61), of whom 120 (60 in each group) were included in the full analysis set; all participants were assessed for safety endpoints. At 12 weeks, UACR decreased by 17·7% (95% CI 5·0 to 28·6) in the ASP8232 group and increased by 2·3% (-11·4 to 18·1) in the placebo group; the placebo-adjusted difference between groups was -19·5% (95% CI -34·0 to -1·8; p=0·033). 39 (61%) patients in the ASP8232 group and 34 (56%) patients in the placebo group had a treatment-emergent adverse event, of which 16 in the ASP8232 group and four in the placebo group were drug-related. The most frequently reported adverse events that were possibly drug-related in the ASP8232 group were renal impairment (five patients) and decreased eGFR (three patients); in the placebo group, no single drug-related treatment-emergent adverse event was reported by more than one participant. INTERPRETATION: ASP8232 is effective in reducing albuminuria in patients with diabetic kidney disease and is safe and well tolerated. These findings warrant further research to ascertain the effect of ASP8232 on delaying progression of diabetic kidney disease. FUNDING: Astellas.

Klotho controls the brain-immune system interface in the choroid plexus.

Located within the brain's ventricles, the choroid plexus produces cerebrospinal fluid and forms an important barrier between the central nervous system and the blood. For unknown reasons, the choroid plexus produces high levels of the protein klotho. Here, we show that these levels naturally decline with aging. Depleting klotho selectively from the choroid plexus via targeted viral vector-induced knockout in Klothoflox/flox mice increased the expression of multiple proinflammatory factors and triggered macrophage infiltration of this structure in young mice, simulating changes in unmanipulated old mice. Wild-type mice infected with the same Cre recombinase-expressing virus did not show such alterations. Experimental depletion of klotho from the choroid plexus enhanced microglial activation in the hippocampus after peripheral injection of mice with lipopolysaccharide. In primary cultures, klotho suppressed thioredoxin-interacting protein-dependent activation of the NLRP3 inflammasome in macrophages by enhancing fibroblast growth factor 23 signaling. We conclude that klotho functions as a gatekeeper at the interface between the brain and immune system in the choroid plexus. Klotho depletion in aging or disease may weaken this barrier and promote immune-mediated neuropathogenesis.

In vivo evidence for an interplay of FGF23/Klotho/PTH axis on the phosphate handling in renal proximal tubules.

Phosphate homeostasis is primarily maintained in the renal proximal tubules, where the expression of sodium/phosphate cotransporters (Npt2a and Npt2c) is modified by the endocrine actions of both fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). However, the specific contribution of each regulatory pathway in the proximal tubules has not been fully elucidated in vivo. We have previously demonstrated that proximal tubule-specific deletion of the FGF23 coreceptor Klotho results in mild hyperphosphatemia with little to no change in serum levels of FGF23, 1,25(OH)2D3, and PTH. In the present study, we characterized mice in which the PTH receptor PTH1R was specifically deleted from the proximal tubules, either alone or in combination with Klotho ( PT-PTH1R-/- and PT-PTH1R/KL-/-, respectively). PT-PTH1R-/- mice showed significant increases in serum FGF23 and PTH levels, whereas serum phosphate levels were maintained in the normal range, and Npt2a and Npt2c expression in brush border membrane (BBM) did not change compared with control mice. In contrast, PT-PTH1R/KL-/- mice displayed hyperphosphatemia and an increased abundance of Npt2a and Npt2c in the renal BBM, along with increased circulating FGF23 levels. While serum calcium was normal, 1,25(OH)2D3 levels were significantly decreased, leading to extremely high levels of PTH. Collectively, mice with a deletion of PTH1R alone in proximal tubules results in only minor changes in phosphate regulation, whereas deletion of both PTH1R and Klotho leads to a severe disturbance, including hyperphosphatemia with increased sodium/phosphate cotransporter expression in BBM. These results suggest an important interplay between the PTH/PTH1R and FGF23/Klotho pathways to affect renal phosphate handling in the proximal tubules.

