Evaluation of biomarkers of cell cycle arrest and inflammation in prediction of dialysis or recovery after kidney transplantation.

Early prediction of delayed graft function (DGF) after kidney transplantation would facilitate patient management. Cell cycle arrest and inflammation are implicated in the pathogenesis of DGF. We assessed the utility of two novel acute kidney injury (AKI) biomarkers, urinary tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7), and five inflammatory markers to predict DGF following deceased-donor kidney transplantation. Serial urine concentrations of TIMP-2 and IGFBP7 were measured immediately after transplantation in 56 recipients along with vascular endothelial growth factor-A (VEGF-A), macrophage migration inhibitory factor (MIF), monocyte chemotactic protein-1 (MCP-1), trefoil factor 3 (TFF3) and chemokine (C-X-C) ligand 16 (CXCL16). Delayed graft function (DGF) was defined as requirement for dialysis within 7 days. Integrated discrimination improvement analysis was used to evaluate whether these biomarkers enhanced prediction of DGF independently of a validated clinical risk prediction model. DGF occurred in 22 patients (39%). At 4 h after kidney reperfusion, the area under the receiver operator characteristic curve (AUC) for VEGF-A was good (AUC > 0.80); for TIMP-2, IGFBP7 and [TIMP-2] × [IGFBP7] fair (AUCs 0.70-0.79); and for MIF, MCP-1, TFF3 and CXCL16 poor (AUC < 0.70). Only TIMP-2 and VEGF significantly enhanced the DGF prediction at 4 and 12 h. The cell cycle arrest marker urinary TIMP-2 and the inflammatory biomarker VEGF-A are potentially useful adjuncts to clinical data for early prediction of DGF.

Adiponectin binds C1q and activates the classical pathway of complement.

The adipose-specific protein adiponectin binds to a number of target molecules, including damaged endothelium and the surface of apoptotic cells. However, the significance of this binding remains unclear. This study demonstrates the binding of purified C1q to recombinant adiponectin under physiological conditions, and the dependence of this upon Ca(++) and Mg(++). Binding was enhanced by metaperiodate-mediated destruction of glucosylgalactosyl sugars on adiponectin. Adiponectin was bound by the globular domain of the A chain of collagenase-digested C1q, and C1q binding induced deposition of C4 and C3 through activation of the classical complement pathway. After Western blotting, affinity-purified adiponectin from human serum bound C1q, whereas adiponectin in whole serum did not, unless pre-treated with metaperiodate. These results suggest adiponectin is member of the pattern-recognition family of defence collagens, able to bind target molecules and activate complement. It may therefore play an important role in innate immunity and autoimmune phenomena.

Review article: Adiponectin: its role in kidney disease.

The multifactorial glycoprotein, adiponectin has demonstrable insulin-sensitizing, anti-atherogenic and anti-inflammatory properties. However, despite the prevalence of both insulin-resistance and vascular disease in patients with end-stage kidney disease, levels of adiponectin are high. Adiponectin circulates in different sizes (the high-molecular-weight (HMW) isoform is thought to be the most insulin-sensitizing type) and binds to two receptors, adiponectin receptors (AdipoR) 1 and 2. The adiponectin/receptor system appears to be upregulated in end-stage kidney disease possibly as an appropriate counter-regulatory response to the uraemic milieu. In contrast, adiponectin and its HMW isoform, AdipoR mRNA expression on peripheral blood mononuclear cells decrease after kidney transplantation, likely secondary to immunosuppression and/or an improvement in glomerular filtration rate and the uraemic environment. Adiponectin has also been detected in the urine of patients with proteinuric kidney disease. The presence of AdipoR on an immortal cell line of proximal tubular epithelial cells (HK-2) and an increased amount of intact HMW isoform in the urine of patients with various forms of proteinuria lead us to speculate about the potential role of urinary adiponectin. This review will also discuss the structure and function of adiponectin and its potential relevance to patients with kidney disease and the different factors that may influence the metabolism of this protein in kidney failure.

Adiponectin is present in the urine in its native conformation, and specifically reduces the secretion of MCP-1 by proximal tubular cells.

