V. Esnault

KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron

Familial hyperkalemic hypertension (FHHt) is a Mendelian form of arterial hypertension that is partially explained by mutations in WNK1 and WNK4 that lead to increased activity of the Na+-Cl− cotransporter (NCC) in the distal nephron. Using combined linkage analysis and whole-exome sequencing in two families, we identified KLHL3 as a third gene responsible for FHHt. Direct sequencing of 43 other affected individuals revealed 11 additional missense mutations that were associated with heterogeneous phenotypes and diverse modes of inheritance. Polymorphisms at KLHL3 were not associated with blood pressure. The KLHL3 protein belongs to the BTB-BACK-kelch family of actin-binding proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes. KLHL3 is coexpressed with NCC and downregulates NCC expression at the cell surface. Our study establishes a role for KLHL3 as a new member of the complex signaling pathway regulating ion homeostasis in the distal nephron and indirectly blood pressure.

Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy

BACKGROUND Autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) are specific markers for primary membranous nephropathy (pMN) and anti-PLA2R1 serum levels may be useful to monitor disease activity. So far, a recombinant cell-based indirect immunofluorescence assay (RC-IFA) using recombinant PLA2R1 as a substrate has been widely available but lacks a finely graduated assessment of antibody concentrations. METHODS In order to setup a standardized ELISA, the extracellular domain of human PLA2R1 was expressed in HEK293. The purified protein was used to form the solid-phase in an ELISA which was then employed to analyze sera from 200 patients with primary MN, 27 patients with secondary MN, 230 patients with other glomerular diseases, 316 patients with systemic autoimmune diseases, and from 291 healthy blood donors. RESULTS At a set specificity of 99.9% the sensitivity of the anti-PLA2R1 IgG ELISA was found to be 96.5%. A similar sensitivity (98.5%) was obtained when binding of only subclass IgG4 was analyzed. The calibrated assay showed a good class correlation with the results of the RC-IFA, was robust and could be stored for several months without any loss of quality. CONCLUSION The results demonstrate that the new test system is qualified for routine use and that it has an almost perfect agreement with both, the clinical characterization of the patients and the results generated with RC-IFA.

Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy

The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy. However, the value of anti-PLA2R1 antibody titers in predicting patient outcomes is unknown. Here, we screened serum samples from 50 patients positive for PLA2R1 for immunoreactivity against a series of PLA2R1 deletion mutants covering the extracellular domains. We identified reactive epitopes in the cysteine-rich (CysR), C-type lectin domain 1 (CTLD1), and C-type lectin domain 7 (CTLD7) domains and confirmed the reactivity with soluble forms of each domain. We then used ELISAs to stratify 69 patients positive for PLA2R1 by serum reactivity to one or more of these domains: CysR (n=23), CysRC1 (n=14), and CysRC1C7 (n=32). Median ELISA titers measured using the full-length PLA2R1 antigens were not statistically different between subgroups. Patients with anti-CysR-restricted activity were younger (P=0.008), had less nephrotic range proteinuria (P=0.02), and exhibited a higher rate of spontaneous remission (P=0.03) and lower rates of renal failure progression (P=0.002) and ESRD (P=0.01) during follow-up. Overall, 31 of 69 patients had poor renal prognosis (urinary protein/creatinine ratio >4 g/g or eGFR<45 ml/min per 1.73 m(2) at end of follow-up). High anti-PLA2R1 activity and epitope spreading beyond the CysR epitope were independent risk factors of poor renal prognosis in multivariable Cox regression analysis. Epitope spreading during follow-up associated with disease worsening (n=3), whereas reverse spreading from a CysRC1C7 profile back to a CysR profile associated with favorable outcome (n=1). We conclude that analysis of the PLA2R1 epitope profile and spreading is a powerful tool for monitoring disease severity and stratifying patients by renal prognosis.

Prediction of membranous nephropathy recurrence after transplantation by monitoring of anti-PLA2R1 (M-type phospholipase A2 receptor) autoantibodies: a case series of 15 patients

BACKGROUND The predictive value of anti-M-type phospholipase A2 receptor (PLA2R1) autoantibodies for membranous nephropathy (MN) recurrence after renal transplantation remains controversial. METHODS Our aim was to monitor anti-PLA2R1 IgG4 activity using a sensitive enzyme-linked immunosorbent assay in 15 kidney transplant recipients with MN, and to test the correlation between antibody titres and MN recurrence. RESULTS Five patients never exhibited anti-PLA2R1 antibodies, and one of them relapsed. Ten patients (67%) had IgG4 anti-PLA2R1 antibodies at the time of transplantation and during follow-up. The presence of IgG4 anti-PLA2R1 antibodies at the time of kidney transplantation does not imply MN recurrence (P = 0.600, n = 15). However, a positive IgG4 anti-PLA2R1 activity during follow-up (>Month 6) was a significant risk factor for MN relapse (P = 0.0048, n = 10). Indeed, four patients had persistent IgG4 anti-PLA2R1 activity after transplantation and relapsed. Among them, one was successfully treated with rituximab. Another had persistently high IgG4 anti-PLA2R1 activity and exhibited a histological relapse but no proteinuria while on treatment with renin-angiotensin system inhibitors. In contrast, the six other patients who did not relapse exhibited a decrease of their IgG4 anti-PLA2R1 activity following transplant immunosuppression, including two with proteinuria due to biopsy-proven differential diagnoses. A weak transplant immunosuppressive regimen was also a risk factor of MN recurrence (P = 0.0048, n = 10). Indeed, the six patients who received both an induction therapy and a combined treatment with calcineurin inhibitors/mycophenolate exhibited a decrease of IgG4 anti-PLA2R1 activity and did not relapse, while the four patients who did not receive this strong immunosuppressive treatment association had persistently high IgG4 anti-PLA2R1 activity and relapsed. CONCLUSION The monitoring of IgG4 anti-PLA2R1 titres during follow-up helps to predict MN recurrence, and a strong immunosuppressive treatment of anti-PLA2R1 positive patients may prevent recurrence.