Interrelated role of Klotho and calcium-sensing receptor in parathyroid hormone synthesis and parathyroid hyperplasia.

The pathogenesis of parathyroid gland hyperplasia is poorly understood, and a better understanding is essential if there is to be improvement over the current strategies for prevention and treatment of secondary hyperparathyroidism. Here we investigate the specific role of Klotho expressed in the parathyroid glands (PTGs) in mediating parathyroid hormone (PTH) and serum calcium homeostasis, as well as the potential interaction between calcium-sensing receptor (CaSR) and Klotho. We generated mouse strains with PTG-specific deletion of Klotho and CaSR and dual deletion of both genes. We show that ablating CaSR in the PTGs increases PTH synthesis, that Klotho has a pivotal role in suppressing PTH in the absence of CaSR, and that CaSR together with Klotho regulates PTH biosynthesis and PTG growth. We utilized the tdTomato gene in our mice to visualize and collect PTGs to reveal an inhibitory function of Klotho on PTG cell proliferation. Chronic hypocalcemia and ex vivo PTG culture demonstrated an independent role for Klotho in mediating PTH secretion. Moreover, we identify an interaction between PTG-expressed CaSR and Klotho. These findings reveal essential and interrelated functions for CaSR and Klotho during parathyroid hyperplasia.

Associations of Fibroblast Growth Factor 23 with Markers of Inflammation and Leukocyte Transmigration in Chronic Kidney Disease.

BACKGROUND: Patients with chronic kidney disease (CKD) show elevated levels of inflammatory markers and have an increased risk of infections as well as cardiovascular morbidity. Recent studies have implied effects of fibroblast growth factor 23 (FGF23) on inflammation in CKD. We analyzed potential correlations between levels of FGF23 with pro-inflammatory chemokines and markers of leukocyte transmigration in CKD patients. METHODS: One hundred three patients with CKD 2-5ND and 54 healthy controls, had biochemical markers in blood and urine analyzed according to routine protocol. Pro-inflammatory cytokines were analyzed by Milliplex technique and leukocyte CD11b adhesion molecule expression was measured by flow cytometry. FGF23 levels were measured with ELISA technique. Treatment of leukocytes from healthy blood donors with FGF23 was performed in vitro and effects analyzed by flow cytometry. RESULTS: Tumor necrosis factor-alpha, RANTES and interleukin (IL)-12 levels were significantly higher (p = 0.001, p < 0.001, and p < 0.001) in patients with CKD. Elevated FGF23 levels in the CKD group correlated to glomerular filtration rate, parathyroid hormone, urinary albumin excretion and phosphate as well as to IL-12 and RANTES. CD11b expression on resting granulocytes and monocytes, and on activated monocytes, was associated with FGF23. In vitro treatment of leukocytes with FGF23 reduced CD11b expression in resting as well as in formyl-methyinoyl-leucyl-phenylalanine-stimulated granulocytes (p = 0.03). CONCLUSION: FGF23 levels are associated with various inflammatory markers such as pro-inflammatory cytokines and adhesion molecules on innate immune cells. However, further studies are warranted to define the direct role of FGF23 in modulation of the innate immune system in CKD.

NPT-IIb Inhibition Does Not Improve Hyperphosphatemia in CKD.

