[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Background:
Cystinosis is an autosomal recessive disorder marked by intralysosomal cystine accumulation. Patients present with generalized proximal tubular dysfunction called renal Fanconi syndrome. Urinary carnitine loss results in plasma and muscle carnitine deficiency, but no clinical signs of carnitine deficiency have been described. Also, the optimal dose of carnitine supplementation is undefined. This study aimed to determine whether currently recommended carnitine doses result in adequate correction of plasma carnitine.
Five cystinosis patients with renal Fanconi syndrome, aged 2-18 years, were included. L-carnitine was prescribed 50 mg/kg/day since diagnosis: median 36 (range 18-207) months. Total and free plasma and urine carnitine and carnitine profiles were measured at study onset, after stopping L-carnitine for 3 months and 3 months after reintroducing L-carnitine 50 mg/kg/day.
At study onset, plasma free carnitine was normal in all patients, total carnitine (1/5), acetylcarnitine (3/5), and several short- and medium-chain acylcarnitines ≤10 carbons (5/5) were increased indicating carnitine over-supplementation. Three months after cessation, carnitine profiles normalized and 3/5 patients showed plasma carnitine deficiency. Three months after reintroduction, plasma free carnitine normalized in all patients, however, carnitine profiles were disturbed in 4/5 patients. Urine free carnitine, acetylcarnitine, and acylcarnitines ≤10 carbons were increased in all patients independent of carnitine supplementation.
Administration of recommended doses L-carnitine (50 mg/kg/day) resulted in over-supplementation. Although the drug is considered to be rather safe, long-term effects of over-supplementation remain unknown warranting cautious use of high doses. Plasma carnitine profile might be used as a monitor, to prevent overdosing.
[Show abstract][Hide abstract] ABSTRACT: Promising renal replacement therapies include the development of a bio-artificial 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.
Experimental Cell Research 02/2014; 323(1). DOI:10.1016/j.yexcr.2014.02.011 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Cystinosis is an autosomal recessive disease caused by intralysosomal cystine accumulation, treated with cysteamine. Recently, new adverse effects of cysteamine were reported. Skin biopsies showed microvascular proliferation (angioendotheliomatosis). To examine the mechanism of angioendotheliomatosis associated with cysteamine toxicity, we examined the effect of cysteamine on human dermal microvascular endothelial cells (HDMVEC).
After cysteamine exposure (range 0-3.0 mM) during 24 h, cell viability was measured using water soluble tetrazolium salt-1 (WST-1) in both control HDMVEC and fibroblasts. Cell proliferation and apoptosis rate were measured in HDMVEC by bromodeoxyuridine (BrdU) incorporation and caspase 3 and caspase 7 activity, respectively. Intracellular glutathione (GSH) was measured in HDMVEC after cysteamine exposure of 0, 0.1 or 1.0 mM. Medium and cysteamine were refreshed every 6 h to mimic the in vivo situation. Next, cell viability in HDMVEC was measured after 24 h of GSH exposure (range 0-10.0 mM).
HDMVEC viability and proliferation increased after cysteamine exposure 0.03-3.0 mM (p < 0.01) and 0.03-1.0 mM (p = 0.01) respectively; cell viability in fibroblasts was not affected by incubation with cysteamine. Apoptosis remained unaffected by incubation with 0-1.0 mM cysteamine, 3.0 mM caused increased apoptosis. Intracellular GSH was significantly increased after incubation with cysteamine 0.1 mM (p = 0.02) and 1.0 mM (p < 0.01). HDMVEC viability increased after exposure to GSH 1.0-5.0 mM (p < 0.01).
Cysteamine concentrations, similar to those described in plasma of cystinosis patients, stimulate HDMVEC viability and proliferation and increase intracellular GSH content. We postulate that this mechanism might underlie angioendotheliomatosis induced by cysteamine.