The effects of amlodipine and enalapril on renal function in adults with hypertension and nondiabetic nephropathies: A 3-year, randomized, multicenter, double-blind, placebo-controlled study

BACKGROUND Placebo-controlled trials have found that angiotensin-converting enzyme inhibitors (ACEIs) decrease proteinuria and slow the progression of nondiabetic nephropathies. However, head-to-head comparisons of ACEIs and calcium channel blockers (CCBs) have shown conflicting results. Indeed, a recent metaanalysis concluded that there is still uncertainty about the greater renoprotection seen with ACEIs or angiotensin II receptor blockers in nondiabetic patients with renal disease, particularly when using true glomerular filtration rate (GFR) as the primary outcome. OBJECTIVE The objective of this 3-year, randomized, multicenter, double-blind, placebo-controlled study was to compare true GFR decline (measured by yearly 51Cr-EDTA blood clearance) in nondiabetic, nonnephrotic adult hypertensive patients with estimated creatinine clearance of 20 to 60 mL/min.1.73 m(2), when randomized to a CCB (amlodipine, 5-10 mg/d) or an ACEI (enalapril, 5-20 mg/d). METHODS Patients (aged 18-80 years) entered a 4-week placebo run-in washout period and previous antihypertensive drugs were tapered off over 2 weeks. Add-on treatments were atenolol (50-100 mg/d), loop diuretics (furosemide, 20-500 mg/d or torsemide, 5-200 mg/d), alpha-blockers (prazosin, 2.5-5 mg/d or doxazosin, 1-16 mg/d), and centrally acting drugs (rilmenidine, 1-2 mg/d or methyldopa, 250-500 mg/d). The primary end point was true GFR measured by yearly (51)Cr-EDTA blood clearance. Secondary end points included a clinical composite of renal events and tolerability collected by a full clinical and laboratory evaluation at each study visit. Post hoc analyses for the change in GFR, proteinuria, and time to clinical events were also planned on baseline proteinuria subgroups (<1 and >or=1 g/d) before unblinding the database. RESULTS Three hundred eighteen patients entered the run-in period and 263 patients (156 men/107 women; mean age, 58 years) were randomized to receive either amlodipine (5 mg/d, n=132) or enalapril (5 mg/d, n=131). Blood pressure declined from 165/102 mm Hg to 138/84 mm Hg and 138/85 mm Hg with amlodipine and enalapril, respectively (no between-group significance). Only 20.8% of the patients randomized to ACEI treatment received diuretics at the last observation. No statistically significant difference was found between amlodipine and enalapril in GFR decline (-4.92 and -3.98 mL/min.1.73 m(2), respectively, at last observation) and composite secondary end point after a median follow-up of 2.9 years, including in the subgroup of patients with proteinuria >1 g/d at baseline. Protein excretion rate decreased significantly from baseline in patients taking enalapril plus diuretics (median -270 mg/d; P<0.001) but not in patients taking amlodipine plus diuretics (-25 mg/d at last observation). CONCLUSION In this cohort of nondiabetic, nonnephrotic hypertensive patients, no statistically significant difference in true GFR decline was found over 3 years between amlodipine-treated patients and enalapril-treated patients with main add-on treatment with ss-blockers, including in the subgroup of patients with proteinuria >1 g/d.

Diuretic uptitration with half dose combined ACEI + ARB better decreases proteinuria than combined ACEI + ARB uptitration