INTRODUCTION: Serum phosphate levels are insufficiently controlled in many patients with end-stage renal disease (ESRD), and novel therapeutic strategies are needed. Blocking intestinal phosphate absorption mediated by sodium-dependent phosphate co-transporter type 2b (NPT-IIb) holds promise; thus, we evaluated the efficacy, safety, tolerability, and pharmacokinetics of the novel and specific small molecule NPT-IIb inhibitor ASP3325 for the first time in humans. METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 1a single (n = 88) and multiple (n = 36) ascending dose study in healthy subjects, and a randomized, open-label, uncontrolled, phase 1b study in hyperphosphatemic ESRD patients on hemodialysis (single oral dose, n = 5; multiple oral doses, n = 17). Primary efficacy measures were urinary phosphate and fecal phosphorous excretion (healthy subjects) and serum phosphate level (ESRD patients). RESULTS: No time- or dose-dependent changes in urinary phosphate or fecal phosphorous excretion were observed following single/multiple ASP3325 doses for 7 days in healthy subjects. In ESRD patients, ASP3325 administered 3 times daily for 2 weeks before or after a meal did not reduce serum phosphate levels. ASP3325 was safe and well tolerated in both populations. CONCLUSION: NPT-IIb inhibition with ASP3325 was not effective in reducing serum phosphate levels in ESRD patients. The relevance of NPT-IIb in humans and feasibility of oral NPT-IIb inhibitors for treatment of hyperphosphatemia in ESRD remain uncertain.

Klotho expression in osteocytes regulates bone metabolism and controls bone formation.

Osteocytes within the mineralized bone matrix control bone remodeling by regulating osteoblast and osteoclast activity. Osteocytes express the aging suppressor Klotho, but the functional role of this protein in skeletal homeostasis is unknown. Here we identify Klotho expression in osteocytes as a potent regulator of bone formation and bone mass. Targeted deletion of Klotho from osteocytes led to a striking increase in bone formation and bone volume coupled with enhanced osteoblast activity, in sharp contrast to what is observed in Klotho hypomorphic (kl/kl) mice. Conversely, overexpression of Klotho in cultured osteoblastic cells inhibited mineralization and osteogenic activity during osteocyte differentiation. Further, the induction of chronic kidney disease with high-turnover renal osteodystrophy led to downregulation of Klotho in bone cells. This appeared to offset the skeletal impact of osteocyte-targeted Klotho deletion. Thus, our findings establish a key role of osteocyte-expressed Klotho in regulating bone metabolism and indicate a new mechanism by which osteocytes control bone formation.

Tissue expression and source of circulating αKlotho.

αKlotho (Klotho), a type I transmembrane protein and a coreceptor for Fibroblast Growth Factor-23, was initially thought to be expressed only in a limited number of tissues, most importantly the kidney, parathyroid gland and choroid plexus. Emerging data may suggest a more ubiquitous Klotho expression pattern which has prompted reevaluation of the restricted Klotho paradigm. Herein we systematically review the evidence for Klotho expression in various tissues and cell types in humans and other mammals, and discuss potential reasons behind existing conflicting data. Based on current literature and tissue expression atlases, we propose a classification of tissues into high, intermediate and low/absent Klotho expression. The functional relevance of Klotho in organs with low expression levels remain uncertain and there is currently limited data on a role for membrane-bound Klotho outside the kidney. Finally, we review the evidence for the tissue source of soluble Klotho, and conclude that the kidney is likely to be the principal source of circulating Klotho in physiology.

Klotho expression in long bones regulates FGF23 production during renal failure.

Circulating levels of bone-derived fibroblast growth factor 23 (FGF23) increase early during acute and chronic kidney disease and are associated with adverse outcomes. Membrane-bound Klotho acts as a permissive coreceptor for FGF23, and its expression was recently found in osteoblasts/osteocytes. We hypothesized that Klotho in bone cells is part of an autocrine feedback loop that regulates FGF23 expression during renal failure. Thus, we induced renal failure in mice with targeted deletion of Klotho in long bones. Uremic wild-type (KLfl/fl ) and knockout (Prx1-Cre;KLfl/fl ) mice both responded with reduced body weight, kidney atrophy, hyperphosphatemia, and increased bone turnover. Importantly, long bones of Prx1-Cre;KLfl/fl mice but not their axial skeleton failed to increase FGF23 expression as observed in uremic KLfl/fl mice. Consequently, Prx1-Cre;KLfl/fl mice had significantly lower serum FGF23 and parathyroid hormone levels, and higher renal 1-α-hydroxylase expression, serum 1,25-dihydroxyvitamin D, and calcium levels than KLfl/fl mice. These results were confirmed in two independent models of renal failure, adenine diet induced and 5/6 nephrectomy. Moreover, FGF23-treated bone cells required Klotho to increase FGF23 mRNA and ERK phosphorylation. In summary, our novel findings show that Klotho in bone is crucial for inducing FGF23 production upon renal failure. We propose the presence of an autocrine feedback loop in which Klotho senses the need for FGF23.-Kaludjerovic, J., Komaba, H., Sato, T., Erben, R. G., Baron, R., Olauson, H., Larsson, T. E., Lanske, B. Klotho expression in long bones regulates FGF23 production during renal failure.

