Propagation, Culture, and Characterization of Renal Fibroblasts.

The renal fibroblast, and phenotypically related myofibroblast, are universally present in all forms of progressive kidney disease. The in vitro study of the fibroblast, its behaviour, and factors affecting its activity is therefore key to understanding both its role and significance. In this protocol, we describe a reproducible method for selective propagation and culture of primary renal fibroblasts from kidney cortex. Techniques for their isolation, subculture, characterization, and cryogenic storage and retrieval are described in detail.

Isolation of Rat Glomeruli and Propagation of Mesangial Cells to Study the Kidney in Health and Disease.

Whole organ molecular analysis of the kidney potentially misses important factors involved in the pathogenesis of the glomerular disease. Organ-wide analysis therefore needs to be augmented by techniques that isolate enriched populations of glomeruli. Herein, we describe how differential sieving can be used to isolate a suspension of rat glomeruli from fresh tissue. Secondly, we also show how these can be used for the propagation of primary mesangial cell cultures. These protocols provide a practical approach for protein and RNA isolation for downstream analysis. These techniques are readily applicable to studies in isolated glomeruli in both experimental animal models and human kidney tissue.

Magnetic Resonance Imaging (MRI) Analysis of Tissue Sodium Concentration in Chronic Kidney Disease.

Human body sodium is regulated by the kidneys and extrarenal mechanisms. Stored skin and muscle tissue sodium accumulation is associated with kidney function decline, hypertension, and a pro-inflammatory and cardiovascular disease profile. In this chapter, we describe the use of sodium-hydrogen magnetic resonance imaging (23Na/1H MRI) to dynamically quantify tissue sodium concentration in the lower limb of humans. Real-time quantification of tissue sodium is calibrated against known sodium chloride aqueous concentrations. This method may be useful for investigating in vivo (patho-)physiological conditions associated with tissue sodium deposition and metabolism (including in relation to water regulation) to enlighten our understanding of sodium physiology.

Effect of the phosphate binder sucroferric oxyhydroxide in dialysis patients on endogenous calciprotein particles, inflammation, and vascular cells.

BACKGROUND: Calciprotein particles (CPPs), colloidal mineral-protein nanoparticles, have emerged as potential mediators of phosphate toxicity in dialysis patients, with putative links to vascular calcification, endothelial dysfunction and inflammation. We hypothesized that phosphate binder therapy with sucroferric oxyhydroxide (SO) would reduce endogenous CPP levels and attenuate pro-calcific and pro-inflammatory effects of patient serum towards human vascular cells in vitro. METHODS: This secondary analysis of a randomised controlled crossover study compared the effect of 2-week phosphate binder washout with high-dose (2000 mg/day) and low-dose (250 mg/day) SO therapy in 28 haemodialysis patients on serum CPP levels, inflammatory cytokine/chemokine arrays and human aortic smooth muscle cell (HASMC) and coronary artery endothelial cell (HCAEC) bioassays. RESULTS: In our cohort (75% male, 62 ± 12 years) high-dose SO reduced primary (amorphous) and secondary (crystalline) CPP levels {-62% [95% confidence interval (CI) -76 to -44], P < .0001 and -38% [-62 to -0.14], P < .001, respectively} compared with washout. Nine of 14 plasma cytokines/chemokines significantly decreased with high-dose SO, with consistent reductions in interleukin-6 (IL-6) and IL-8. Exposure of HASMC and HCAEC cultures to serum of SO-treated patients reduced calcification and markers of activation (IL-6, IL-8 and vascular cell adhesion protein 1) compared with washout. Serum-induced HASMC calcification and HCAEC activation was ameliorated by removal of the CPP-containing fraction from patient sera. Effects of CPP removal were confirmed in an independent cohort of chronic kidney disease patients. CONCLUSIONS: High-dose SO reduced endogenous CPP formation in dialysis patients and yielded serum with attenuated pro-calcific and inflammatory effects in vitro.

Effect of lanthanum carbonate on serum calciprotein particles in patients with stage 3-4 CKD-results from a placebo-controlled randomized trial.