AIM: To determine whether adiponectin detected in urine is present in its native form and if adiponectin receptors (AdipoR) present and functional in proximal tubular (HK-2) cells. BACKGROUND: Adiponectin is a protein with anti-inflammatory, anti-atherogenic and insulin-sensitizing properties. It has previously been detected antigenically in the urine in several forms of renal disease. METHODS: We compared the isoform distribution of urinary adiponectin in patients with proteinuric and non-proteinuric renal disease with that of matched controls using chromatography and enzyme-linked immunosorbent assay. We examined whether AdipoR were present in HK-2 cells by real-time reverse transcription polymerase chain reaction. Their functionality was investigated by determining the effect of recombinant adiponectin on adenosine monophosphate-activated protein kinase phosphorylation using western blotting, and on the secretion of monocyte chemotactic protein-1 and C3 using enzyme-linked immunosorbent assays. RESULTS: Adiponectin in the urine is physiologically intact and largely present as the low molecular weight isoform. Subjects with urinary protein >150 mg/L excreted significantly more adiponectin and its high and low molecular weight isoforms than those with <150 mg/L. mRNA for AdipoR were present in HK-2 cells, with levels of mRNA for AdipoR1 being 20 times greater than those for AdipoR2. Ligation of AdipoR on proximal tubular cells increased phosphorylation of adenosine monophosphate-activated protein kinase, and downregulated the secretion of the inflammatory cytokine monocyte chemotactic protein-1, but not of C3. CONCLUSION: Physiologically relevant isoforms of adiponectin are present in the urine of normal subjects and those with proteinuria. In addition, functional receptors for adiponectin are present in HK-2 cells. Abnormal levels of adiponectin in the urine may therefore activate these receptors, potentially resulting in anti-inflammatory activity.

Complement activation contributes to leukocyte recruitment and neuropathic pain following peripheral nerve injury in rats.

Complement activation triggers inflammation and has been implicated in neurological diseases associated with pain. However, the role of complement in neuropathic pain has not been clearly defined. In this study, we tested whether complement is activated by partial ligation of the rat sciatic nerve, a widely used model of neuropathic pain, and whether complement activation or inhibition in peripheral nerve influences leukocyte recruitment and neuropathic pain. We found that C3 deposition significantly increased from 6 h to 7 days in the injured nerve and was associated with the extent of thermal hyperalgesia and mechanical allodynia. However, no deposition of the membrane attack complex was detected. Complement activation by endoneurial injection of aggregated rat immunoglobulin G into normal sciatic nerve produced significant thermal hyperalgesia and mechanical allodynia of the ipsilateral hindpaw at 2-7 days after injection. This was accompanied by increased deposition of C3 and recruitment of macrophages at 7 days following injection. Complement inhibition using systemic injections of soluble complement receptor 1 (AVANT Immunotherapeutics, Inc., Needham, USA) into rats markedly suppressed C3 deposition and T-cell and macrophage recruitment to the injured nerve, and produced significant alleviation of thermal hyperalgesia and mechanical allodynia. These results demonstrate that C3 activation in the nerve contributes to increased infiltration of inflammatory cells and to neuropathic pain behaviors following peripheral nerve injury. Complement inhibition may be a potential therapeutic treatment for neuropathic pain.

The effect of renal transplantation on adiponectin and its isoforms and receptors.

Insulin resistance (IR) and other proatherogenic risk factors associated with end-stage kidney disease (ESKD) are improved by renal transplantation. Adiponectin is a protein with insulin-sensitizing, anti-inflammatory, and antiatherogenic properties. It exists in several isoforms, but the high molecular weight (HMW) isoform correlates best with insulin sensitivity. Paradoxically, the levels of this protein and its HMW isoform are increased in ESKD. We measured the homeostasis model assessment for insulin resistance (HOMA-IR), plasma adiponectin and its isoforms, and messenger RNA for adiponectin receptors (AdipoR1 and AdipoR2) on peripheral blood mononuclear cells in 54 stable transplant recipients, 50 patients established on hemodialysis, and 52 controls; groups were matched for body mass index and sex. HOMA-IR values were significantly higher in patients with ESKD compared with controls (P < .0005) and transplant patients (P < .05) but there was no difference between the latter 2 groups. Adiponectin levels were also higher in patients with ESKD compared with controls (P < .0005), and although levels were lower in the transplant group, they remained higher than in controls (P < .0001). However, although the absolute amount of HMW isoform in transplant patients remained higher than in controls (P < .0001), the proportion was similar, and less than in patients with ESKD (P < .005). The absolute amount of the HMW isoform correlated with superior metabolic indices in all 3 cohorts. AdipoR1 and AdipoR2 messenger RNA levels after transplantation were significantly lower than those of ESKD subjects (P < .0001, P < .01), but transplant patients had less AdipoR1 than controls, although their AdipoR2 levels were higher. AdipoR1 correlated with AdipoR2 in all 3 cohorts. We conclude that HOMA-IR was lower in the transplant group compared with the group on hemodialysis and this coincided with lower total adiponectin levels and absolute amount of the HMW isoform and AdipoR on peripheral blood mononuclear cells. Lower AdipoR after transplantation may be secondary to immunosuppression and/or an improvement in glomerular filtration rate and the uremic milieu.