In vivo evidence for a limited role of proximal tubular Klotho in renal phosphate handling.

Klotho is a transmembrane protein expressed in the renal tubules where it acts as a permissive coreceptor for fibroblast growth factor 23 (FGF23). FGF23 signaling reduces the abundance of CYP27b1 and phosphate cotransporters NPT2a and NPT2c, leading to a decrease in 1,25(OH)2D3 synthesis and a rise in urinary phosphate excretion, respectively. Systemic or whole-nephron deletion of Klotho in mice results in renal FGF23 resistance characterized by high 1,25(OH)2D3 and phosphate levels and premature aging. Expression of Klotho is highest in the distal tubules, whereas 25OH vitamin D 1α hydroxylation and phosphate reabsorption predominantly occur in the proximal tubules. Currently, the segment-specific roles of Klotho in renal tubules are not fully understood. Here we have generated mice with Klotho specifically ablated from the proximal tubules using 3 different Cre mouse strains. All 3 models displayed impaired urinary phosphate excretion and increased abundance of NPT2a in the brush border membrane. Notably, hyperphosphatemia in knockout mice was mild or nonexistent under basal conditions but occurred upon high phosphate loading, indicating the presence of compensatory mechanisms. Effects on 1,25(OH)2D3 varied between mouse strains but were modest overall. Thus, Klotho expressed in the proximal tubules has a defined but limited role in renal phosphate handling in vivo.

FGF-23 and Osteoprotegerin but not Fetuin-A are associated with death and enhance risk prediction in non-dialysis chronic kidney disease stages 3-5.

AIM: Numerous biomarkers have been shown to associate with clinical endpoints in chronic kidney disease (CKD). There is limited evidence whether biomarkers improve risk prediction in relation to clinical outcomes. Our study investigates whether a small suite of key chronic kidney disease-mineral and bone disorder biomarkers could be used to enhance risk assessment in CKD. METHODS: Fetuin-A, fibroblast growth factor-23 and osteoprotegerin were measured on baseline plasma samples from 463 patients recruited to the Chronic Renal Insufficiency Standards Implementation Study. The biomarkers were analysed in relation to progression to end stage kidney disease, death and major cardiovascular events. RESULTS: Over a median follow up of 46 months (interquartile range 21-69), fibroblast growth factor-23 was associated with risk for renal replacement therapy (hazard ratio (HR) 1.35, P = 0.05, 95% confidence interval (CI) 1.001-1.820), cardiovascular events (HR 1.74 P < 0.001, 95% CI 1.303-1.305) and death (HR 1.4 P = 0.005, 95% CI 1.109-1.767). Osteoprotegerin was associated with risk for death (HR 1.06, P = 0.03, 95% CI 1.006-1.117). There was no clear association between Fetuin-A and any of the clinical endpoints. The addition of biomarkers to risk models led to marginal improvement in model discrimination and reclassification. CONCLUSION: Biomarkers are often associated with clinical endpoints, and we observed such associations in our study of patients with advanced CKD. However, the markers analysed in our study were of limited benefit in improving the prediction of these outcomes. Any extra information biomarkers may provide to improve risk prediction in clinical practice needs to be carefully balanced against the potential cost of these tools.