Thomas F. Hiemstra

Mycophenolate Mofetil vs Azathioprine for Remission Maintenance in Antineutrophil Cytoplasmic Antibody–Associated Vasculitis: A Randomized Controlled Trial

CONTEXT Current remission maintenance therapies for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) are limited by partial efficacy and toxicity. OBJECTIVE To compare the effects of mycophenolate mofetil with azathioprine on the prevention of relapses in patients with AAV. DESIGN, SETTING, AND PARTICIPANTS Open-label randomized controlled trial, International Mycophenolate Mofetil Protocol to Reduce Outbreaks of Vasculitides (IMPROVE), to test the hypothesis that mycophenolate mofetil is more effective than azathioprine for preventing relapses in AAV. The trial was conducted at 42 centers in 11 European countries between April 2002 and January 2009 (42-month study). Eligible patients had newly diagnosed AAV (Wegener granulomatosis or microscopic polyangiitis) and were aged 18 to 75 years at diagnosis. INTERVENTIONS Patients were randomly assigned to azathioprine (starting at 2 mg/kg/d) or mycophenolate mofetil (starting at 2000 mg/d) after induction of remission with cyclophosphamide and prednisolone. MAIN OUTCOME MEASURES The primary end point was relapse-free survival, which was assessed using a Cox proportional hazards model. The secondary end points were Vasculitis Damage Index, estimated glomerular filtration rate, and proteinuria. RESULTS A total of 156 patients were assigned to azathioprine (n = 80) or mycophenolate mofetil (n = 76) and were followed up for a median of 39 months (interquartile range, 0.66-53.6 months). All patients were retained in the analysis by intention to treat. Relapses were more common in the mycophenolate mofetil group (42/76 patients) compared with the azathioprine group (30/80 patients), with an unadjusted hazard ratio (HR) for mycophenolate mofetil of 1.69 (95% confidence interval [CI], 1.06-2.70; P = .03). Severe adverse events did not differ significantly between groups. There were 22 severe adverse events in 13 patients (16%) in the azathioprine group and there were 8 severe adverse events in 8 patients (7.5%) in the mycophenolate mofetil group (HR, 0.53 [95% CI, 0.23-1.18]; P = .12). The secondary outcomes of Vasculitis Damage Index, estimated glomerular filtration rate, and proteinuria did not differ significantly between groups. CONCLUSIONS Among patients with AAV, mycophenolate mofetil was less effective than azathioprine for maintaining disease remission. Both treatments had similar adverse event rates. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00307645.

Human Urinary Exosomes as Innate Immune Effectors

Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm-Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.

α-Klotho Expression in Human Tissues

Context: α-Klotho has emerged as a powerful regulator of the aging process. To date, the expression profile of α-Klotho in human tissues is unknown, and its existence in some human tissue types is subject to much controversy. Objective: This is the first study to characterize systemwide tissue expression of transmembrane α-Klotho in humans. We have employed next-generation targeted proteomic analysis using parallel reaction monitoring in parallel with conventional antibody-based methods to determine the expression and spatial distribution of human α-Klotho expression in health. Results: The distribution of α-Klotho in human tissues from various organ systems, including arterial, epithelial, endocrine, reproductive, and neuronal tissues, was first identified by immunohistochemistry. Kidney tissues showed strong α-Klotho expression, whereas liver did not reveal a detectable signal. These results were next confirmed by Western blotting of both whole tissues and primary cells. To validate our antibody-based results, α-Klotho-expressing tissues were subjected to parallel reaction monitoring mass spectrometry (data deposited at ProteomeXchange, PXD002775) identifying peptides specific for the full-length, transmembrane α-Klotho isoform. Conclusions: The data presented confirm α-Klotho expression in the kidney tubule and in the artery and provide evidence of α-Klotho expression across organ systems and cell types that has not previously been described in humans.