The metabolism of isoforms of human adiponectin: studies in human subjects and in experimental animals.

OBJECTIVE: Little is known of the metabolism of different isoforms of adiponectin. We therefore (a) characterised the size distribution of human adiponectin in relation to gender, body composition and following a challenge with a fat meal or oral glucose in humans, and (b) studied the metabolism of isoforms of human adiponectin in rabbits. METHOD: Electrophoresis, blotting and chromatography were used to characterise human adiponectin in 36 healthy subjects, including 15 with at least two first-degree relatives with type 2 diabetes, before and after consumption of a fatty meal or glucose. The metabolism of column-fractionated human adiponectin was studied in rabbits, some of which were coinjected with insulin. RESULTS: Females had a higher proportion of high molecular weight (HMW) and hexameric adiponectin (P = 0.002 and 0.004 respectively), and a lower proportion of trimers (P < 0.0001) than males. Females also showed a strong negative relationship between body fat measures and the proportion of HMW adiponectin. There were no differences in isoforms between insulin-resistant and -sensitive subjects, or following oral glucose or a fat meal. Adiponectin in rabbits had an extravascular/intravascular ratio of 0.71, and a half-life (T1/2) of 14.3 h. Metabolism was not influenced by insulin or reduction of sulphydryl bonds. HMW and trimeric isoforms had a significantly different T1/2 of 13.0 and 17.5 h respectively (P < 0.05), and these isoforms did not interconvert in vivo. CONCLUSIONS: Human adiponectin is present as trimers, hexamers and HMW forms. Females had a higher proportion and absolute amount of HMW species compared with males, and female, but not male, subjects showed a strong negative relationship between measures of body fat, and the proportion of HMW species. These isoforms did not respond to challenge in man with a fatty meal or oral glucose, and in the rabbit, to injected insulin. HMW adiponectin was more rapidly metabolised than the trimeric form, but both were stable in vivo, and did not interconvert. We conclude that human adiponectin is much longer-lived than is the case with other hormones, a finding with positive implications for the potential to supplement levels of adiponectin in man.

Insulin resistance and postprandial triglyceride levels in primary renal disease.

BACKGROUND: Renal failure is associated with a range of metabolic abnormalities including insulin resistance and dyslipidemia. We examined the role of creatinine clearance (CrCl) and body composition in the development of insulin resistance in patients with primary renal disease and a variable degree of renal failure. We also determined the effect of a high-fat meal on postprandial triglyceride levels in a subgroup of these patients. METHODS: Forty-four patients with primary renal disease (men, 25; women, 19; age, 21-75 years) were compared to 44 controls matched for age, sex, and body composition. Renal biochemistry, plasma glucose, insulin, lipids, and nonesterified fatty acids were measured in the fasting state. Insulin sensitivity was calculated using the Homeostasis Model Assessment for Insulin Resistance (HOMA-R), and pancreatic beta-cell secretory capacity by HOMA- beta . Fourteen normotriglyceridemic subjects from each group consumed an 80-g fat meal to examine their postprandial metabolic response. RESULTS: Although there was no significant difference between HOMA-R for the controls and the entire patient group ( P = .06), HOMA-R was significantly higher in patients with CrCl less than 60 mL/min than those with CrCl greater than 60 mL/min or control subjects ( P < .01 for each pair). Exponential analysis of the relationship between CrCl and HOMA-R and - beta showed a line of best fit that was superior to that obtained by linear regression analysis ( P < .01 and P < .005, respectively). HOMA-R in renal patients was correlated with several parameters of body composition, including central fat (kilogram) ( P < .005). There was no difference in body fat parameters or HOMA-R for the patient and control subgroups undergoing a fat meal challenge. However, the patient subgroup showed a greater postprandial incremental rise in plasma triglycerides compared to controls ( P < .02). CONCLUSION: Patients with renal disease exhibit metabolic features typically associated with the metabolic syndrome. Insulin resistance increased with decline in renal function and was significantly higher in patients with CrCl less than 60 mL/min compared to subjects with CrCl greater than 60 mL/min or carefully matched controls. Renal patients also showed significant postprandial hypertriglyceridemia.

