Publications by authors named "Tim D Hewitson"

75 Publications

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

Hemodial Int 2021 Mar 28. Epub 2021 Mar 28.

Department of Nephrology, The Royal Melbourne Hospital, Parkville, Australia.

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.
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http://dx.doi.org/10.1111/hdi.12924DOI Listing
March 2021

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.

Kidney Int Rep 2020 Sep 29;5(9):1432-1447. Epub 2020 Jun 29.

Department of Nephrology, The Royal Melbourne Hospital, Parkville, Victoria, Australia.

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%,  = 0.02). In subgroup analysis, this effect was confined to those receiving SC (-83.4%, 95% CI -95.7% to -36.8%,  = 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  = 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.
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http://dx.doi.org/10.1016/j.ekir.2020.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486191PMC
September 2020

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

BMC Nephrol 2020 07 16;21(1):279. Epub 2020 Jul 16.

Department of Nephrology, The Royal Melbourne Hospital, 300 Grattan St, Parkville, Victoria, 3050, Australia.

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.
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http://dx.doi.org/10.1186/s12882-020-01928-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7364566PMC
July 2020

Calciprotein particles: mineral behaving badly?

Curr Opin Nephrol Hypertens 2020 07;29(4):378-386

Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Hospital, Aachen, Germany.

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.
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http://dx.doi.org/10.1097/MNH.0000000000000609DOI Listing
July 2020

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

Intern Med J 2020 06 5;50(6):685-690. Epub 2020 May 5.

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Victoria, Australia.

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.
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http://dx.doi.org/10.1111/imj.14748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318648PMC
June 2020

Diagnostic Tests for Vascular Calcification.

Adv Chronic Kidney Dis 2019 11;26(6):445-463

Department of Nephrology, The Royal Melbourne Hospital, Parkville, VIC, Australia; Department of Medicine, University of Melbourne, Parkville, VIC, Australia.

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.
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http://dx.doi.org/10.1053/j.ackd.2019.07.001DOI Listing
November 2019

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

J Am Soc Nephrol 2019 11 11;30(11):2191-2207. Epub 2019 Sep 11.

Department of Biochemistry and Molecular Biology, and

Background: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated 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 (ATR) to ameliorate renal fibrogenesis and . Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (ATR), which is expressed on myofibroblasts along with RXFP1 and ATR, is unknown.

Methods: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts , and in three models of renal- or cardiomyopathy-induced fibrosis .

Results: The ATR blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin RXFP1 and . 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 ATRs but that constitutive ATR-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 ATR agonist (compound 21).

Conclusions: These findings have significant implications for the concomitant use of RXFP1 or ATR agonists with ATR blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.
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http://dx.doi.org/10.1681/ASN.2019060597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830801PMC
November 2019

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

Cell Mol Life Sci 2019 Dec 26;76(23):4705-4724. Epub 2019 Jul 26.

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Australia.

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.
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http://dx.doi.org/10.1007/s00018-019-03241-yDOI Listing
December 2019

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

J Circ Biomark 2018 Jan-Dec;7:1849454418794582. Epub 2018 Aug 16.

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, VIC, Australia.

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 (), 0 (), 30 (), 240 () 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 ( median 448 pg/mL vs. median 576 pg/mL; < 0.01). There was a nonsignificant sPi decline at ( 0.94 ± 0.12 mmol/L vs. 0.83 ± 0.22 mmol/L). Exercise led to a reduction in blood glucose by with median glucose levels at , , , and 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.
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http://dx.doi.org/10.1177/1849454418794582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100126PMC
August 2018

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

BMC Nephrol 2018 05 15;19(1):113. Epub 2018 May 15.

Department of Nephrology, The Royal Melbourne Hospital, 300 Grattan St, Parkville, Victoria, 3050, Australia.

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.
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http://dx.doi.org/10.1186/s12882-018-0910-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5952622PMC
May 2018

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

Semin Dial 2018 09 7;31(5):487-499. Epub 2018 May 7.

Department of Nephrology, The Royal Melbourne Hospital, Parkville, Vic., Australia.

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.
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http://dx.doi.org/10.1111/sdi.12710DOI Listing
September 2018

Biochemical transformation of calciprotein particles in uraemia.

Bone 2018 05 27;110:355-367. Epub 2018 Feb 27.