[Show abstract][Hide abstract] ABSTRACT: A multicentre 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 are 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial disorders are a heterogeneous group of disorders affecting energy production of the body. Different consensus diagnostic criteria for mitochondrial disorders in childhood are available - Wolfson, Nijmegen and modified Walker criteria. Due to the extreme complexity of mitochondrial disorders in children, we decided to develop a diagnostic algorithm, applicable in clinical practice in Estonia, in order to identify patients with mitochondrial disorders among pediatric neonatology and neurology patients. Additionally, it was aimed to evaluate the live-birth prevalence of mitochondrial disorders in childhood. During the study period (2003-2009), a total of 22 children were referred to a muscle biopsy in suspicion of mitochondrial disorder based on the preliminary biochemical, metabolic and instrumental investigations. Enzymatic and/or molecular analysis confirmed mitochondrial disease in 5 of them - an SCO2 gene (synthesis of cytochrome c oxidase, subunit 2) defect, 2 cases of pyruvate dehydrogenase complex deficiency and 2 cases of combined complex I and IV deficiency. The live-birth prevalence for mitochondrial defects observed in our cohort was 1/20,764 live births. Our epidemiological data correlate well with previously published epidemiology data on mitochondrial diseases in childhood from Sweden and Australia, but are lower than in Finland.
[Show abstract][Hide abstract] ABSTRACT: Brody disease is a rare inherited myopathy due to reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA)1 activity caused by mutations in ATP2A1, which causes delayed muscle relaxation and silent cramps. So far the disease has mostly been diagnosed by measurement of SERCA1 activity. Since mutation analysis became more widely available, it has appeared that not all patients with reduced SERCA1 activity indeed have ATP2A1 mutations, and a distinction between Brody disease (with ATP2A1 mutations) and Brody syndrome (without ATP2A1 mutations) was proposed. We aim to compare the clinical features of patients with Brody disease and those with Brody syndrome and detect clinical features which help to distinguish between the two. In addition, we describe the Brody syndrome phenotype in more detail. We therefore performed a literature review on clinical features of both Brody disease and Brody syndrome and a cross-sectional clinical study consisting of questionnaires, physical examination, and a review of medical files in 17 Brody syndrome patients in our centre. The results showed that Brody disease presents with an onset in the 1st decade, a generalized pattern of muscle stiffness, delayed muscle relaxation after repetitive contraction on physical examination, and autosomal recessive inheritance. Patients with Brody syndrome more often report myalgia and experience a considerable impact on daily life. Future research should focus on the possible mechanisms of reduction of SERCA activity in Brody syndrome and other genetic causes, and on evaluation of treatment options.
[Show abstract][Hide abstract] ABSTRACT: The haemolytic uraemic syndrome (HUS) is characterised by haemolytic anaemia, thrombocytopenia and acute renal failure. The majority of cases are seen in childhood and are preceded by an infection with Shiga-like toxin producing Escherichia coli (STEC-HUS; so-called typical HUS). Non-STEC or atypical HUS (aHUS) is seen in 5 to 10% of all cases and occurs at all ages. These patients have a poorer outcome and prognosis than patients with STEC-HUS. New insights into the pathogenesis of aHUS were revealed by the identification of mutations in genes encoding proteins of the alternative pathway of the complement system in aHUS patients. Specific information of the causative mutation is important for individualised patient care with respect to choice and efficacy of therapy, the outcome of renal transplantation, and the selection of living donors. This new knowledge about the aetiology of the disease has stimulated the development of more specific treatment modalities. Until now, plasma therapy was used with limited success in aHUS, but recent clinical trials have demonstrated that patients with aHUS can be effectively treated with complement inhibitors, such as the monoclonal anti-C5 inhibitor eculizumab.