Fab fragments of ovine antibody to colchicine enhance its clearance in the rat.

CONTEXT: Colchicine is an anti-inflammatory alkaloid used for the treatment of acute gout, but has a narrow therapeutic index. Colchicine overdoses are relatively rare, but have high mortality requiring rapid treatment. OBJECTIVE: To evaluate the ability of a newly available ovine fragment antigen-binding (Fab) antibody to colchicine (ColchiFab(™)) to protect rats against renal and other injury 24 h after colchicine ingestion. MATERIALS AND METHODS: Rats were gavaged with colchicine (5 mg/kg), then 2 h later injected intraperitoneally with 5 ml of sterile saline, or Fab anti-colchicine, a newly available ovine antibody to colchicine. Samples of blood were taken at 1, 2, 5 and 24 h after gavage, and urine was collected from 5 to 24 h after gavage. Concentrations of colchicine in tissue, blood and urine were measured by liquid chromatography/mass spectrometry, concentrations of Fab anti-colchicine, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 or KIM-1 by enzyme-linked immunosorbent assay or ELISA, while concentrations of creatine kinase and creatinine (Cr) were measured enzymatically. RESULTS: Colchicine equilibrated rapidly throughout the body and increased serum creatine kinase. Fab anti-colchicine also rapidly redistributed to the blood and remained at high concentrations over 24 h. Fab anti-colchicine caused a rapid 7.1-fold increase in serum colchicine level, followed by excretion of both colchicine and Fab anti-colchicine through the urine. This was associated with the accumulation of colchicine in the kidney, a reversal of colchicine-induced diarrhoea, and increasing urinary NGAL level; from 168 ± 48 to 477 ± 255 ng/mmol Cr [mean ± standard deviation or SD]. DISCUSSION: Fab anti-colchicine greatly increased the clearance of colchicine, although increasing NGAL level suggested the presence of mild kidney damage. CONCLUSION: These data suggest clinical utility for Fab anti-colchicine in the treatment of colchicine overdose.

Kinetic Estimation of GFR Improves Prediction of Dialysis and Recovery after Kidney Transplantation.

BACKGROUND: The early prediction of delayed graft function (DGF) would facilitate patient management after kidney transplantation. METHODS: In a single-centre retrospective analysis, we investigated kinetic estimated GFR under non-steady-state conditions, KeGFR, in prediction of DGF. KeGFR(sCr) was calculated at 4h, 8h and 12h in 56 recipients of deceased donor kidneys from initial serum creatinine (sCr) concentrations, estimated creatinine production rate, volume of distribution, and the difference between consecutive sCr values. The utility of KeGFR(sCr) for DGF prediction was compared with, sCr, plasma cystatin C (pCysC), and KeGFR(pCysC) similarly derived from pCysC concentrations. RESULTS: At 4h, the KeGFR(sCr) area under the receiver operator characteristic curve (AUC) for DGF prediction was 0.69 (95% CI: 0.56-0.83), while sCr was not useful (AUC 0.56, (CI: 0.41-0.72). Integrated discrimination improvement analysis showed that the KeGFR(sCr) improved a validated clinical prediction model at 4h, 8h, and 12h, increasing the AUC from 0.68 (0.52-0.83) to 0.88 (0.78-0.99) at 12h (p = 0.01). KeGFR(pCysC) also improved DGF prediction. In contrast, sCr provided no improvement at any time point. CONCLUSIONS: Calculation of KeGFR from sCr facilitates early prediction of DGF within 4 hours of renal transplantation.

Clusterin in kidney transplantation: novel biomarkers versus serum creatinine for early prediction of delayed graft function.