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Victoria, Australia; Department of Medicine - Royal Melbourne Hospital, University of Melbourne, Melbourne, Victoria, Australia.

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.
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http://dx.doi.org/10.1016/j.bone.2018.02.023DOI Listing
May 2018

Fetuin-A in the peritoneal effluent of patients with encapsulating peritoneal sclerosis-more than a protein?

Kidney Int 2017 11;92(5):1289-1290

Department of Nephrology, The Royal Melbourne Hospital, Parkville, Victoria, Australia; Department of Medicine-Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria, Australia.

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http://dx.doi.org/10.1016/j.kint.2017.07.028DOI Listing
November 2017

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

Int J Biochem Cell Biol 2017 11 15;92:63-78. Epub 2017 Sep 15.

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Victoria, Australia; Department of Medicine, The Royal Melbourne Hospital, University of Melbourne, Melbourne, Victoria, Australia.

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)-β 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-β1 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.
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http://dx.doi.org/10.1016/j.biocel.2017.09.009DOI Listing
November 2017

A novel fluorescent probe-based flow cytometric assay for mineral-containing nanoparticles in serum.

Sci Rep 2017 07 18;7(1):5686. Epub 2017 Jul 18.

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Victoria, Australia.

Calciprotein particles, nanoscale aggregates of insoluble mineral and binding proteins, have emerged as potential mediators of phosphate toxicity in patients with Chronic Kidney Disease. Although existing immunochemical methods for their detection have provided compelling data, these approaches are indirect, lack specificity and are subject to a number of other technical and theoretical shortcomings. Here we have developed a rapid homogeneous fluorescent probe-based flow cytometric method for the detection and quantitation of individual mineral-containing nanoparticles in human and animal serum. This method allows the discrimination of membrane-bound from membrane-free particles and different mineral phases (amorphous vs. crystalline). Critically, the method has been optimised for use on a conventional instrument, without the need for manual hardware adjustments. Using this method, we demonstrate a consistency in findings across studies of Chronic Kidney Disease patients and commonly used uraemic animal models. These studies demonstrate that renal dysfunction is associated with the ripening of calciprotein particles to the crystalline state and reveal bone metabolism and dietary mineral as important modulators of circulating levels. Flow cytometric analysis of calciprotein particles may enhance our understanding of mineral handling in kidney disease and provide a novel indicator of therapeutic efficacy for interventions targeting Chronic Kidney Disease-Mineral Bone Disorder.
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http://dx.doi.org/10.1038/s41598-017-05474-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515983PMC
July 2017

Soluble klotho may be a marker of phosphate reabsorption.

Clin Kidney J 2017 Jun 28;10(3):397-404. Epub 2017 Feb 28.

Department of Nephrology, Royal Melbourne Hospital, Parkville, Australia.

Membrane-bound α-klotho functions as a co-receptor with fibroblast growth factor receptor at the renal tubule conferring specificity to fibroblast growth factor-23 (FGF-23), allowing it to inhibit tubular phosphate reabsorption at physiological concentrations. α-klotho also exists as a soluble protein. However, the complex interrelationships between soluble α-klotho (sKl), FGF-23 and phosphate reabsorption are poorly understood, with little known about the links between sKl, FGF-23 and phosphate reabsorption in chronic kidney disease (CKD). This study addresses this issue in a cohort of patients with and without CKD. We conducted a single-centre, cross-sectional study of contemporaneously obtained samples of blood and 24-h urine biochemistry along with sKl and intact FGF-23 (iFGF-23) from non-dialysis-dependent CKD patients and healthy volunteers. Pearson's correlation coefficients were used to determine correlations between natural log-transformed (Ln) sKl and iFGF-23 with other parameters of interest. Backward multivariate analysis was undertaken to evaluate the relationship between mineral parameters. One hundred and sixteen participants (77 with CKD and 39 healthy volunteers) were studied, of which 74 (63.8%) were male. The median age was 61 (interquartile range 49-71) years. Those with CKD had lower sKl (408 versus 542 pg/mL), higher iFGF-23 (94 versus 41 pg/mL), higher fractional excretion of phosphate (25.05 versus 10.98%) and lower daily urinary phosphate excretion (UPE) (24.8 versus 32.3 mmol/L) compared with healthy volunteers (all P 0.002). Age correlated inversely and estimated glomerular filtration rate (eGFR) correlated positively with phosphate reabsorption and Ln(sKl), while the opposite was seen with Ln(iFGF23). Upon multivariate analysis, eGFR, Ln(sKl) and parathyroid hormone were independently associated with phosphate reabsorption, whereas Ln(iFGF-23) was not, after adjustment for age. Abnormalities in phosphate regulatory pathways are disturbed early in CKD. While iFGF-23 is associated with phosphate excretion on univariate analyses, sKl demonstrates a significant association with phosphate reabsorption independent of iFGF-23, and this relationship deserves further exploration.
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http://dx.doi.org/10.1093/ckj/sfw146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466110PMC
June 2017