BACKGROUND: Calciprotein particles (CPP) are colloidal aggregates of calcium phosphate and the mineral-binding protein fetuin-A, and are potential mediators of cardiovascular disease in chronic kidney disease (CKD). Emerging evidence suggests non-calcium-containing phosphate binders may reduce serum CPP in patients with kidney failure who require dialysis; however, it is unclear whether similar interventions are effective in patients with earlier stages of CKD. METHODS: The IMpact of Phosphate Reduction On Vascular End-points in CKD (IMPROVE-CKD) was a multi-centre, placebo-controlled, randomized trial of lanthanum carbonate on cardiovascular markers in 278 participants with stage 3b/4 CKD. In this pre-specified exploratory analysis, primary (CPP-I) and secondary CPP (CPP-II) were measured in a sub-cohort of participants over 96 weeks. Treatment groups were compared using linear mixed-effects models and the relationship between serum CPP and pulse wave velocity (PWV) and abdominal aortic calcification (AAC) was examined. RESULTS: A total of 253 participants had CPP data for baseline and at least one follow-up timepoint and were included in this analysis. The mean age was 62.4 ± 12.6 years, 32.0% were female and the mean estimated glomerular filtration rate (eGFR) was 26.6 ± 8.3 mL/min/1.73 m2. Baseline median serum CPP-I was 14.9 × 104 particles/mL [interquartile range (IQR) 4.6-49.3] and median CPP-II was 3.3 × 103 particles/mL (IQR 1.4-5.4). There was no significant difference between treatment groups at 96 weeks in CPP-I [22.8% (95% confidence interval -39.2, 36.4), P = 0.65] or CPP-II [-18.3% (95% confidence interval -40.0, 11.2), P = 0.20] compared with a placebo. Serum CPP were not correlated with baseline PWV or AAC, or with the progression of either marker. CONCLUSIONS: Lanthanum carbonate was not associated with a reduction of CPP at 96 weeks when compared with a placebo in a CKD cohort.

Effect of a medium cut-off dialyzer on protein-bound uremic toxins and mineral metabolism markers in patients on hemodialysis.

INTRODUCTION: Hemodialysis (HD) with medium cut-off (MCO) dialyzers may expand molecular clearance, predominantly larger middle molecules (molecular weight 25-60 kDa). However, the impact of MCO dialyzers on long-term clearance of various other components of the uremic milieu is unknown. The tRial Evaluating Mid cut-Off Value membrane clearance of Albumin and Light chains in HemoDialysis patients (REMOVAL-HD) provided an opportunity to assess the effect of MCO dialyzers on protein-bound uremic toxins and novel markers of mineral metabolism. METHODS: This exploratory sub-study of REMOVAL-HD evaluated changes in protein-bound solutes (total and free indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) and mineral metabolism markers (intact fibroblast growth factor-23 [iFGF23], fetuin-A and endogenous calciprotein particles [CPP-1 and CPP-2]). Mid-week, pre-HD serum samples were collected at baseline and after 12 and 24 weeks of MCO use in stable adult patients. Change from baseline to Week 12 and 24 was estimated using linear mixed effects models. FINDINGS: Eighty-nine participants were studied (mean age 67 ± 15 years, 38% female, 51% diabetic, median urine output 200 ml/24 h). Serum iFGF23 was reduced at Week 12 compared to baseline (-26.8% [95%CI -39.7, -11.1], p = 0.001), which was sustained at Week 24 (-21.7% [95%CI -35.7, -4.5], p = 0.012). There was no significant change in serum IS, PCS, fetuin-A, CPP-1, or CPP-2. DISCUSSION: The use of a MCO dialyzer over 24 weeks was associated with a sustained reduction in FGF23, while other measured components of the uremic milieu were not significantly altered. Further studies are required to determine whether FGF23 reduction is associated with improved patient outcomes.

Effect of Sevelamer on Calciprotein Particles in Hemodialysis Patients: The Sevelamer Versus Calcium to Reduce Fetuin-A-Containing Calciprotein Particles in Dialysis (SCaRF) Randomized Controlled Trial.