BACKGROUND AND OBJECTIVES: Current methods for rapid detection of delayed graft function (DGF) after kidney transplantation are unreliable. Urinary clusterin is a biomarker of kidney injury but its utility for prediction of graft dysfunction is unknown. METHODS: In a single-center, prospective cohort study of renal transplant recipients (N=81), urinary clusterin was measured serially between 4 hr and 7 days after transplantation. The utility of clusterin for prediction of DGF (hemodialysis within 7 days of transplantation) was compared with urinary interleukin (IL)-18, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1, serum creatinine, and clinical variables. RESULTS: At 4 hr after reperfusion, anuria was highly specific, but of low sensitivity for detection of DGF. At 4 hr, receiver operating characteristic analysis suggested that urinary clusterin, IL-18, kidney injury molecule-1, and NGAL concentration were predictive of DGF. After adjusting for preoperative clinical variables and anuria, clusterin and IL-18 independently enhanced the clinical model for prediction of DGF. Kidney injury molecule-1 only modestly improved the prediction of DGF, whereas NGAL, serum creatinine, and the creatinine reduction ratio did not improve on the clinical model. At 12 hr, the creatinine reduction ratio independently predicted DGF. CONCLUSION: Both urinary clusterin and IL-18 are useful biomarkers and may allow triaging of patients with DGF within 4 hr of transplantation. Relative performance of biomarkers for prediction of graft function is time-dependant. Early and frequent measurements of serum creatinine and calculation of the creatinine reduction ratio also predict DGF within 12 hr of reperfusion.

Biomarkers of calcineurin inhibitor nephrotoxicity in transplantation.

Over 35 years of use has demonstrated the revolutionary therapeutic benefits of calcineurin inhibitors (CNI) in not only preventing transplant rejection, but also the renal and nonrenal toxicity of CNI. Acute reversible and insidious irreversible forms of CNI nephrotoxicity have been identified, with ischemia from an imbalance between vasoconstrictors and vasodilators playing an important role. The ongoing search to define toxicity pathways has been enriched by 'Omics' studies. Changes in proteins including those involved in activation of pro-inflammatory responses, oxidative stress, ER stress and the unfolded protein response have been identified, and these may serve as biomarkers of toxicity. However, the current standard of CNI toxicity, histology, lacks specificity, which creates challenges for biomarker validation. This review focuses on progress in nephrotoxic pathway identification of CNI and biomarker validation.

A comparison of the ability of levels of urinary biomarker proteins and exosomal mRNA to predict outcomes after renal transplantation.

BACKGROUND: mRNA for biomarkers of kidney injury extracted from urinary exosomes may reflect or predict levels of the corresponding protein after transplantation and clinical outcomes. METHODS: Urinary exosomes were isolated from patients following renal transplantation, from healthy controls, and patients with CKD. Expression of exosomal mRNA for the injury biomarkers neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), and cystatin C was compared with the concentrations of corresponding urinary proteins, 18S RNA and serum creatinine. RESULTS: All biomarker protein concentrations increased after transplantation, and urinary NGAL and IL-18 at 24 and 168 h correlated with the day 7 creatinine reduction ratio (CRR). Exosomal18S RNA increased after transplantation, but exosomal mRNA for NGAL, IL-18 and cystatin C did not correlate with the day 7 CRR, or urinary biomarker concentrations at any time after transplantation. Exosomal NGAL mRNA was lower 4 h after transplantation than in control exosomes. In contrast, exosomal mRNA for cystatin C was unchanged after transplantation and in CKD, although urinary cystatin C temporarily increased following transplantation. Urinary KIM-1 increased after transplantation, but exosomal mRNA for KIM-1 remained undetectable. In CKD 18S RNA was raised, and exosomal mRNA for NGAL, IL-18 and cystatin C was detected in all patients. While urinary NGAL was greater in CKD than control subjects, exosomal NGAL mRNA was unchanged. Exosomal IL-18 mRNA was increased in CKD, but not IL-18 protein. CONCLUSIONS: After renal transplantation, urinary NGAL and IL-18 levels reflect the day 7 CRR. However, while mRNA for these biomarkers is present in exosomes, their levels do not reflect or predict urinary biomarker levels or the CRR. This likely reflects the fact that packaging of mRNA in exosomes is selective, and is not necessarily representative of mRNA in the parent cells responsible for biomarker production.

Kidney biomarkers in MCPA-induced acute kidney injury in rats: reduced clearance enhances early biomarker performance.

