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.

Ischaemia induces paradoxical changes in axonal excitability in end-stage kidney disease.

Peripheral neuropathy is present in 65% of patients with end-stage kidney disease (ESKD). No cause is yet established: nerve excitability studies have shown that axons are chronically depolarized, primarily owing to hyperkalaemia, but in vitro studies have suggested a role for axonal Na+/K+ pump dysfunction. To investigate Na+/K+ pump activity in vivo, lower limb ischaemia was induced in five ESKD patients and six healthy controls by a sphygmomanometer cuff, inflated to 200 mm Hg and maintained for 13 min. The peroneal nerve was stimulated at the fibular neck and excitability parameters were recorded from tibialis anterior (TA) and extensor digitorum brevis (EDB) before, during and after the ischaemic period. Baseline excitability studies in ESKD patients demonstrated reductions in threshold electrotonus and superexcitability and increased refractoriness, consistent with membrane depolarization. During ischaemia, threshold increased in ESKD patients [by +23.6 +/- 5.0% (TA); +32.1 +/- 7.3% (EDB)] in contrast to the persistent threshold reduction observed in normal controls [-2.4 +/- 5.2% (TA); -13.0 +/- 8.2% (EDB); P < 0.01]. These changes were accompanied by increased refractoriness and reduced superexcitability in both ESKD and control groups, consistent with ischaemic depolarization. Conversely, there was reduction in strength-duration time constant towards the end of ischaemia. Following release of ischaemia, the marked increase in threshold observed in normal controls was not evident in ESKD patients, but the rapid return of threshold to baseline argues against significant Na+/K+ pump dysfunction. The abnormal pattern of response to ischaemia in the ESKD patients was not fully explained by the hyperkalaemic membrane depolarization and suggests that another dialysable factor affects nerve excitability in ESKD patients, most likely H(+) ions, but that this factor only becomes evident during ischaemia. Blockade of persistent Na+ conductances by H+ would also explain the reduction in strength-duration time constant observed during ischaemia.

Neuropathy, axonal Na+/K+ pump function and activity-dependent excitability changes in end-stage kidney disease.

OBJECTIVE: To investigate the mechanisms underlying peripheral neuropathy and to provide insights into axonal Na(+)/K(+) pump function in patients with end-stage kidney disease (ESKD). METHODS: Nerve excitability was assessed in 10 ESKD patients before and after a single session of haemodialysis and in 29 age-matched control subjects. Changes in excitability were recorded at baseline and following maximal voluntary contraction (MVC) of abductor pollicis brevis (APB) for 60s. Serum concentrations of putative neurotoxins including potassium, urea, parathyroid hormone and beta-2-microglobulin were also measured. RESULTS: Baseline excitability values were consistent with axonal depolarisation prior to dialysis. Following maximal voluntary contraction (MVC), there was an increase in threshold, which was associated with reduced strength-duration time constant and increased superexcitability, consistent with axonal hyperpolarisation. These changes were quantitatively similar for patients and controls, arguing against any significant reduction in the axonal Na(+)/K(+) pump in ESKD. Following dialysis, activity-dependent changes were less in ESKD, which suggests greater Na(+)/K(+) pump activity prior to dialysis, the opposite of the changes expected with reduced Na(+)/K(+) pump function. The reduced post-contraction threshold change in post-dialysis recordings is likely to be secondary to relative hyperpolarisation of the axonal membrane following dialysis and reduction in K(+) concentration. CONCLUSIONS: Our findings suggest that Na(+)/K(+) pump function is not impaired in patients with ESKD. SIGNIFICANCE: Pre-dialysis excitability changes in ESKD patients may be explained on the basis of hyperkalaemia. Alteration in Na(+)/K(+) pump function does not appear to be a contributing factor to the development of neuropathy in ESKD patients.

Altered motor nerve excitability in end-stage kidney disease.

Although multiple toxins have been implicated in the development of uraemic neuropathy, no causative agent has been identified. In the present study, the excitability properties of lower limb motor nerves in patients with end-stage kidney disease treated with haemodialysis were measured before, during and after a standard 5 h haemodialysis session, in an attempt to explore the pathophysiology of uraemic neuropathy. Compound muscle action potentials were recorded from tibialis anterior and extensor digitorum brevis, following stimulation of the common peroneal nerve in 14 patients. Measures of excitability were assessed in relation to changes in serum levels of potential neurotoxins, including potassium, calcium, urea, uric acid, parathyroid hormone and beta-2-microglobulin. Before dialysis, measures of nerve excitability were significantly abnormal in the patient group for axons innervating tibialis anterior and extensor digitorum brevis, consistent with axonal depolarization: refractoriness was increased and superexcitability and depolarizing threshold electrotonus were reduced. Pre-dialysis excitability abnormalities were strongly correlated with serum K+. Correlation was also noted between the severity of symptoms and excitability abnormalities. Haemodialysis normalized the majority of nerve excitability parameters. In conclusion, lower limb motor axons in uraemic patients are depolarized before dialysis. The correlation between serum K+ and excitability measures indicates that hyperkalaemia is primarily responsible for uraemic depolarization, and a likely contributing factor to the development of neuropathy.