BACKGROUND Residual proteinuria is a strong modifiable risk factor for renal failure progression. We previously showed that the antiproteinuric effect of combined half doses of angiotensin-converting enzyme inhibitor (ACEI) and angiotensin receptor blocker (ARB) is increased by raising diuretic dosage. Methods. We tested whether uptitration of loop diuretics on top of combined half doses of ACEI and ARB would better decrease proteinuria than uptitration to combined full doses of ACEI and ARB in a randomized, crossover, three periods of 6-week controlled study. Eighteen patients with stable proteinuria over 1 g/day with combined ramipril at 5 mg/day and valsartan at 80 mg/day in addition to conventional antihypertensive treatments were randomized to receive combined ramipril at 5 mg/day and valsartan at 80 mg/day, or combined ramipril at 10 mg/day and valsartan at 160 mg/day, or combined ramipril at 5 mg/day, valsartan at 80 mg/day and increased furosemide dosage in random order. The primary end point was the mean urinary protein/creatinine ratio in two 24-hour urine collections at the end of the three treatment periods. Secondary end points included mean 24-hour proteinuria, home systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MAP) and estimated glomerular filtration rate (eGFR) levels. RESULTS The geometric mean urinary protein/creatinine ratio was lower with combined ramipril at 5 mg/day, valsartan at 80 mg/day and increased furosemide dosage compared to combined ramipril at 5 mg/day and valsartan at 80 mg/day, but also to combined ramipril at 10 mg/day and valsartan at 160 mg/day. These differences remained significant after adjustment for SBP, DBP or MAP and 24-hour natriuresis but not after adjustment on eGFR. Diuretic dosage uptitration did not increase the number of home systolic blood pressure measurements below 100 mmHg, but led to a statistically significant increase in the number of symptomatic hypotension episodes. CONCLUSIONS A cautious uptitration of loop diuretic dosage in addition to combined half doses of ACEI and ARB better decrease proteinuria in patients with CKD and high residual proteinuria than uptitration to full dose of combined ACEI and ARB. This antiproteinuric effect of diuretics was partly explained by an eGFR decrease, suggesting the contribution of haemodynamic modifications, whose safety on the long term still need to be addressed.

Carbapenem-resistant Klebsiella pneumoniae is associated with poor outcome in hemodialysis patients.

BACKGROUND Hemodialysis (HD) units have become a source of resistant bacteria. One of the most alarming developments is the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP). Risk factors and outcomes of CRKP isolation in HD patients have not been previously studied. METHODS A nested case-control study was conducted in maintenance HD patients between January 1st 2006 and June 30th 2009. CRKP-positive patients were matched with randomly selected CRKP-negative HD patients. Demographics, clinical and laboratory data were collected for 24 months prior to the specimen collection. Multivariate analyses identified independent risk factors for CRKP. A prospective follow-up determined CRKP-associated outcome. RESULTS Demographics associated with CRKP acquisition in HD patients were age between 65 and 75 and having no living offspring. Clinical conditions associated with CRKP were previous hospitalization, temporary HD catheter and previous isolation of vancomycin-resistant enterococcus. CRKP-related outcome was poor: median survival of one month and a hazard ratio [95% CI] of 5.9 [3.2-11.0] for mortality. CONCLUSIONS Temporary HD catheters and previous treatment for VRE may predict subsequent CRKP isolation. A microbiological diagnosis of CRKP in HD patients is highly associated with imminent mortality. Meticulous measures to control the spread of CRKP bacteria among HD patients appear particularly warranted.

Vitamin E–Coated and Heparin-Coated Dialyzer Membranes for Heparin-Free Hemodialysis: A Multicenter, Randomized, Crossover Trial

BACKGROUND Hemodialysis requires effective anticoagulation to avoid blood circuit clotting. In patients at high risk for bleeding, several alternative methods have been developed. STUDY DESIGN Multicenter, prospective, randomized, crossover study evaluating the noninferiority of vitamin E-coated compared with heparin-coated dialyzers in a 4-hour heparin-free hemodialysis strategy. SETTINGS & PARTICIPANTS 32 adult long-term hemodialysis patients from 2 French hemodialysis units with well-functioning fistulas or double-lumen catheters. INTERVENTION Patients were randomly allocated to a first period using either vitamin E- or heparin-coated dialyzers. After a washout period of 2 hemodialysis sessions, each patient was switched to the alternative dialyzer for a second period. Each study period started with 2 hemodialysis sessions with reduced heparin dose (50% and 25% of usual heparin dose, respectively, for sessions 1 and 2) followed by 2 heparin-free sessions. OUTCOMES The primary end point was the percentage of successful study periods, defined as no circuit-clotting event leading to premature interruption of any of the 4 dialysis sessions. Secondary end points included total number and cumulative duration of hemodialysis sessions without clotting, number of saline solution flushes, dialysis circuit bubble trap status and dialyzer membrane status by visual inspection, and dialysis adequacy. RESULTS The percentage of success with vitamin E-coated dialyzers (25/32 study periods [78%]) was not inferior to that with heparin-coated dialyzers (26/32 study periods [81%]). Visual inspection showed equal numbers of clean dialysis circuit bubble traps (vitamin E-coated, 34/121; heparin-coated, 32/120), whereas clean fiber bundles were more frequently noted with the vitamin E-coated compared with heparin-coated dialyzers (25/121 vs 2/120; P=0.002). LIMITATIONS Results may not extrapolate to critically ill patients. Differences in dialyzer transparency may account for visual inspection scores. CONCLUSIONS The success rate of 4-hour heparin-free hemodialysis sessions is lower than that previously claimed in uncontrolled studies. Vitamin E-coated and heparin-coated dialyzers exposed patients to similar and unacceptable high failure rates. Further studies are required to improve heparin-free hemodialysis.