For improved early detection and assessment of severe acute kidney damage following accidental or intentional ingestion of the herbicide MCPA, we compared a panel of 14 novel kidney injury biomarkers with plasma creatinine. Male Wistar rats received four different oral doses of MCPA and plasma and urine biomarker levels were measured at 8, 24 and 48 h after MCPA exposure. Diagnostic performances using absolute levels, urine levels normalized to urine creatinine or urinary excretion rate were determined by ROC analysis. Plasma creatinine remained the best early biomarker for predicting histological changes at 48 h. The performance of plasma cystatin C in mirroring kidney function was similar to that of plasma creatinine. While urine concentrations were generally less predictive, normalization by urine creatinine greatly improved the performance of several biomarkers. This may be due to an apparent amplification of the biomarker signal on normalizing to creatinine, in the presence of a declining glomerular filtration rate prior to reaching steady state. Normalized 8 h osteopontin and albumin concentrations outperformed other normalized biomarkers in predicting histological changes at later times. Normalized urinary kidney injury molecule-1 at 48 h also correlated well with the degree of kidney damage.

Clinical use of biomarkers for toxicant-induced acute kidney injury.

Toxicant-induced acute kidney injury (ToxAKI) causes substantial morbidity and retards drug development. ToxAKI is relatively underexplored compared with ischemia-reperfusion injury in clinical biomarker studies. We highlight the rationale for novel AKI biomarkers in management of ToxAKI, and review the contemporary evidence supporting their clinical use. Directly-acting nephrotoxins, such as cisplatin, aminoglycosides, vancomycin and radiocontrast, remain widely used and highlight how novel biomarkers can either improve the detection of changes in glomerular filtration rate or directly signal cellular injury and structural damage. Serum cystatin C has already improved clinical risk prediction and drug dosing although its clinical use for early diagnosis awaits validation. The use of novel functional and structural biomarkers to stage ToxAKI and aid prognosis requires robust validation and better understanding of the relationship between biomarkers, morbidity and mortality. Biomarkers that illustrate the probable mechanisms and phase of ToxAKI may guide mechanism-specific diagnosis and therapy.

Glycosylation of human adiponectin affects its conformation and stability.

The collagenous region of adiponectin is glycosylated in vitro with glucosylgalactosyl moieties on four conserved lysines. We investigated the glycosylation of human adiponectin in vivo. Sugar vicinyl hydroxides on adiponectin were oxidized with 10 or 1 mM metaperiodate, and the result analyzed by two-dimensional electrophoresis and immunoblotting. Only 10 mM metaperiodate caused significant changes in electrophoretic mobility and an altered susceptibility to proteinase K digestion. Such treatment also increased the susceptibility of hexamers and high molecular weight (HMW) isoforms to dissociation by SDS. By contrast, untreated low molecular weight (LMW) isoforms were readily dissociated by low concentrations of SDS. Reduced HMW isoforms were able to partially reassemble following the removal of dithiothreitol, and this process was unaffected by metaperiodate. The presence of sialic acid was detected by Maackia amurensis Lectin II blotting, and by oxidation with 1 mM metaperiodate, followed by detection with Emerald Green 300 fluorescent dye. Quantitation of sugars on affinity-purified adiponectin from nine human plasmas showed that dimers of HMW isoforms contained a 1.3-fold greater amount of total sugar than LMW isoforms. However, both contained similar amounts of sialic acid. We conclude that glucosylgalactosyl residues contribute to the conformation of HMW human plasma adiponectin. In addition, the HMW isoform contains greater amounts of glucosylgalactosyl residues than the LMW isoform, and these sugars are important in determining its stability in vivo.

Up-regulation of adiponectin, its isoforms and receptors in end-stage kidney disease.

BACKGROUND: Despite the favourable effects of adiponectin on the vasculature and insulin resistance (IR), levels are increased in patients with end-stage kidney disease (ESKD), in whom both IR and atherosclerosis are prevalent. METHODS: To investigate this paradox, we examined the distribution of adiponectin isoforms, the expression of adiponectin receptor (AdipoR) mRNA on peripheral blood mononuclear cells (PBMC) in 41 patients with ESKD on haemodialysis and 41 matched controls, and its function by adenosine monophosphate-activated protein kinase (AMPK) phosphorylation of AdipoR on PBMC. We also compared the expression of AdipoR on PBMC with that on muscle and subcutaneous and visceral fat in 10 patients undergoing elective cholecystectomy. RESULTS: The proportion of the high molecular weight (HMW) isoform of adiponectin was increased in the dialysis group (P = 0.001), even though these patients were significantly insulin resistant compared with controls (P = 0.006). AdipoR1 and AdipoR2 on PBMC were also increased in patients with ESKD (P < 0.05 and P = 0.007, respectively), but levels did not correlate with IR, the HMW isoform or other anthropometric measurements. There was a strong correlation between AdipoR1 and AdipoR2 on PBMC in ESKD and in subcutaneous and visceral fat in controls. However, there was no relationship between AdipoR in PBMC, muscle or visceral fat. Phosphorylation of AMPK by recombinant adiponectin showed that AdipoR on PBMC, from controls and ESKD patients, were equally functional. CONCLUSIONS: IR in ESKD is not explained by the change in isoformic distribution, or by AdipoR down-regulation or dysfunction. Rather, this receptor-ligand axis is up-regulated and may be a beneficial response to the inflammatory milieu of ESKD.

