Eculizumab Rescue Therapy in Patients With Recurrent Atypical Hemolytic Uremic Syndrome After Kidney Transplantation.

Introduction: Since 2016, kidney transplantation in patients with atypical hemolytic uremic syndrome (aHUS) in the Netherlands is performed without eculizumab prophylaxis. Eculizumab is given in case of posttransplant aHUS recurrence. Eculizumab therapy is monitored in the CUREiHUS study. Methods: All participating kidney transplant patients who received eculizumab therapy for a suspected posttransplant aHUS recurrence were evaluated. Overall recurrence rate was monitored prospectively at Radboud University Medical Center. Results: In the period from January 2016 until October 2020, we included 15 (12 females, 3 males; median age 42 years, range 24-66 years) patients with suspected aHUS recurrence after kidney transplantation in this study. The time interval to recurrence showed a bimodal distribution. Seven patients presented early after transplantation (median 3 months, range 0.3-8.8 months), with typical aHUS features: rapid loss of estimated glomerular filtration rate (eGFR) and laboratory signs of thrombotic microangiopathy (TMA). Eight patients presented late (median 46 months, range 18-69 months) after transplantation. Of these, only 3 patients had systemic TMA, whereas 5 patients presented with slowly deteriorating eGFR without systemic TMA. Treatment with eculizumab resulted in improvement or stabilization of eGFR in 14 patients. Eculizumab discontinuation was tried in 7 patients; however, it was successful only in 3. At the end of the follow-up (median 29 months, range 3-54 months after start of eculizumab), 6 patients had eGFR <30 ml/min per 1.73 m2. Graft loss had occurred in 3 of them. Overall, aHUS recurrence rate without eculizumab prophylaxis was 23%. Conclusions: Rescue treatment of posttransplant aHUS recurrence is effective; however, some patients suffer from irreversible loss of kidney function, likely caused by delayed diagnosis and treatment and/or too aggressive discontinuation of eculizumab. Physicians should be aware that recurrence of aHUS can present without evidence of systemic TMA.

Hyperhomocysteinemia: a trigger for complement-mediated TMA?

A 34-year-old man of North African descent was referred to the emergency department because of malignant hypertension (220/113 mmHg), acute visual disturbances and acute kidney failure (serum creatinine 14.0 mg/dL). Blood analysis was compatible with thrombotic microangiopathy (TMA). Kidney biopsy confirmed this diagnosis with histological changes including intimal edema, arteriolar thrombi, and severe tubulointerstitial damage. Fundoscopy showed hypertensive retinopathy stage IV. Subsequent biochemical screening revealed normal complement testing and a marked elevation in homocysteine concentration (161 µmol/L; normal value 7-15 µmol/L). Other secondary causes of TMA were excluded. Further genetic testing for cobalamin C (cblC) deficiency showed no pathogenic mutations in the MMACHC gene. However, a homozygous c.665C>T polymorphism (NM_005957.4) in the methylenetetrahydrofolate reductase (MTHFR) gene was found explaining the severe hyperhomocysteinemia due to reduced activity of MTHFR. Additional genetic testing for alternative complement pathway proteins showed mutations in the genes encoding factor H and factor B, both categorized as possibly pathogenic using mutation prediction software. This is the first described case of TMA in a patient with severe hyperhomocysteinemia caused by a genetic defect other than cblC. We postulate that endothelial damage due to hyperhomocysteinemia and hypertension could have triggered the TMA episode in this patient with two possible predisposing pathogenic mutations in the alternative complement pathway. Furthermore, our case demonstrates the need for complete full diagnostic testing in patients with TMA.

Heme as Possible Contributing Factor in the Evolvement of Shiga-Toxin Escherichia coli Induced Hemolytic-Uremic Syndrome.