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.

Inflammation, insulin resistance, and adiposity: a study of first-degree relatives of type 2 diabetic subjects.

OBJECTIVE: Inflammatory markers such as C-reactive protein (CRP) are associated with insulin resistance, adiposity, and type 2 diabetes. Whether inflammation causes insulin resistance or is an epiphenomenon of obesity remains unresolved. We aimed to determine whether first-degree relatives of type 2 diabetic subjects differ in insulin sensitivity from control subjects without a family history of diabetes, whether first-degree relatives of type 2 diabetic subjects and control subjects differ in CRP, adiponectin, and complement levels, and whether CRP is related to insulin sensitivity independently of adiposity. RESEARCH DESIGN AND METHODS: We studied 19 young normoglycemic nonobese first-degree relatives of type 2 diabetic subjects and 22 control subjects who were similar for age, sex, and BMI. Insulin sensitivity (glucose infusion rate [GIR]) was measured by the euglycemic-hyperinsulinemic clamp. Dual-energy X-ray absorptiometry determined total and abdominal adiposity. Magnetic resonance imaging measured abdominal adipose tissue volumes. RESULTS: First-degree relatives of type 2 diabetic subjects had a 20% lower GIR than the control group (51.8 +/- 3.9 vs. 64.9 +/- 4.6 micromol x min(-1) x kg fat-free mass(-1), P = 0.04). However, first-degree relatives of subjects with type 2 diabetes and those without a family history of diabetes had normal and comparable levels of CRP, adiponectin, and complement proteins. When the cohort was examined as a whole, CRP was inversely related to GIR (r = -0.33, P = 0.04) and adiponectin (r = -0.34, P = 0.03) and positively related to adiposity (P < 0.04). However, CRP was not related to GIR independently of fat mass. In contrast to C3 (r = 0.41, P = 0.009) and factor B (r = 0.43, P = 0.005), CRP was unrelated to factor D. CONCLUSIONS: The insulin-resistant state is not associated with changes in inflammatory markers or complement proteins in subjects at high risk of type 2 diabetes. Our study confirms a strong relationship between CRP and fat mass. Increasing adiposity and insulin resistance may interact to raise CRP levels.

Adult nephrotic syndrome: non-specific strategies for treatment.

Irrespective of aetiology, the nephrotic syndrome presents a range of potentially serious complications. These include thrombo-embolism, infection and hyperlipidaemia. Despite the prevalence of the nephrotic state among renal patients, there has been little prospective analysis of the therapeutic approach to these potentially life-threatening events even though their pathogenesis has been examined in some detail. Most of these complications are more prevalent once the albumin concentration falls below 20 g/L and it is recognized that restoration of serum albumin significantly diminishes their frequency. However, this may be difficult to achieve, especially in adults. The problems of thrombo-embolism and infection are of immediate concern but, in persistent cases, the additional issues of hyperlipidaemia and loss of bone density also require consideration for therapy. Thus, in addition to specific attempts to reduce proteinuria, it is recommended that high-risk nephrotic patients receive anticoagulation, pneumococcal vaccination and lipid lowering therapy. Strategies for the preservation of bone density should also be considered, particularly in patients who receive high-dose corticosteroids. Among a range of non-specific treatments for proteinuria, angiotensin-converting enzyme inhibitors appear best in terms of efficacy and safety. Prospective trials are required to clarify the longitudinal impact of these generic strategies on the protection of the persistently nephrotic patient.

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.

The development of cardiac fibrosis in low tissue factor mice is gender-dependent and is associated with differential regulation of urokinase plasminogen activator.