INTRODUCTION: Calciprotein particles (CPPs) are potentially modifiable mediators of phosphate toxicity in patients with kidney disease. We compared the effects of calcium carbonate (CC) and the non-calcium-based phosphate binder sevelamer on CPP levels in patients undergoing hemodialysis (HD). We hypothesized that treatment with sevelamer would achieve greater reductions in amorphous calcium phosphate-containing CPP (CPP-1) and hydroxyapatite-containing CPP (CPP-2) owing to reduced calcium loading and anti-inflammatory pleiotropic effects. METHODS: We conducted an open-label, randomized controlled trial (RCT) in which 31 stable prevalent HD patients were allocated to receive either sevelamer hydrochloride (SH), sevelamer carbonate (SC), or CC for 24 weeks. Dual primary endpoints were the between groups differences in serum CPP-1 and CPP-2 levels at 24 weeks in SH + SC-treated versus CC-treated patients. Effects on aortic pulse wave velocity (aPWV), inflammatory cytokines (interleukin-6 and -8), and effects across individual treatment arms were also assessed. RESULTS: Serum CPP-1, but not CPP-2, levels were lower in those randomly assigned to the sevelamer (SH + SC) group compared with the CC group at 24 weeks (-70%, 95% confidence interval [CI] -90% to -15%, P = 0.02). In subgroup analysis, this effect was confined to those receiving SC (-83.4%, 95% CI -95.7% to -36.8%, P = 0.01). aPWV and interleukin-8 levels were also lower in those who received sevelamer compared with CC at 24 weeks (-2.0 m/s, 95% CI -2.9 to -1.1; -57%, 95% CI -73% to -30%, respectively, both P = 0.01). Conventional markers of mineral metabolism remained stable across all treatment groups. DISCUSSION: Compared with treatment with CC, use of sevelamer for 24 weeks was associated with lower serum CPP-1 levels and a reduction in aPWV and systemic inflammation.

Vascular calcification in skin and subcutaneous tissue in patients with chronic and end-stage kidney disease.

BACKGROUND: Vascular calcification (VC) is well described in large- and medium-sized vessels in patients with chronic kidney disease (CKD), especially in those with end-stage kidney disease (ESKD) on dialysis. Medial calcification is particularly prevalent in this population and contributes to arterial stiffness and increased cardiovascular mortality and morbidity. Apart from in the setting of calciphylaxis, few studies have assessed skin and subcutaneous calcification and associations with abnormalities of bone and mineral metabolism in patients with CKD. METHODS: We performed a single-centre observational study to evaluate incisional skin tissue samples from three anatomical sites in patients with different stages of CKD undergoing elective surgery. We compared these samples to skin samples of a control cohort without CKD. Staining for calcification was performed with von Kossa method. A subgroup of skin samples were assessed by RT-PCR for upregulation of pro-calcific gene transcripts for tissue non-specific alkaline phosphatase (TNAP) and Runt-related transcription factor 2 (RUNX2). RESULTS: Forty-five patients were evaluated, 34 with CKD (including ESKD) and 11 control patients. VC was identified in 15 skin samples (13 CKD/ESKD and 2 controls). VC was present in the dermal and subcutaneous tissues of the neck, abdomen and arm samples. Two different histological types of VC were identified: speckled medial calcification and internal elastic lamina calcification. Presence of perieccrine calcification was identified in 14 samples, 10 with concurrent VC. There were no significant differences in serum parathyroid hormone, phosphate or calcium in patients with or without VC. Expression of TNAP or RUNX2 was not increased in samples from patients with ESKD or those with histological evidence of calcification. CONCLUSION: This study reports the novel finding of dermal and subcutaneous calcification in multiple anatomical locations in 38% of patients with advanced CKD/ESKD undergoing elective surgery but free from calciphylaxis.

Calciprotein particles: mineral behaving badly?

PURPOSE OF REVIEW: Calciprotein particles (CPP) are formed in supersaturated solutions of calcium, phosphate and the mineral-binding protein fetuin-A. CPP have garnered considerable interest as potential mediators of mineral stress, but little consideration has been given to their origin, clearance and role in metabolism. RECENT FINDINGS: CPP are made whilst buffering the mineral absorbed from the intestine after a meal or during remodelling of bone matrix. The postprandial rise in circulating CPP rise may be sensed by osteoblasts/osteocytes in bone, stimulating the secretion of the master phosphatonin fibroblast growth factor 23. Amorphous calcium phosphate-containing CPP are rapidly cleared by endothelial cells in the liver whereas crystalline apatite-containing CPP are filtered by phagocytic cells of the reticuloendothelial system. Impaired excretory function in kidney disease may lead to accumulation of CPP and its precursors with possible pathological sequalae. Inability to stabilize CPP in fetuin-A-deficiency states can result in intraluminal precipitation and inflammatory cascades if other mineralisation regulatory networks are compromised. SUMMARY: CPP allow efficient transport and clearance of bulk calcium phosphate as colloids without risk of precipitation. As circulating factors, CPP may couple dietary mineral exposure with endocrine control of mineral metabolism in bone, signalling the need to dispose of excess phosphate from the body.