Shiga-toxin (Stx)-producing Escherichia coli hemolytic-uremic syndrome (STEC-HUS) is one of the most common causes of acute kidney injury in children. Stx-mediated endothelial injury initiates the cascade leading to thrombotic microangiopathy (TMA), still the exact pathogenesis remains elusive. Interestingly, there is wide variability in clinical presentation and outcome. One explanation for this could be the enhancement of TMA through other factors. We hypothesize that heme, as released during extensive hemolysis, contributes to the etiology of TMA. Plasma levels of heme and its scavenger hemopexin and degrading enzyme heme-oxygenase-1 (HO-1) were measured in 48 STEC-HUS patients. Subsequently, the effect of these disease-specific heme concentrations, in combination with Stx, was assessed on primary human glomerular microvascular endothelial cells (HGMVECs). Significantly elevated plasma heme levels up to 21.2 µM were found in STEC-HUS patients compared to controls and were inversely correlated with low or depleted plasma hemopexin levels (R2 -0.74). Plasma levels of HO-1 are significantly elevated compared to controls. Interestingly, especially patients with high heme levels (n = 12, heme levels above 75 quartile range) had high plasma HO-1 levels with median of 332.5 (86-720) ng/ml (p = 0.008). Furthermore, heme is internalized leading to a significant increase in reactive oxygen species production and stimulated both nuclear translocation of NF-κB and increased levels of its target gene (tissue factor). In conclusion, we are the first to show elevated heme levels in patients with STEC-HUS. These increased heme levels mediate endothelial injury by promoting oxidative stress and a pro-inflammatory and pro-thrombotic state. Hence, heme may be a contributing and driving factor in the pathogenesis of STEC-HUS and could potentially amplify the cascade leading to TMA.

Diagnosis and treatment of C3 glomerulopathy in a center of expertise.

C3 glomerulopathy is a rare renal disease that has been distinguished as a renal disease for about 10 years. It is caused by an excessive activation of the alternative complement pathway in the circulation, which leads to deposition of complement factor C3 in glomeruli. It is diagnosed based on clinical presentation, histological patterns in a kidney biopsy and tests of the complement pathways. It can closely resemble immune complexmediated glomerulonephritis and postinfectious glomerulonephritis. Renal failure develops in up to half of all patients within 10 years after presentation. A curative treatment is not available. Treatment relies on renoprotective measures, occasional immunosuppressive medication and experimental novel complement inhibitors. Because the disease is rare, its care and cure are concentrated in centers of expertise. Here we provide an overview of the state-ofthe-art diagnosis and treatment of C3 glomerulopathy in a center of expertise in the Netherlands.

Genotype-phenotype correlations of low-frequency variants in the complement system in renal disease and age-related macular degeneration.

Genetic alterations in the complement system have been linked to a variety of diseases, including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), and age-related macular degeneration (AMD). We performed sequence analysis of the complement genes complement factor H (CFH), complement factor I (CFI), and complement C3 (C3) in 866 aHUS/C3G and 697 AMD patients. In total, we identified 505 low-frequency alleles, representing 121 unique variants, of which 51 are novel. CFH contained the largest number of unique low-frequency variants (n = 64; 53%), followed by C3 (n = 32; 26%) and CFI (n = 25; 21%). A substantial number of variants were found in both patients groups (n = 48; 40%), while 41 (34%) variants were found only in aHUS/C3G and 32 (26%) variants were AMD specific. Genotype-phenotype correlations between the disease groups identified a higher frequency of protein altering alleles in short consensus repeat 20 (SCR20) of factor H (FH), and in the serine protease domain of factor I (FI) in aHUS/C3G patients. In AMD, a higher frequency of protein-altering alleles was observed in SCR3, SCR5, and SCR7 of FH, the SRCR domain of FI, and in the MG3 domain of C3. In conclusion, we observed a substantial overlap of variants between aHUS/C3G and AMD; however, there is a distinct clustering of variants within specific domains.

REducing STEroids in Relapsing Nephrotic syndrome: the RESTERN study- protocol of a national, double-blind, randomised, placebo-controlled, non-inferiority intervention study.

INTRODUCTION: Oral corticosteroids are the first-line treatment for idiopathic childhood nephrotic syndrome. Most children experience several relapses, needing repeated courses of corticosteroid therapy. This exposes them to side effects and long-term complications. For most patients, long-term prognosis is for complete resolution of the disease over time and maintenance of normal kidney function. Therefore, it is vital to focus on minimising adverse events of the disease and its therapy. Unfortunately, no randomised controlled trials are available to determine the optimal corticosteroid treatment of an infrequent relapse of nephrotic syndrome. Recent studies show that treatment schedules for the first episode can safely be shortened to 2 months. The hypothesis of the REducing STEroids in Relapsing Nephrotic syndrome (RESTERN) study is that a 4-week reduction of alternate-day steroids after inducing remission is effective and safe, reduces steroid exposure by 35% on average and is therefore preferable. METHODS AND ANALYSIS: The RESTERN study is a nationwide, double-blind, randomised, placebo-controlled, non-inferiority intervention study. Children aged 1-18 years with a relapse of steroid-sensitive nephrotic syndrome are eligible for this study. Study subjects (n=144) will be randomly assigned to either current standard therapy in the Netherlands or a reduced prednisolone schedule. The primary outcome of the RESTERN study is the time to first relapse after the final prednisolone dose. The secondary outcomes are the number or relapses, progression to frequent relapsing or steroid dependent nephrotic syndrome and the cumulative dosage of prednisolone during the study period. ETHICS AND DISSEMINATION: This non-inferiority trial will be performed in accordance with the Declaration of Helsinki and has been approved by the medical ethical committee of Arnhem-Nijmegen and the Dutch Competent Authority (Central Committee on Research Involving Human Subjects, CCMO). After completion of this study, results will be published in national and international peer-reviewed scientific journals. Papers will be published according to CCMO guidelines. The final report will be made available to trial participants. TRIAL REGISTRATION NUMBER: NTR5670, EudraCT no 2016-002430-76.