FGF23 is synthesised locally by renal tubules and activates injury-primed fibroblasts.

Sci Rep 2017 06 13;7(1):3345. Epub 2017 Jun 13.

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Victoria, Australia.

In kidney disease, higher circulating levels of the mineral-regulating hormone fibroblast growth factor (FGF)-23 are predictive of disease progression but direct pathogenic effects on the kidney are unknown. We sought evidence of local renal synthesis in response to unilateral ureteric obstruction in the mouse, and pro-fibrotic actions of FGF23 on the fibroblast in vitro. Acute tubulointerstitial injury due to unilateral ureteric obstruction stimulated renal FGF23 synthesis by tubules, and downregulated inactivating proprotein convertases, without effects on systemic mineral metabolism. In vitro, FGF23 had divergent effects on fibroblast activation in cells derived from normal and obstructed kidneys. While FGF23 failed to stimulate fibrogenesis in normal fibroblasts, in those primed by injury, FGF23 induced pro-fibrotic signalling cascades via activation of TGF-β pathways. Effects were independent of α-klotho. Tubule-derived FGF23 may amplify myofibroblast activation in acute renal injury, and might provide a novel therapeutic target in renal fibrosis.
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http://dx.doi.org/10.1038/s41598-017-02709-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469734PMC
June 2017

Changes in Markers of Mineral Metabolism After Living Kidney Donation.

Transplant Direct 2017 Apr 28;3(4):e150. Epub 2017 Mar 28.

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Victoria, Australia.; Department of Medicine (RMH), The University of Melbourne, Parkville, Victoria, Australia.

Background: Living kidney donors (LKDs) experience reduction in kidney function, however serum phosphate (sPi) levels are lower compared to patients with chronic kidney disease matched for reduced kidney function. Mineral metabolism adaptations that occur in LKDs have not been adequately investigated. To evaluate the effect of nephrectomy on markers of mineral metabolism in LKDs compared to healthy volunteers (HV) over 12 months.

Methods: Mineral parameters were evaluated in twenty-one adult LKDs and twenty HVs. Parameters included sPi, intact parathyroid hormone, fibroblast growth factor-23 (FGF23), soluble Klotho (sKl) and urinary phosphate, measured prior to donation (T), 1 month (T), 6 months (T) and 12 months (T) post-kidney donation. Statistical analyses were conducted on normalized variables and changes were assessed using 2-way analysis of variance.

Results: Mean ages of LKDs and HVs were 54.1 ± 14.7 and 52.6 ± 8.0 years, respectively. There were no baseline clinical or biochemical differences between LKDs and HVs. In LKDs at T, serum creatinine increased (from 75 ± 12 to 114 ± 22 μmol/L), FGF23 increased (52 ± 15 to 70 ± 19 pg/mL) and sKl decreased (564 [469-662] to 424 [375-523] pg/mL), all less than 0.001. Changes were sustained at T. After donation, LKDs consistently demonstrated lower sPi compared with T, with the maximal sPi change at T (-0.19 mmol/L difference, < 0.001). Other markers of mineral metabolism were unchanged in LKDs. There were no mineral differences in HVs over 12 months.

Conclusions: Prospective evaluation of mineral metabolism parameters in LKDs provides valuable insight into compensatory mechanisms after reduction in kidney function. Further reduction of sPi at T despite early alterations in FGF23 and sKl suggest adaptation of mineral metabolism continues long-term in LKDs.
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http://dx.doi.org/10.1097/TXD.0000000000000660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381743PMC
April 2017

Mineral adaptations following kidney transplantation.

Transpl Int 2017 May 5;30(5):463-473. Epub 2017 Mar 5.