Monitoring skin temperature at the wrist in hospitalised patients may assist in the detection of infection.

BACKGROUND: Measuring temperature has always been a key observation in the diagnosis of infection. No studies have examined the usefulness of measuring temperature at the wrist to detect infection. AIM: We sought to determine whether a watch measuring wrist temperature could accurately identify patients who are infected. METHODS: Prospective cross-sectional pilot study of temperature monitoring in an unselected patients in a tertiary referral adult nephrology unit. RESULTS: One hundred and four data recording sessions revealed 88 useful data sets, with recording failures in the others. Patients were retrospectively classified as having no infection (Group A, n = 60), clinically diagnosed infection with less than 24 h of treatment with antibiotics (Group B, n = 5), and clinically diagnosed infection with greater than 24 h on antibiotics (Group C, n = 23). There was a significantly higher average maximum temperature in Group B (mean (SEM)) 38°C (0.6) compared with Groups A (36.1°C (0.1)) and C (36.3°C (0.3)). Based on receiver operating characteristics (ROC) a cut-off temperature of ≥37.5°C gave sensitivity 80% and specificity 98%. Mean electrodermal activity was significantly higher in Groups B and C. CONCLUSIONS: ROC of peripheral skin temperature measurements suggest that such a device may identify many patients requiring treatment for infection. This proof of principle study showed value in using a wearable device in the detection of infection and its potential as an early warning or monitoring device.

Diagnostic Tests for Vascular Calcification.

Vascular calcification (VC) is the heterogeneous endpoint of multiple vascular insults, which varies by arterial bed, the layer of the arterial wall affected, and is propagated by diverse cellular and biochemical mechanisms. A variety of in vivo and ex vivo techniques have been applied to the analysis of VC in preclinical studies, but clinical examination has principally relied on a number of noninvasive and invasive imaging modalities for detection and quantitation. Most imaging methods suffer from suboptimal spatial resolution, leading to the inability to distinguish medial from intimal VC and insufficient sensitivity to detect microcalcifications that are indicative of active mineral deposition and of vulnerable plaques which may be prone to rupture. Serum biomarkers lack specificity for VC and cannot discriminate pathology. Overall, uncertainties surrounding the sensitivity and specificity of different VC testing modalities, the absence of a clear cause-effect relationship, and lack of any evidence-based diagnostic or therapeutic protocols in relation to VC testing in chronic kidney disease has yielded weak or ungraded recommendations for their use in clinical practice. While VC is recognized as a key manifestation of chronic kidney disease-mineral and bone disorder and those with an increasing burden of VC are considered to be at higher cardiovascular risk, routine screening is not currently recommended.

AT1R-AT2R-RXFP1 Functional Crosstalk in Myofibroblasts: Impact on the Therapeutic Targeting of Renal and Cardiac Fibrosis.

BACKGROUND: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo. Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT1R), which is expressed on myofibroblasts along with RXFP1 and AT2R, is unknown. METHODS: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal- or cardiomyopathy-induced fibrosis in vivo. RESULTS: The AT1R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo. Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT1Rs but that constitutive AT1R-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT2R agonist (compound 21). CONCLUSIONS: These findings have significant implications for the concomitant use of RXFP1 or AT2R agonists with AT1R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.

αKlotho-FGF23 interactions and their role in kidney disease: a molecular insight.

Following the serendipitous discovery of the ageing suppressor, αKlotho (αKl), several decades ago, a growing body of evidence has defined a pivotal role for its various forms in multiple aspects of vertebrate physiology and pathology. The transmembrane form of αKl serves as a co-receptor for the osteocyte-derived mineral regulator, fibroblast growth factor (FGF)23, principally in the renal tubules. However, compelling data also suggest that circulating soluble forms of αKl, derived from the same source, may have independent homeostatic functions either as a hormone, glycan-cleaving enzyme or lectin. Chronic kidney disease (CKD) is of particular interest as disruption of the FGF23-αKl axis is an early and common feature of disease manifesting in markedly deficient αKl expression, but FGF23 excess. Here we critically discuss recent findings in αKl biology that conflict with the view that soluble αKl has substantive functions independent of FGF23 signalling. Although the issue of whether soluble αKl can act without FGF23 has yet to be resolved, we explore the potential significance of these contrary findings in the context of CKD and highlight how this endocrine pathway represents a promising target for novel anti-ageing therapeutics.