Mild intracellular acidification by dexamethasone attenuates mitochondrial dysfunction in a human inflammatory proximal tubule epithelial cell model.

Septic acute kidney injury (AKI) associates with poor survival rates and often requires renal replacement therapy. Glucocorticoids may pose renal protective effects in sepsis via stimulation of mitochondrial function. Therefore, we studied the mitochondrial effects of dexamethasone in an experimental inflammatory proximal tubule epithelial cell model. Treatment of human proximal tubule epithelial cells with lipopolysaccharide (LPS) closely resembles pathophysiological processes during endotoxaemia, and led to increased cytokine excretion rates and cellular reactive oxygen species levels, combined with a reduced mitochondrial membrane potential and respiratory capacity. These effects were attenuated by dexamethasone. Dexamethasone specifically increased the expression and activity of mitochondrial complex V (CV), which could not be explained by an increase in mitochondrial mass. Finally, we demonstrated that dexamethasone acidified the intracellular milieu and consequently reversed LPS-induced alkalisation, leading to restoration of the mitochondrial function. This acidification also provides an explanation for the increase in CV expression, which is expected to compensate for the inhibitory effect of the acidified environment on this complex. Besides the mechanistic insights into the beneficial effects of dexamethasone during renal cellular inflammation, our work also supports a key role for mitochondria in this process and, hence, provides novel therapeutic avenues for the treatment of AKI.

Intranuclear Aggregates Precede Clinical Onset in Oculopharyngeal Muscular Dystrophy.

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) has long been characterized by a combination of bilateral ptosis and dysphagia and subsequent limb girdle weakness. The role of the typical intranuclear inclusion in the pathophysiology is unresolved. OBJECTIVE: The aim of this study was to describe the clinical and histopathological features of oculopharyngeal muscular dystrophy (OPMD). We examined this in a Dutch cohort including presymptomatic Ala-expanded-PABPN1 carriers and late symptomatic patients. METHODS: We performed a prospective, observational study in OPMD patients and adult children of genetically confirmed OPMD patients. The study includes a structured history, a detailed neurological examination, muscle histology and biochemical analysis. Forty patients and 18 adult children participated in this study, among whom were six presymptomatic mutation carriers. One patient died during the study and had given permission to autopsy. RESULTS: In addition to the characteristic OPMD symptoms including ptosis and dysphagia, other symptoms such as limb girdle and axial weakness, and external ophthalmoplegia were frequently observed. Intranuclear aggregates were observed in the biopsies of presymptomatic carriers. Biochemical analysis of the biopsies of the presymptomatic carriers showed no mitochondrial dysfunction. The autopsy showed that muscle weakness correlated with histopathological findings in five different muscles in an individual patient. CONCLUSIONS: The main findings of this nationwide study are the presence of intranuclear aggregates before clinical onset and the absence of mitochondrial changes in Ala-expanded-PABPN1 carriers. This indicates that the expression of Ala-expanded-PABPN1 causes the formation of nuclear aggregates before the onset of muscle weakness. Normal results of biochemical analysis in presymptomatic carriers suggest that possible mitochondrial dysfunction occurs later. Furthermore we confirmed that limb girdle weakness occurs frequently in Dutch OPMD patients. This study thus expands the OPMD research towards characterization of presymptomatic carriers.

Bioengineered kidney tubules efficiently excrete uremic toxins.

The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs.

Regularized MANOVA (rMANOVA) in untargeted metabolomics.