Glyceraldehyde 3-phosphate dehydrogenase is required for band 3 (anion exchanger 1) membrane residency in the mammalian kidney

The mammalian kidney isoform of the essential chloride-bicarbonate exchanger AE1 differs from its erythrocyte counterpart, being shorter at its N terminus. It has previously been reported that the glycolytic enzyme GAPDH interacts only with erythrocyte AE1, by binding to the portion not found in the kidney isoform. (Chu H, Low PS. Biochem J 400:143–151, 2006). We have identified GAPDH as a candidate binding partner for the C terminus of both AE1 and AE2. We show that full-length AE1 and GAPDH coimmunoprecipitated from both human and rat kidney as well as from Madin-Darby canine kidney (MDCK) cells stably expressing kidney AE1, while in human liver, AE2 coprecipitated with GAPDH. ELISA and glutathione S-transferase (GST) pull-down assays using GST-tagged C-terminal AE1 fusion protein confirmed that the interaction is direct; fluorescence titration revealed saturable binding kinetics with Kd 2.3 ± 0.2 μM. Further GST precipitation assays demonstrated that the D902EY residues in the D902EYDE motif located within the C terminus of AE1 are important for GAPDH binding. In vitro GAPDH activity was unaffected by C-terminal AE1 binding, unlike in erythrocytes. Also, differently from red cell N-terminal binding, GAPDH-AE1 C-terminal binding was not disrupted by phosphorylation of AE1 in kidney AE1-expressing MDCK cells. Importantly, small interfering RNA knockdown of GAPDH in these cells resulted in significant intracellular retention of AE1, with a concomitant reduction in AE1 at the cell membrane. These results indicate differences between kidney and erythrocyte AE1/GAPDH behavior and show that in the kidney, GAPDH is required for kidney AE1 to achieve stable basolateral residency.

Vitamin K Antagonists Predispose to Calciphylaxis in Patients with End-Stage Renal Disease

Background/Aims: Calciphylaxis is associated with a poor prognosis in dialysis patients, and its pathogenesis remains incompletely understood. Although the use of vitamin K antagonists (VKA) has been implicated, previous reports are conflicting. We aimed to determine if vitamin K antagonists conferred an increased risk of calciphylaxis in patients on dialysis. Methods: We performed a single-centre, retrospective cohort study of 2,234 patients receiving dialysis, and compared the characteristics of those with and without calciphylaxis. Results: We identified 5 cases of calciphylaxis (all female) between January 2009 and December 2013. Overall, 142 patients (6.4%) were treated with VKA during the study period. Calciphylaxis was more common in the VKA group (4 of 142 patients, OR = 61, 95% CI 6.7-546, p = 0.0001). VKA was withdrawn in all cases and treatment instituted with sodium thiosulphate, cinacalcet and supportive measures. All patients recovered, although there was one sudden cerebrovascular death during follow-up. Conclusion: Treatment with VKA predisposes to the development of calciphylaxis.

Treatment of primary systemic vasculitis with the inosine monophosphate dehydrogenase inhibitor mycophenolic acid.

Despite advances in the treatment of vasculitis, modern therapies fail to induce or maintain remission in a significant proportion of patients. Mycophenolic acid is increasingly used to treat vasculitis syndromes. Here, we consider relevant pharmacokinetic and pharmacodynamic properties of mycophenolate, with emphasis on the impact of renal impairment, and we review the existing evidence for and current trials of mycophenolate in the treatment of primary systemic vasculitides.

Newer therapies for vasculitis

There is a clear unmet need in the therapy of vasculitis reflecting the toxicity and partial efficacy of conventional agents. Vasculitis is a complex area for the evaluation of newer therapies due to the heterogeneity between and within syndromes with multisystem manifestations. Much of the evidence supporting newer therapies comes from small, non-randomised trials and is insufficient to permit firm recommendations. Newer immunosuppressive drugs, including mycophenolic acid and leflunomide, are alternative second-line agents to methotrexate and azathioprine. Plasma exchange appears to have a role in severe renal vasculitis and vasculitis caused by circulating immune complexes, but evidence supporting other indications is weak. In contrast to most other therapies, intravenous immunoglobulin (Ig) does not affect infective risk and is an alternative agent for refractory disease where standard approaches are contraindicated. The role of tumour necrosis factor blockade remains unresolved with important negative studies, but the therapeutic rationale persists and positive non-randomised trials are also under way. Experience with more aggressive immunosuppression, such as, T-cell depletion or autologous stem cell transplantation has been limited to a few centres. B-cell depletion with rituximab is currently attracting most attention with good success rates in small studies of refractory disease. The treatment of vasculitis in the future will become more complex with a wider range of available treatments; their optimal combination, sequencing and tailoring to the individual clinical situation will place unique demands on those delivering vasculitis services.