Tissue factor deficiency and PAR-1 deficiency are protective against renal ischemia reperfusion injury.

Ischemia/reperfusion (IR) injury is a leading cause of acute renal failure and an important contributor to allograft damage. Tissue factor (TF) is up-regulated during IR, and TF inhibition reduces renal injury. However, the underlying mechanisms by which TF contributes to injury have not been elucidated. We postulated that TF contributes to IR injury by production of coagulation proteases and subsequent signaling by protease activated receptor (PARs). We compared renal injury after 25 minutes of bilateral renal ischemia and varying periods of reperfusion in C57BL/6 mice, those expressing low levels of TF (low-TF), hirudin-treated C57BL/6, and mice lacking either PAR-1 or PAR-2. C57BL/6 mice developed severe renal failure and died within 48 hours of reperfusion. In contrast, low-TF, hirudin-treated C57BL/6, and PAR-1-/- mice were protected from renal failure and had reduced mortality, tubular injury, neutrophil accumulation, and lower levels of the chemokines KC and MIP-2. Importantly, PAR-1-/- mice had lower chemokine levels despite up-regulation of TF and fibrin deposition. In addition, treating PAR-1-/- mice with hirudin conferred no additional benefit. Somewhat surprisingly, PAR-2 deficiency did not protect from renal failure. These experiments indicate that increased TF activity after renal IR leads to increased CXC chemokine expression and subsequent neutrophil-mediated injury predominantly by thrombin-dependent PAR-1 signaling.

Human adiponectin binds to bacterial lipopolysaccharide.

Adiponectin has anti-inflammatory and anti-atherogenic properties in addition to its acknowledged roles in insulin sensitivity and energy homeostasis. These properties include the suppression of lipopolysaccharide [LPS]-mediated inflammatory events. We demonstrated that both recombinant and native adiponectin directly bind LPS derived from three different bacteria. The interaction occurred at pH 5.0-6.0 and was inhibited by the presence of Ca(2+) and Mg(2+), but enhanced by the sequestration of these cations. Maximal binding occurred at pH 6.0 in the presence of ethylenediaminetetraacetic acid. Lipid A and C1q were not inhibitory, although LPS, heparin, zymosan, and individual sugars all inhibited the reaction. Periodate-mediated deglycosylation of adiponectin, and reduction and alkylation also inhibited binding. Since adiponectin infiltrates into [relatively] acidic sites of inflammation, it may act as a scavenging anti-inflammatory agent in atherosclerosis and vascular damage where LPS [and other pro-inflammatory molecules] are present.

Should we quantify insulin resistance in patients with renal disease?

Cardiovascular disease is a major cause of morbidity and mortality in dialysis patients. Vascular disease develops before the initiation of dialysis, and it is now recognized that chronic kidney disease (CKD) is an independent risk factor for cardiovascular disease. Death from cardiovascular disease is a more common endpoint of CKD than progression to dialysis. There are multiple mechanisms that contribute to the increased vascular risk of CKD, one of which is the presence of insulin resistance (IR). CKD is characterised by many features of the metabolic syndrome, and features of IR are also observed in dialysis and transplant patients. IR may be quantified by several different methods. One such method is homeostatic model assessment (HOMA) technique, which derives a measurement of IR from fasting plasma glucose and insulin concentrations. The HOMA index has been demonstrated to be an independent predictor of survival in dialysis patients. CKD is characterised by a chronic inflammatory response and abnormalities in the production and regulation of adipose tissue derived proteins, which may contribute to the development of IR. There are a range of interventions including diet and exercise programmes or medications that may influence IR; however, the impact of these interventions in the context of CKD has not been systematically evaluated.