Department of Nephrology, The Royal Melbourne Hospital, Parkville, Vic., Australia.

Klotho is predominantly expressed in the kidney and reported to have antioxidant and antifibrotic properties. Soluble Klotho (sKl), the circulating protein cleaved from membrane-bound Klotho, is reduced significantly with kidney disease and inversely associated with mortality. sKl has not been thoroughly evaluated prospectively after kidney transplantation. Incident kidney transplant recipients (KTRs) were prospectively evaluated pretransplantation, 1, 12 and 52 weeks post-transplantation. Basic biochemistry, sKl and intact FGF23 were measured. Within-subject comparisons were evaluated using repeat-measure anova or Friedman's analysis. Effects of immunosuppression and biochemical parameters on sKl and FGF-23 over time were analysed using mixed-effects modelling. Median serum creatinine (sCr) at 1 week was 116 (92-142) μmol/l, and at 52 weeks, all 29 KTRs had a functioning graft with median sCr of 111 (97-131) μmol/l. Compared with baseline, sKl was increased at 52 weeks following an initial decline at 1 week (P < 0.005 and P < 0.01, respectively), while FGF23 was considerably reduced at 52 weeks (P < 0.001). In a mixed-effects model, an increased sKl was not associated with reduction in immunosuppression or evaluated biochemical parameters. Modest increase in sKl is observed one-year postkidney transplantation with excellent early graft function suggesting factors beyond renal capacity may influence circulating sKl. FGF23 normalization was observed. Longer term evaluation in transplantation, specifically addressing the effects of immunosuppression, is required to understand the pathophysiology of the sKl/FGF23 axis and potential for modification.
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http://dx.doi.org/10.1111/tri.12925DOI Listing
May 2017

Hypoxia in tissue repair and fibrosis.

Cell Tissue Res 2016 09 16;365(3):553-62. Epub 2016 Jul 16.

Department of Nephrology, Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia.

Hypoxia and hypoxia signalling through the transcription factor hypoxia inducible factor-1 (HIF-1), play an important role in normal tissue repair processes. Tissue injury generally produces at least the transient loss of normal vascular perfusion and the resulting hypoxia induces the expression of many genes that allow the tissue to adapt to hypoxia, to start the repair process and, in time, to re-establish oxygen delivery to the tissue. In most cases, transient hypoxia and the activation of the HIF-1 pathway are beneficial and promote the repair process, producing tissue that might not perfectly reform its original architecture but that has its function substantially restored. However, in some cases of chronic injury, chronic hypoxia and pathological repair, the hypoxia pathway might be responsible for driving the process of fibrosis and can lead to excessive scarring and compromised organ function.
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http://dx.doi.org/10.1007/s00441-016-2461-3DOI Listing
September 2016

Estrogens do not protect, but androgens exacerbate, collagen accumulation in the female mouse kidney after ureteric obstruction.

Life Sci 2016 Aug 29;158:130-6. Epub 2016 Jun 29.

Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia; Cardiovascular Disease Program, Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Clayton, Victoria, Australia. Electronic address:

Aims: Controversy surrounds the gender basis of progression in chronic kidney disease. Unfortunately, most experimental studies addressing this question do not distinguish between direct effects of estrogen and indirect activation of estrogen receptors through conversion of testosterone to 17β-estradiol by aromatase. We examined the pathogenesis of renal fibrosis in female aromatase knockout (ArKO) mice, which lack circulating and stored estrogens, while having normal levels of testosterone.

Main Methods: ArKO mice and their wild-type (ArWT) counterparts were subjected to unilateral ureteric obstruction (UUO), with kidney tissue collected at day(D) 0, 3 and 9 post-UUO. Effects of 5α-dihydrotestosterone (DHT) administration on each genotype were also studied. Tissue was assessed biochemically and histochemically for fibrosis. Western blot analysis was used to measure α-smooth muscle actin (α-SMA) expression and TGF-β1 signalling. Matrix metalloproteinase-2 (MMP-2) activity was measured by zymography.

Key Findings: UUO increased collagen content over time (p<0.05 (D3) and p<0.01 (D9) vs day 0), with no difference between genotypes in qualitative (collagen IV staining) and quantitative (hydroxyproline concentration) analyses. Systemic administration of non-aromatizable DHT increased collagen content after 3days of UUO in both genotypes. This was not paralleled by any change in α-SMA (myofibroblast burden) or TGF-β1 signalling but was commensurate with DHT reducing MMP2 activity in both genotypes (p<0.05 vs genotype controls).