Plasma exchange induces vitamin D deficiency

BACKGROUND Plasma exchange is used in the treatment of diseases mediated by pathogenic circulating proteins, or for transplant desensitization. Its non-targeted nature results in the depletion of physiologically important molecules, and it is often complicated by hypocalcaemia. AIM To determine the effects of plasma exchange on vitamin D binding protein (DBP) and associated vitamin D metabolites. DESIGN Single-centre prospective cohort study of 11 patients. METHODS DBP and vitamin D metabolites were measured before and immediately after five plasma exchanges, and 7 and 28 days after discontinuation of plasma exchange. RESULTS Plasma exchange reduced plasma DBP concentration from 196.9 ± 53.2 to 98.5 ± 34 μg/ml (P = 0.0001), 1,25-dihydroxyvitamin D from 103 ± 52 to 42 ± 4 pmol/l (P = 0.003) and 25-hydroxyvitamin D from 49.7 ± 29 to 22 ± 9.4 nmol/l (P = 0.0017), through their removal in effluent. After 7 days, DBP and 1,25-dihydroxyvitamin D were not significantly different from baseline, but 25-hydroxyvitamin D remained significantly lower after 7 days (26.4 ± 9.8 nmol/l, P = 0.02) and 28 days (30.8 ± 15.5 nmol/l, P = 0.048). Corrected calcium decreased from 2.23 ± 0.11 to 1.98 ± 0.08 mmol/l (P = 0.0007) immediately after five treatments. Plasma calcium was significantly associated with 1,25-dihydroxyvitamin D (r(2) = 0.79, P < 0.0001). CONCLUSION Plasma exchange induced an acute reversible decrease in plasma 1,25-dihydroxyvitamin D, DBP, calcium and a sustained decrease in plasma 25-hydroxyvitamin D.

Arterial Expression of the Calcium-Sensing Receptor Is Maintained by Physiological Pulsation and Protects against Calcification

Vascular calcification (VC) is common in chronic kidney disease (CKD) and contributes to cardiovascular mortality. The calcium-sensing receptor (CaSR) is present in human artery, senses extracellular calcium and may directly modulate VC. Objective: to investigate the association between arterial cyclic strain, CaSR expression and VC. Methods and Results: human aortic smooth muscle cells (HAoSMC) were cultured under static or strained conditions, with exposure to CaSR agonists, the calcimimetic R568, and after CaSR silencing and over-expression. High extracellular calcium reduced CaSR expression and promoted osteochondrogenic transformation and calcium deposition. This was partially prevented by cyclic strain and exposure to R568. CaSR silencing enhanced calcification and osteochondrogenic transformation, whereas CaSR over-expression attenuated this procalcific response, demonstrating a central role for the CaSR in the response to cyclic strain and regulation of VC. In arterial explants from CKD patients (n = 11) and controls (n = 9), exposure to R568 did not significantly alter calcium deposition, osteochondrogenic markers or total artery calcium content. Conclusions: physiological mechanical strain is important for arterial homeostasis and may protect arteries from VC. The beneficial effects of cyclic strain may be mediated via the CaSR.

Clinical utility of PKD2 mutation testing in a polycystic kidney disease cohort attending a specialist nephrology out-patient clinic

BackgroundADPKD affects approximately 1:1000 of the worldwide population. It is caused by mutations in two genes, PKD1 and PKD2. Although allelic variation has some influence on disease severity, genic effects are strong, with PKD2 mutations predicting later onset of ESRF by up to 20 years. We therefore screened a cohort of ADPKD patients attending a nephrology out-patient clinic for PKD2 mutations, to identify factors that can be used to offer targeted gene testing and to provide patients with improved prognostic information.Methods142 consecutive individuals presenting to a hospital nephrology out-patient service with a diagnosis of ADPKD and CKD stage 4 or less were screened for mutations in PKD2, following clinical evaluation and provision of a detailed family history (FH).ResultsPKD2 mutations were identified in one fifth of cases. 12% of non-PKD2 patients progressed to ESRF during this study whilst none with a PKD2 mutation did (median 38.5 months of follow-up, range 16–88 months, p < 0.03). A significant difference was found in age at ESRF of affected family members (non-PKD2 vs. PKD2, 54 yrs vs. 65 yrs; p < 0.0001). No PKD2 mutations were identified in patients with a FH of ESRF occurring before age 50 yrs, whereas a PKD2 mutation was predicted by a positive FH without ESRF.ConclusionsPKD2 testing has a clinically significant detection rate in the pre-ESRF population. It did not accurately distinguish those individuals with milder renal disease defined by stage of CKD but did identify a group less likely to progress to ESRF. When used with detailed FH, it offers useful prognostic information for individuals and their families. It can therefore be offered to all but those whose relatives have developed ESRF before age 50.