Many advanced metabolomics experiments currently lead to data where a large number of response variables were measured while one or several factors were changed. Often the number of response variables vastly exceeds the sample size and well-established techniques such as multivariate analysis of variance (MANOVA) cannot be used to analyze the data. ANOVA simultaneous component analysis (ASCA) is an alternative to MANOVA for analysis of metabolomics data from an experimental design. In this paper, we show that ASCA assumes that none of the metabolites are correlated and that they all have the same variance. Because of these assumptions, ASCA may relate the wrong variables to a factor. This reduces the power of the method and hampers interpretation. We propose an improved model that is essentially a weighted average of the ASCA and MANOVA models. The optimal weight is determined in a data-driven fashion. Compared to ASCA, this method assumes that variables can correlate, leading to a more realistic view of the data. Compared to MANOVA, the model is also applicable when the number of samples is (much) smaller than the number of variables. These advantages are demonstrated by means of simulated and real data examples. The source code of the method is available from the first author upon request, and at the following github repository: https://github.com/JasperE/regularized-MANOVA.

Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

Adult-onset congenital thrombotic thrombocytopenic purpura caused by a novel compound heterozygous mutation of the ADAMTS13 gene.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease, characterized by microangiopathic hemolytic anaemia and thrombocytopenia, resulting in neurologic and/or renal abnormalities. We report a 49-year-old patient with a history of thrombotic events, renal failure, and thrombocytopenia. Blood analysis demonstrated no ADAMTS13 activity in the absence of antibodies against ADAMTS13. The complete ADAMTS13 gene was sequenced, and two mutations were identified: one mutation on exon 24 (Arg1060Asp), which had previously been described, and a mutation on exon 27 (Met1260IlefsX34), which has not been reported. For these mutations, compound heterozygosity appears to be necessary to cause TTP, as family members of the patient display only one of the mutations and all displayed normal ADAMTS13 activity.

Eculizumab in Pediatric Dense Deposit Disease.

BACKGROUND AND OBJECTIVES: Dense deposit disease (DDD), a subtype of C3 glomerulopathy, is a rare disease affecting mostly children. Treatment options are limited. Debate exists whether eculizumab, a monoclonal antibody against complement factor C5, is effective in DDD. Reported data are scarce, especially in children. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The authors analyzed clinical and histologic data of five pediatric patients with a native kidney biopsy diagnosis of DDD. Patients received eculizumab as therapy of last resort for severe nephritic or nephrotic syndrome with alternative pathway complement activation; this therapy was given only when the patients had not or only marginally responded to immunosuppressive therapy. Outcome measures were kidney function, proteinuria, and urine analysis. RESULTS: In all, seven disease episodes were treated with eculizumab (six episodes of severe nephritic syndrome [two of which required dialysis] and one nephrotic syndrome episode). Median age at treatment start was 8.4 (range, 5.9-13) years. For three treatment episodes, eculizumab was the sole immunosuppressive treatment. In all patients, both proteinuria and renal function improved significantly within 12 weeks of treatment (median urinary protein-to-creatinine ratio of 8.5 [range, 2.2-17] versus 1.1 [range, 0.2-2.0] g/g, P<0.005, and eGFR of 58 [range, 17-114] versus 77 [range, 50-129] ml/min per 1.73 m(2), P<0.01). A striking finding was the disappearance of leukocyturia within 1 week after the first eculizumab dose in all five episodes with leukocyturia at treatment initiation. CONCLUSIONS: In this case series of pediatric patients with DDD, eculizumab treatment was associated with reduction in proteinuria and increase in eGFR. Leukocyturia resolved within 1 week of initiation of eculizumab treatment. These results underscore the need for a randomized trial of eculizumab in DDD.

Complement activation patterns in atypical haemolytic uraemic syndrome during acute phase and in remission.

Atypical haemolytic uraemic syndrome (aHUS) is associated with (genetic) alterations in alternative complement pathway. Nevertheless, comprehensive evidence that the complement system in aHUS patients is more prone to activation is still lacking. Therefore, we performed a thorough analysis of complement activation in acute phase and in remission of this disease. Complement activation patterns of the aHUS patients in acute phase and in remission were compared to those of healthy controls. Background levels of complement activation products C3b/c, C3bBbP and terminal complement complex (TCC) were measured using enzyme-linked immunosorbent assay (ELISA) in ethylenediamine tetraacetic acid (EDTA) plasma. In vitro-triggered complement activation in serum samples was studied using zymosan-coating and pathway-specific assay. Furthermore, efficiencies of the C3b/c, C3bBbP and TCC generation in fluid phase during spontaneous activation were analysed. Patients with acute aHUS showed elevated levels of C3b/c (P < 0·01), C3bBbP (P < 0·0001) and TCC (P < 0·0001) in EDTA plasma, while values of patients in remission were normal, compared to those of healthy controls. Using data from a single aHUS patient with complement factor B mutation we illustrated normalization of complement activation during aHUS recovery. Serum samples from patients in remission showed normal in vitro patterns of complement activation and demonstrated normal kinetics of complement activation in the fluid phase. Our data indicate that while aHUS patients have clearly activated complement in acute phase of the disease, this is not the case in remission of aHUS. This knowledge provides important insight into complement regulation in aHUS and may have an impact on monitoring of these patients, particularly when using complement inhibition therapy.