Significance: Physiological concentrations of estrogens do not protect the injured kidney from fibrosis progression. Androgens rather than estrogens are the relevant factor involved in regulating disease-related renal scarring in this model.
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http://dx.doi.org/10.1016/j.lfs.2016.06.022DOI Listing
August 2016

The Anti-fibrotic Actions of Relaxin Are Mediated Through a NO-sGC-cGMP-Dependent Pathway in Renal Myofibroblasts In Vitro and Enhanced by the NO Donor, Diethylamine NONOate.

Front Pharmacol 2016 31;7:91. Epub 2016 Mar 31.

Cardiovascular Disease Program, Biomedicine Discovery Institute, Department of Pharmacology, Monash University Clayton, VIC, Australia.

Introduction: The anti-fibrotic hormone, relaxin, has been inferred to disrupt transforming growth factor (TGF)-β1/Smad2 phosphorylation (pSmad2) signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO)-dependent pathway. Here, we determined the extent to which NO, soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP) were directly involved in the anti-fibrotic actions of relaxin using a selective NO scavenger and sGC inhibitor, and comparing and combining relaxin's effects with that of an NO donor.

Methods And Results: Primary renal cortical myofibroblasts isolated from injured rat kidneys were treated with human recombinant relaxin (RLX; 16.8 nM), the NO donor, diethylamine NONOate (DEA/NO; 0.5-5 μM) or the combined effects of RLX (16.8 nM) and DEA/NO (5 μM) over 72 h. The effects of RLX (16.8 nM) and DEA/NO (5 μM) were also evaluated in the presence of the NO scavenger, hydroxocobalamin (HXC; 100 μM) or sGC inhibitor, ODQ (5 μM) over 72 h. Furthermore, the effects of RLX (30 nM), DEA/NO (5 μM) and RLX (30 nM) + DEA/NO (5 μM) on cGMP levels were directly measured, in the presence or absence of ODQ (5 μM). Changes in matrix metalloproteinase (MMP)-2, MMP-9 (cell media), pSmad2 and α-smooth muscle actin (α-SMA; a measure myofibroblast differentiation) (cell layer) were assessed by gelatin zymography and Western blotting, respectively. At the highest concentration tested, both RLX and DEA/NO promoted MMP-2 and MMP-9 levels by 25-33%, while inhibiting pSmad2 and α-SMA expression by up to 50% (all p < 0.05 vs. untreated and vehicle-treated cells). However, 5μM of DEA/NO was required to produce the effects seen with 16.8 nM of RLX over 72 h. The anti-fibrotic effects of RLX or DEA/NO alone were completely abrogated by HXC and ODQ (both p < 0.01 vs. RLX alone or DEA/NO alone), but were significantly enhanced when added in combination (all p < 0.05 vs. RLX alone). Additionally, the direct cGMP-promoting effects of RLX, DEA/NO and RLX+DEA/NO (which all increased cGMP levels by 12-16-fold over basal levels; all p < 0.01 vs. vehicle-treated cells) were significantly inhibited by pre-treatment of ODQ (all p < 0.05 vs. the respective treatments alone).

Conclusion: These findings confirmed that RLX mediates its TGF-β1-inhibitory and gelatinase-promoting effects via a NO-sGC-cGMP-dependent pathway, which was additively augmented by co-administration of DEA/NO.
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http://dx.doi.org/10.3389/fphar.2016.00091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815292PMC
April 2016

Antifibrotic Actions of Serelaxin - New Roles for an Old Player.

Trends Pharmacol Sci 2016 06 17;37(6):485-497. Epub 2016 Mar 17.

Department of Nephrology, Royal Melbourne Hospital, Parkville, Victoria, Australia; Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria, Australia.

Fibrosis represents a failed wound healing response to tissue injury. It is characterized by the accumulation of excess connective tissue and is a significant cause of organ failure, morbidity, and mortality. Fibrotic disorders accompany a wide spectrum of conditions including both systemic and organ-specific diseases, for which there is currently no effective cure. Serelaxin, the recombinant form of the major stored and circulating form of human relaxin, has emerged as a pleiotropic drug with rapidly occurring antifibrotic actions. This review discusses the effectiveness of serelaxin as an antifibrotic, and how it augments the actions of several other therapeutics leading to its potential use not only as a monotherapy but also as an adjunct therapy with other antifibrotic agents.
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http://dx.doi.org/10.1016/j.tips.2016.02.007DOI Listing
June 2016

Qualitative and Quantitative Analysis of Histone Deacetylases in Kidney Tissue Sections.