High-intensity physical exercise increases serum α-klotho levels in healthy volunteers.

The recently discovered klotho proteins have roles in a diverse range of metabolic processes with the oldest protein, α-klotho, implicated in various cellular pathways in energy, glucose, and phosphate metabolism. Circulating soluble klotho (sKl), derived from membrane α-klotho cleavage, not only has effects on ion channels and insulin signaling pathways, but is inversely associated with mortality. Effects of physical exercise on sKl have not been well studied. The effect of a single high-intensity standardized exercise on sKl and serum phosphate (sPi) levels in healthy adults was investigated. A standard Bruce protocol treadmill exercise was undertaken by 10 fasting healthy volunteers. sKl, sPi, and blood glucose levels were measured in samples collected 1-week prior, immediately pre (Tpre), 0 (Tpost), 30 (T30), 240 (T240) min, and 1-week after exercise. Median (interquartile range) age of participants was 47.5 (44-51) years; five (50%) were male. All study participants achieved at least 90% predicted maximum heart rate (MHR). sKl increased acutely after exercise (Tpre median 448 pg/mL vs. Tpost median 576 pg/mL; p < 0.01). There was a nonsignificant sPi decline at T30 (Tpre 0.94 ± 0.12 mmol/L vs. T30 0.83 ± 0.22 mmol/L). Exercise led to a reduction in blood glucose by T240 with median glucose levels at Tpre, Tpost, T30, and T240 of 6.0, 6.5, 6.3, and 5.7 mmol/L, respectively. In conclusion, a single high-intensity exercise session is associated with a transient increase in sKl, a delayed reduction in blood glucose, and a nonsignificant decrease in sPi levels in healthy adults. The evaluation of long-term effects of cardiovascular fitness programs on sKl and sPi in healthy individuals and disease cohorts are required to identify potential lifestyle modifications to help improve chronic disease management and long-term outcomes.

Longitudinal changes in bone and mineral metabolism after cessation of cinacalcet in dialysis patients with secondary hyperparathyroidism.

BACKGROUND: The calcimimetic agent cinacalcet is effective for the management of secondary hyperparathyroidism (SHPT) in dialysis patients. Changes to reimbursement of cinacalcet in Australia provided an opportunity to assess effects of medication cessation on biochemical and clinical outcomes in dialysis patients, including changes to novel biomarkers such as calciprotein particles (CPP). CPP are nanoparticles of mineral and protein in the circulation associated with increased vascular calcification in patients with chronic kidney disease. METHODS: Dialysis patients from a single center who ceased cinacalcet between August 2015 and March 2016 were included in a prospective observational study. Bloods were taken at the time of cessation of cinacalcet and at 1, 6 and 12 months. Clinical and biochemical outcomes were compared with an age- and gender-matched cohort of cinacalcet-naïve dialysis patients. RESULTS: Sixty-two patients participated in the study. Mean age was 69.6 ± 13.2 years. Biochemical changes over 12 months following cessation of cinacalcet included an increase in serum parathyroid hormone (PTH) (42.2 [IQR 27.8-94.6] pmol/L to 114.8 [83.9-159.1] pmol/L [p < 0.001]), serum calcium (2.31 ± 0.21 mmol/L to 2.46 ± 0.14 mmol/L [p < 0.001]) and primary CPP (CPP-I) (p = 0.002). Changes in CPP were associated with an increase in PTH (p = 0.007), calcium (p = 0.002) and ferritin (p = 0.02) but a reduction in serum albumin (p = 0.001). Over the 12-month period, there were two fractures, five cardiovascular events, one episode of calciphylaxis, and one parathyroidectomy, with a mortality rate of 19% (n = 13). CONCLUSION: Uniquely we report the effects of cinacalcet withdrawal in a real world setting with demonstrated increases in PTH, serum calcium and CPP subsets, novel CKD-MBD related factors, over a 12-month period.

Current and potential therapeutic strategies for the management of vascular calcification in patients with chronic kidney disease including those on dialysis.