Biotechnological challenges of bioartificial kidney engineering.

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.

A novel method for direct measurement of complement convertases activity in human serum.

Complement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function leads directly or indirectly to pathological conditions, including higher infection rate, kidney diseases, autoimmune- or neurodegenerative diseases and ischaemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and, thus, convertase formation occurs under non-physiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. The use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of haemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong the half-life of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay.

A morphological and functional comparison of proximal tubule cell lines established from human urine and kidney tissue.

Promising renal replacement therapies include the development of a bioartificial kidney using functional human kidney cell models. In this study, human conditionally immortalized proximal tubular epithelial cell (ciPTEC) lines originating from kidney tissue (ciPTEC-T1 and ciPTEC-T2) were compared to ciPTEC previously isolated from urine (ciPTEC-U). Subclones of all ciPTEC isolates formed tight cell layers on Transwell inserts as determined by transepithelial resistance, inulin diffusion, E-cadherin expression and immunocytochemisty. Extracellular matrix genes collagen I and -IV α1 were highly present in both kidney tissue derived matured cell lines (p<0.001) compared to matured ciPTEC-U, whereas matured ciPTEC-U showed a more pronounced fibronectin I and laminin 5 gene expression (p<0.01 and p<0.05, respectively). Expression of the influx carrier Organic Cation Transporter 2 (OCT-2), and the efflux pumps P-glycoprotein (P-gp), Multidrug Resistance Protein 4 (MRP4) and Breast Cancer Resistance Protein (BCRP) were confirmed in the three cell lines using real-time PCR and Western blotting. The activities of OCT-2 and P-gp were sensitive to specific inhibition in all models (p<0.001). The highest activity of MRP4 and BCRP was demonstrated in ciPTEC-U (p<0.05). Finally, active albumin reabsorption was highest in ciPTEC-T2 (p<0.001), while Na(+)-dependent phosphate reabsorption was most abundant in ciPTEC-U (p<0.01). In conclusion, ciPTEC established from human urine or kidney tissue display comparable functional PTEC specific transporters and physiological characteristics, providing ideal human tools for bioartificial kidney development.

Uremic toxins inhibit renal metabolic capacity through interference with glucuronidation and mitochondrial respiration.

During chronic kidney disease (CKD), drug metabolism is affected leading to changes in drug disposition. Furthermore, there is a progressive accumulation of uremic retention solutes due to impaired renal clearance. Here, we investigated whether uremic toxins can influence the metabolic functionality of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) with the focus on UDP-glucuronosyltransferases (UGTs) and mitochondrial activity. Our results showed that ciPTEC express a wide variety of metabolic enzymes, including UGTs. These enzymes were functionally active as demonstrated by the glucuronidation of 7-hydroxycoumarin (7-OHC; K(m) of 12±2µM and a V(max) of 76±3pmol/min/mg) and p-cresol (K(m) of 33±13µM and a V(max) of 266±25pmol/min/mg). Furthermore, a wide variety of uremic toxins, including indole-3-acetic acid, indoxyl sulfate, phenylacetic acid and kynurenic acid, reduced 7-OHC glucuronidation with more than 30% as compared with controls (p<0.05), whereas UGT1A and UGT2B protein expressions remained unaltered. In addition, our results showed that several uremic toxins inhibited mitochondrial succinate dehydrogenase (i.e. complex II) activity with more than 20% as compared with controls (p<0.05). Moreover, indole-3-acetic acid decreased the reserve capacity of the electron transport system with 18% (p<0.03). In conclusion, this study shows that multiple uremic toxins inhibit UGT activity and mitochondrial activity in ciPTEC, thereby affecting the metabolic capacity of the kidney during CKD. This may have a significant impact on drug and uremic retention solute disposition in CKD patients.

A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders.

A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large variations in observed enzyme activities, we show that inter-laboratory comparison of patient sample test results is possible by using normalization against a control sample.