Methods Mol Biol 2016 ;1397:279-289

Epigenomic Medicine, Baker IDI Heart & Diabetes Institute, 75 Commercial Road, Melbourne, VIC, 3004, Australia.

Fluorescent microscope imaging technologies are increasing in their applications and are being used on a wide scale. However methods used to quantify the level of fluorescence intensity are often not utilized-perhaps given the result may be immediately seen, quantification of the data may not seem necessary. However there are a number of reasons given to quantify fluorescent images including the importance of removing potential bias in the data upon observation as well as quantification of large numbers of images gives statistical power to detect subtle changes in experiments. In addition discreet localization of a protein could be detected without selection bias that may not be detectable by eye. Such data will be deemed useful when detecting the levels of HDAC enzymes within cells in order to develop more effective HDAC inhibitor compounds for use against multiple diseased states. Hence, we discuss a methodology devised to analyze fluorescent images using Image J to detect the mean fluorescence intensity of the 11 metal-dependent HDAC enzymes using murine kidney tissue sections as an example.
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http://dx.doi.org/10.1007/978-1-4939-3353-2_19DOI Listing
October 2016

Laser Capture Microdissection of Archival Kidney Tissue for qRT-PCR.

Methods Mol Biol 2016 ;1397:251-265

Department of Nephrology, The Royal Melbourne Hospital, 300 Grattan Street, Parkville, Melbourne, VIC, 3050, Australia.

Whole-organ molecular analysis of the kidney potentially misses important factors involved in the pathogenesis of disease in glomeruli and tubules. Organ wide analysis can however be augmented by using laser capture microdissection (LCM) to isolate morphologically similar cells and nephron structures from a heterogeneous tissue section via direct visualization of the cells. The protocol here provides a practical approach utilizing LCM in combination with RNA isolation techniques for downstream analysis. This technique is readily applicable to study mRNA expression in isolated glomeruli and tubules in both experimental animal models and human kidney biopsy material.
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http://dx.doi.org/10.1007/978-1-4939-3353-2_17DOI Listing
October 2016

Propagation and Culture of Human Renal Fibroblasts.

Methods Mol Biol 2016 ;1397:11-23

Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia.

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 behavior, 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 human renal fibroblasts from the human kidney cortex. Techniques for their isolation, subculture, characterization, and cryogenic storage and retrieval are described in detail.
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http://dx.doi.org/10.1007/978-1-4939-3353-2_2DOI Listing
October 2016

Relationship between timed and spot urine collections for measuring phosphate excretion.

Int Urol Nephrol 2016 Jan 14;48(1):115-24. Epub 2015 Nov 14.

Department of Nephrology, The Royal Melbourne Hospital, Parkville, VIC, 3052, Australia.

Background: Twenty-four hour urinary phosphate excretion (UPE) reflects intestinal phosphate absorption in steady state and may be more informative than serum phosphate (sPi) when assessing phosphate homoeostasis clinically. Timed urine collections are cumbersome and prone to collection errors. Spot urine phosphate/creatinine ratio (uPiCr) may be a useful, simple surrogate for 24-h UPE, but requires further validation. This study aimed to determine the relationship between uPiCr and 24-h UPE.

Methods: This single-centre cross-sectional study examined contemporaneous serum, spot urine and 24-h urine. Serum biochemistry was analysed. Urine phosphate concentration (uPi) and creatinine concentration (uCr) were measured in spot and 24-h urine collections. Spearman's rank correlation coefficients and Bland-Altman plots were used to assess agreement between spot uPiCr and UPE. Backward multivariate analysis was undertaken for UPE prediction.