Tissue factor (TF) initiates the protease coagulation cascade in response to tissue injury. Homozygous deficiency of murine TF results in embryonic lethality, which is rescued by low-level expression of human TF. These low-TF mice have been shown to develop cardiac fibrosis. We tested the hypothesis that the development of cardiac fibrosis in low-TF mice results from dysregulated protease expression and is affected by gender. Mice were divided into the age groups 2-5, 6-12, 13-18 and 19+ weeks. Fibrosis was assessed by trichrome staining. Protease expression was measured in male and female mice by RT-PCR for mRNA and zymography, ELISA or immunoblot for protein. Urokinase plasminogen activator (uPA) activity was determined by zymography and chromogenic substrate assay. A marked gender effect was noted for the development of fibrosis, with interstitial collagen deposition occurring from 9 weeks in male low-TF mice, but not until 19 weeks in low-TF females. This delayed onset in females was accompanied by delayed up-regulation of molecular markers of injury. Matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 expression were up-regulated in the hearts of male low-TF mice from 6 to 12 weeks and in females from 19 weeks. MMP/TIMP dysregulation was not seen prior to cardiac fibrosis and did not appear to explain the gender differences. However, uPA expression and activity were down-regulated prior to cardiac fibrosis in low-TF females, but were up-regulated in age-matched males. This suggests that the down-regulation of uPA in female low-TF mice protects them from more severe cardiac fibrosis.

Darbepoetin alfa administered monthly maintains haemoglobin concentrations in patients with chronic kidney disease not receiving dialysis: a multicentre, open-label, Australian study.

AIM: Darbepoetin alfa, an erythropoiesis-stimulating protein, has a longer serum half-life than recombinant human erythropoietin, allowing less-frequent administration. This study aimed to demonstrate that once-monthly (QM) darbepoetin alfa administration would maintain haemoglobin (Hb) concentrations in subjects with chronic kidney disease (CKD) not receiving dialysis who had previously been administered darbepoetin alfa every 2 weeks (Q2W). METHODS: This was a multicentre study in which subjects with CKD receiving stable Q2W darbepoetin alfa doses and with stable Hb (100-130 g/L) were started on QM darbepoetin alfa dosing. The initial QM darbepoetin alfa dose was equivalent to the cumulative darbepoetin alfa dose administered during the month preceding enrollment. Darbepoetin alfa doses were titrated to maintain Hb concentrations between 100 and 130 g/L. The primary endpoint was the proportion of subjects maintaining mean Hb >or= 100 g/L during the evaluation period (weeks 21-33). RESULTS: Sixty-six subjects were enrolled in the study and all received at least one dose of darbepoetin alfa; 55 (83%) had mean Hb >or= 100 g/L during evaluation. Mean (SD) Hb concentrations at baseline and during the evaluation period were 119 (8.7) g/L and 114 (9.8) g/L, respectively. The median QM darbepoetin alfa dose at baseline and during the evaluation period was 80 microg. Darbepoetin alfa was considered to be well-tolerated. CONCLUSION: Patients with CKD not receiving dialysis who are receiving darbepoetin alfa Q2W can be safely and effectively extended to darbepoetin alfa QM. Dosing QM may simplify anaemia management for patients and health-care providers.

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.

Outcome of overseas commercial kidney transplantation: an Australian perspective.

Lack of donors has led to a worldwide increase in commercial kidney transplantation programs where recipients acquire kidneys either from executed prisoners or live non-related donors. Commercial transplantation is prohibited by legislation in Australia. Our centres have had 16 patients who have travelled overseas to receive a commercial kidney transplant; five have subsequently died. As has been found previously, patients who received commercial transplants were more likely to develop infections such as HIV, hepatitis B virus, cytomegalovirus and fungal infections. Previous reports have found that patient and graft survival were comparable to local results, whereas we found that patient and graft survival were worse than transplantation within Australia. Patients considering the option of overseas commercial donation should be advised that heightened risks to life and graft survival exist.

The effect of pH and nucleophiles on complement activation by human proximal tubular epithelial cells.

BACKGROUND: Activation of urinary complement proteins in situ by proximal tubular epithelial cells (PTEC) may contribute to the mediation of tubulointerstitial injury in patients with significant proteinuria. However, the mechanism involved is unclear, and the role of changes in urinary pH and in the concentrations of urea or ammonia requires further clarification. METHODS: The protein fraction of urine samples from nine patients with proteinuria >1.5 g/day was purified. A cell ELISA involving cultured HK-2 PTEC was used to investigate the capacity of urinary protein to promote the deposition of both C3 and C9 on the cell surface. The effect of variations in pH (5.5-8.0) and in the concentration of urea and ammonia was also examined. C3 was purified and used to further investigate the mechanism of complement deposition. RESULTS: Urine samples from the majority of patients induced deposition of C3 and C9 on the surface of HK-2 cells via the alternative pathway. This process was maximal at acidic pH values. Preincubation of urinary complement or serum with urea or ammonia inhibited C3 deposition. Purified C3 incubated with HK-2 cells showed no evidence of activation in the absence of other complement components. CONCLUSIONS: These data suggest that bicarbonate protects against complement-mediated damage in the lumen by increasing the local pH, rather than by inhibiting the generation of ammonia. PTEC appear to activate complement through provision of a 'protected site' on their surface, rather than by the activation of C3 by convertase-like protease(s).