Patients with CKD have accelerated vascular stiffening contributing significantly to excess cardiovascular morbidity and mortality. Much of the arterial stiffening is thought to involve vascular calcification (VC), but the pathogenesis of this phenomenon is complex, resulting from a disruption of the balance between promoters and inhibitors of calcification in a uremic milieu, along with derangements in calcium and phosphate metabolic pathways. Management of traditional cardiovascular risk factors to reduce VC may be influential but has not been shown to significantly improve mortality. Control of mineral metabolism may potentially reduce the burden of VC, although using conventional approaches of restricting dietary phosphate, administering phosphate binders, and use of active vitamin D and calcimimetics, remains controversial because recommended biochemical targets are hard to achieve and clinical relevance hard to define. Increasing time on dialysis is perhaps another therapy with potential effectiveness in this area. Despite current treatments, cardiovascular morbidity and mortality remain high in this group. Novel therapies for addressing VC include magnesium and vitamin K supplementation, which are currently being investigated in large randomized control trials. Other therapeutic targets include crystallization inhibitors, ligand trap for activin receptors and BMP-7. This review summarizes current treatment strategies and therapeutic targets for the future management of VC in patients with CKD.

Biochemical transformation of calciprotein particles in uraemia.

Calciprotein particles (CPP) have emerged as nanoscale mediators of phosphate-induced toxicity in Chronic Kidney Disease (CKD). Uraemia favors ripening of the particle mineral content from the amorphous (CPP-I) to the crystalline state (CPP-II) but the pathophysiological significance of this transformation is uncertain. Clinical studies suggest an association between CPP ripening and inflammation, vascular dysfunction and mortality. Although ripening has been modelled in vitro, it is unknown whether particles synthesised in serum resemble their in vivo counterparts. Here we show that in vitro formation and ripening of CPP in uraemic serum is characterised by extensive physiochemical rearrangements involving the accretion of mineral, loss of surface charge and transformation of the mineral phase from a spherical arrangement of diffuse domains of amorphous calcium phosphate to densely-packed lamellar aggregates of crystalline hydroxyapatite. These physiochemical changes were paralleled by enrichment with small soluble apolipoproteins, complement factors and the binding of fatty acids. In comparison, endogenous CPP represent a highly heterogeneous mixture of particles with characteristics mostly intermediate to synthetic CPP-I and CPP-II, but are also uniquely enriched for carbonate-substituted apatite, DNA fragments, small RNA and microbe-derived components. Pathway analysis of protein enrichment predicted the activation of cell death and pro-inflammatory processes by endogenous CPP and synthetic CPP-II alike. This comprehensive characterisation validates the use of CPP-II generated in uraemic serum as in vitro equivalents of their endogenous counterparts and provides insight into the nature and pathological significance of CPP in CKD, which may act as vehicles for various bioactive ligands.

FGF23 activates injury-primed renal fibroblasts via FGFR4-dependent signalling and enhancement of TGF-ß autoinduction.

Bone-derived fibroblast growth factor 23 (FGF23) is an important endocrine regulator of mineral homeostasis with effects transduced by cognate FGF receptor (FGFR)1-α-Klotho complexes. Circulating FGF23 levels rise precipitously in patients with kidney disease and portend worse renal and cardiovascular outcomes. De novo expression of FGF23 has been found in the heart and kidney following injury but its significance remains unclear. Studies showing that exposure to chronically high FGF23 concentrations activates hypertrophic gene programs in the cardiomyocyte has spawned intense interest in other pathological off-target effects of FGF23 excess. In the kidney, observational evidence points to a concordance of ectopic renal FGF23 expression and the activation of local transforming growth factor (TGF)-ß signalling. Although we have previously shown that FGF23 activates injury-primed renal fibroblasts in vitro, our understanding of the mechanism underpinning these effects was incomplete. Here we show that in the absence of α-Klotho, FGF23 augments pro-fibrotic signalling cascades in injury-primed renal fibroblasts via activation of FGFR4 and upregulation of the calcium transporter, transient receptor potential cation channel 6. The resultant rise in intracellular calcium and production of mitochondrial reactive oxygen species induced expression of NFAT responsive-genes and enhanced TGF-ß1 autoinduction through non-canonical JNK-dependent pathways. Reconstitution with transmembrane α-Klotho, or its soluble ectodomain, restored classical Egr signalling and antagonised FGF23-driven myofibroblast differentiation. Thus, renal FGF23 may amplify local myofibroblast activation in injury and perpetuate pro-fibrotic signalling. These findings strengthen the rationale for exploring therapeutic inhibition of FGFR4 or restoration of α-Klotho as upstream regulators of off-target FGF23 effects.