Results: One hundred and sixteen participants (77 with kidney disease and 39 healthy volunteers) were studied. Seventy-four (63.8 %) were male. Median (IQR) age was 61(49-71) years. Median (IQR) spot uPiCr and total UPE were 1.7 (1.3-2.2) mmol/mmol and 25.8 (19.9-35.0) mmol/d, respectively. There was no correlation between spot uPiCr and 24-h UPE (R = 0.064, P = 0.51) but spot uPi significantly correlated with 24-h UPE (R = 0.385, P < 0.001). Although spot and 24-h measures of phosphate handling correlated (all P < 0.001), Bland-Altman analysis revealed bias between collection techniques. UPE prediction model using the independent variables of eGFR, sPi, albumin and spot uPi provided R (2) = 0.443.

Conclusion: No direct correlation was noted between spot uPiCr and 24-h UPE. Normalization of uPi to uCr on spot urine samples may be inappropriate when evaluating urinary phosphate excretion in adults and thus, a spot uPiCr is not a suitable surrogate for measuring UPE. A UPE prediction model utilising spot urine biochemistry cannot be advocated at present.
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http://dx.doi.org/10.1007/s11255-015-1149-zDOI Listing
January 2016

Animal Models to Study Links between Cardiovascular Disease and Renal Failure and Their Relevance to Human Pathology.

Front Immunol 2015 17;6:465. Epub 2015 Sep 17.

Department of Nephrology, Royal Melbourne Hospital (RMH) , Melbourne, VIC , Australia.

The close association between cardiovascular pathology and renal dysfunction is well documented and significant. Patients with conventional risk factors for cardiovascular disease like diabetes and hypertension also suffer renal dysfunction. This is unsurprising if the kidney is simply regarded as a "modified blood vessel" and thus, traditional risk factors will affect both systems. Consistent with this, it is relatively easy to comprehend how patients with either sudden or gradual cardiac and or vascular compromise have changes in both renal hemodynamic and regulatory systems. However, patients with pure or primary renal dysfunction also have metabolic changes (e.g., oxidant stress, inflammation, nitric oxide, or endocrine changes) that affect the cardiovascular system. Thus, cardiovascular and renal systems are intimately, bidirectionally and inextricably linked. Whilst we understand several of these links, some of the mechanisms for these connections remain incompletely explained. Animal models of cardiovascular and renal disease allow us to explore such mechanisms, and more importantly, potential therapeutic strategies. In this article, we review various experimental models used, and examine critically how representative they are of the human condition.
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http://dx.doi.org/10.3389/fimmu.2015.00465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585255PMC
October 2015

Diurnal variation and short-term pre-analytical stability of serum soluble α-klotho in healthy volunteers: a pilot study.

Ann Clin Biochem 2015 Jul 21;52(Pt 4):506-9. Epub 2014 Nov 21.

Department of Nephrology, The Royal Melbourne Hospital (RMH), Parkville, Australia Department of Medicine (RMH), The University of Melbourne, Melbourne, Australia.

Aim: To investigate the diurnal variability and pre-analytical stability of soluble α-klotho in serum.

Background: Recent evidence suggests that the cleaved extracellular domain of the α-klotho receptor, soluble α-klotho, affects phosphate homeostasis, ion channel regulation and antifibrotic/antioxidant pathways. However, soluble α-klotho measurements have yielded inconsistent results when related to renal function, markers of mineral metabolism and patient outcomes. Pre-analytical factors such as biological variation and analyte stability may affect the interpretation of soluble α-klotho results but are yet to be formally assessed.

Methods: Clotted blood samples were collected from 10 healthy adult volunteers at three time-points during the day to assess diurnal change. Additional samples were collected and allowed to stand at room temperature for 30, 60 and 120 min, prior to centrifugation and analysis to evaluate analyte stability. Serum soluble α-klotho was measured using a validated commercial enzyme-linked immunosorbent assay.

Results: Delayed processing of samples had no significant effect on serum soluble α-klotho concentrations over a 2-h period. Serum soluble α-klotho concentrations remained stable over morning, midday and afternoon time-points in this pilot study.

Conclusion: Serum soluble α-klotho demonstrates short-term pre-analytical stability and minimal diurnal variability in this pilot study. Larger studies are warranted to confirm these findings.
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http://dx.doi.org/10.1177/0004563214563415DOI Listing
July 2015

Development of second generation peptides modulating cellular adiponectin receptor responses.

Front Chem 2014 17;2:93. Epub 2014 Oct 17.

Department of Biomedical Sciences, Creighton University NE, USA.

The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM-low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.
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http://dx.doi.org/10.3389/fchem.2014.00093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201147PMC
November 2014
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