The Netherlands Journal of Medicine 04/2012; 70(3):121-9. · 1.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autosomal dominant polycystic kidney disease is caused by loss-of-function mutations in the PKD1 or PKD2 genes encoding respectively polycystin-1 and polycystin-2. Polycystin-2 stimulates the inositol trisphosphate (IP(3)) receptor (IP(3)R), a Ca(2+)-release channel in the endoplasmic reticulum (ER). The effect of ER-located polycystin-1 is less clear. Polycystin-1 has been reported both to stimulate and to inhibit the IP(3)R. We now studied the effect of polycystin-1 and of polycystin-2 on the IP(3)R activity under conditions where the cytosolic Ca(2+) concentration was kept constant and the reuptake of released Ca(2+) was prevented. We also studied the interdependence of the interaction of polycystin-1 and polycystin-2 with the IP(3)R. The experiments were done in conditionally immortalized human proximal-tubule epithelial cells in which one or both polycystins were knocked down using lentiviral vectors containing miRNA-based short hairpins. The Ca(2+) release was induced in plasma membrane-permeabilized cells by various IP(3) concentrations at a fixed Ca(2+) concentration under unidirectional (45)Ca(2+)-efflux conditions. We now report that knock down of polycystin-1 or of polycystin-2 inhibited the IP(3)-induced Ca(2+) release. The simultaneous presence of the two polycystins was required to fully amplify the IP(3)-induced Ca(2+) release, since the presence of polycystin-1 alone or of polycystin-2 alone did not result in an increased Ca(2+) release. These novel findings indicate that ER-located polycystin-1 and polycystin-2 operate as a functional complex. They are compatible with the view that loss-of-function mutations in PKD1 and in PKD2 both cause autosomal dominant polycystic kidney disease.
[Show abstract][Hide abstract] ABSTRACT: Background:
The hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy leading to acute kidney injury in children. In most cases it is triggered by an infection caused by Shiga-like toxin-producing Escherichia coli (STEC). Endothelial damage plays a central role in the pathogenesis of disease. Hemophilia A is a genetic disorder leading to factor VIII (FVIII) deficiency, an important factor in the coagulation system.
Here we describe a hemophilia A patient who developed HUS due to a STEC O26 infection. The patient developed not only acute kidney injury, but also severe gastro-intestinal and neurological complications. Increased amounts of recombinant FVIII (rFVIII) had to be administered during the acute phase of the disease to reach acceptable blood levels of FVIII, in order to control the hemorrhagic colitis and to prevent severe neurological complications.
The patient's treatment schedule of rFVIII during the HUS period was a serious challenge, and we cannot exclude that it contributed to the severity of the HUS by enhancing the thrombotic microangiopathic process. The role of factor VIII administration in the severe outcome of this disease is discussed.
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial disorders are a heterogeneous group of often multisystemic and early fatal diseases caused by defects in the oxidative phosphorylation (OXPHOS) system. Given the complexity and intricacy of the OXPHOS system, it is not surprising that the underlying molecular defect remains unidentified in many patients with a mitochondrial disorder. Here, we report the clinical features and diagnostic workup leading to the elucidation of the genetic basis for a combined complex I and IV OXPHOS deficiency secondary to a mitochondrial translational defect in an infant who presented with rapidly progressive liver failure, encephalomyopathy, and severe refractory lactic acidemia. Sequencing of the GFM1 gene revealed two inherited novel, heterozygous mutations: a.539delG (p.Gly180AlafsX11) in exon 4 which resulted in a frameshift mutation, and a second c.688G > A (p.Gly230Ser) mutation in exon 5. This missense mutation is likely to be pathogenic since it affects an amino acid residue that is highly conserved across species and is absent from the dbSNP and 1,000 genomes databases. Review of literature and comparison were made with previously reported cases of this recently identified mitochondrial disorder encoded by a nuclear gene. Although limited in number, nuclear gene defects causing mitochondrial translation abnormalities represent a new, rapidly expanding field of mitochondrial medicine and should potentially be considered in the diagnostic investigation of infants with progressive hepatoencephalomyopathy and combined OXPHOS disorders.