Nomy Levin-Iaina

Late Diagnosis of Primary Hyperoxaluria Type 2 in the Adult: Effect of a Novel Mutation in GRHPR Gene on Enzymatic Activity and Molecular Modeling

PURPOSE Genetic causes of nephrolithiasis are underestimated. Primary hyperoxaluria type 2 is a rare autosomal recessive disease caused by mutations in the GRHPR gene, leading to an accumulation of oxalate and L-glycerate with recurrent kidney stone formation and nephrocalcinosis, and the later development of renal failure and systemic oxalate depositions. We studied the effects of a novel GRHPR mutation on GRHPR enzymatic activity and molecular modeling. MATERIALS AND METHODS Genomic DNA from a 50-year-old male with a late diagnosis of primary hyperoxaluria type 2 was extracted, analyzed and compared with the established human GRHPR gene sequence. Restriction enzyme analysis of the patient, 30 healthy controls and 30 patients with nephrolithiasis of various causes was done to confirm the presence of the mutation. GRHPR activity was analyzed by site directed mutagenesis of WT and mutant clones. We studied the effects of the mutation on enzymatic molecular modeling. RESULTS We found the novel homozygous single missense mutation A975G in exon 9, creating an amino acid change from asparagine to aspartic acid in position 312. No mutations were detected in restriction enzyme analysis in all 30 healthy controls and 30 patients with nephrolithiasis of various causes. Transfected cells with the mutant clone showed abolished GRHPR activity. Molecular modeling studies revealed that the mutation was likely to disrupt the correct folding of the GRHPR substrate binding domain, hence affecting the enzyme active site. CONCLUSIONS Primary hyperoxaluria type 2 should be considered in patients at adult stone clinics who have had a history of nephrolithiasis since childhood, especially in those with consanguineous parents. Biochemical analysis followed by mutation identification should be the approach for making the definitive diagnosis of primary hyperoxaluria type 2.

The emerging role of NO and IGF‐1 in early renal hypertrophy in STZ‐induced diabetic rats

Diabetic nephropathy (DN) is a major complication of diabetes mellitus, and the most common cause of end‐stage renal disease. DN is characterized by early hyperfiltration and renal hypertrophy, which are associated with increased renal insulin‐like growth factor‐1 (IGF‐1) levels. The relationship between IGF‐1 and nitric oxide (NO) in DN is not established. The aim of this study was to investigate the effects of NO system modulation on the IGF‐1‐mediated hypertrophy and hyperfiltration during the first week after diabetes induction.

Renalase, hypertension, and kidney - the discussion continues.

Hypertension and cardiovascular complications are very common in chronic kidney disease (CKD). Overactivation of sympathetic nervous system is also widely recognized in CKD. Renalase may play an important role in the control of blood pressure (BP) by its regulatory function of catecholamine metabolism. Renalase could be synthesized not only by the kidney but also by cardiomyocytes, liver, and adipose tissue. It probably exerts a hypotensive action, at least in animal models. Whether it metabolizes catecholamines remains to be proved. Another issue that remains to be resolved is the relationship between renalase and renal natriuresis and phosphaturia. In this review, the updated experimental and clinical data on renalase are presented and possible interactions with the endothelium are discussed. Renalase is “a new postulated therapeutic target.” Proof of concept studies are needed to define the pathophysiological link between the kidney, sympathetic tone, BP, and cardiovascular complications.

Truncating mutations in the chloride/proton ClC-5 antiporter gene in Seven Jewish Israeli families with Dent's 1 disease.

Dent’s disease is an X-linked hereditary renal tubular disorder characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets and progressive renal failure. About 60% of patients have mutations in the CLCN5 gene (Dent 1), which encodes a kidney-specific chloride/proton antiporter, and 15% of patients have mutations in the OCRL1 gene (Dent 2). The aim of the study was to identify CLCN5 mutations in Jewish Israeli families with Dent‘s disease and to characterize the associated clinical syndromes. We studied 17 patients from 14 unrelated Israeli families with a clinical diagnosis of Dent’s disease. LMWP was detected in all patients. Most of the affected individuals had hypercalciuria and nephrocalcinosis. Renal stones were found in 1 patient, and renal insufficiency developed in 2 patients. We identified six different truncating CLCN5 mutations that were segregated with the disease in 7 families: three nonsense mutations (Arg28stop, Arg467stop and Arg637stop), one deletion mutation (505delA) and two novel mutations, consisted of one deletion mutation (1493delG) and one insertion mutation (409insC). All the mutations cause premature termination of protein translation and result in a non-functional truncated protein. The clinical characteristics of patients with different mutations were, in general, similar.

Iron metabolism in hemodialyzed patients - a story half told?

Introduction All living organisms have evolved sophisticated mechanisms to maintain appropriate iron levels in their cells and within their body. Recently our understanding of iron metabolism has dramatically increased. Overt labile plasma iron (LPI) represents a component of non-transferrin bound iron (NTBI) that is both redox active and chelatable, capable of permeating into organs and inducing tissue iron overload. The LPI measures the iron-specific capacity of a given sample to produce reactive oxygen species. We studied for the first time NTBI correlations with markers of iron status and inflammation in prevalent hemodialyzed patients. Material and methods Complete blood count, urea, serum lipids, fasting glucose, creatinine, ferritin, serum iron, total iron binding capacity (TIBC) were studied by standard laboratory method. The NTBI was assessed commercially available kits from Aferrix Ltd in Tel Aviv, Israel. A test result of 0.6 units of LPI or more indicates a potential for iron-mediated production of reactive oxygen species in the sample. Results Patients with LPI units ≥ 0.6 had higher serum iron, erythropoiesis stimulating agents (ESA) dose, ferritin, high-sensitivity C-reactive protein (hsCRP), hepcidin and lower hemojuvelin. In hemodialyzed patients NTBI correlated with hsCRP (r = 0.37, p < 0.01), ferritin (r = 0.41, p < 0.001), IL-6 (r = 0.43, p < 0.001). In multivariate analysis predictors of NTBI were hemoglobin and alkaline phosphatase, explaining 58% of the variability Conclusions Elevated NTBI in HD may be due to disturbed iron metabolism. Anemia and liver function might also contribute to the presence of NTBI in this population.

Zonulin, Iron Status, and Anemia in Kidney Transplant Recipients: Are They Related?

BACKGROUND In patients after kidney transplantation, anemia is relatively common and is associated with impaired kidney function, subclinical inflammatory state, and immunosuppressive treatment. Zonulin-prehaptoglobin-2, a newly discovered protein, is necessary for integrity of intracellular tight junctions in the gut. Taking into consideration iron metabolism, including its absorption in the gut, we designed a cross-sectional study to look for the possible interactions among zonulin, iron status, and anemia in kidney transplant recipients. METHODS The study was performed on 72 stable kidney transplant recipients and 22 healthy volunteers. Zonulin, iron status, and inflammatory markers were assessed with the use of commercially available kits. RESULTS Zonulin was significantly lower in kidney allograft recipients than in healthy volunteers (P < .001). Zonulin correlated with systolic blood pressure (r = -0.33; P < .05), thyroid-binding globulin (r = 0.24; P < .05), hematocrit (r = 0.28; P < .005), hemoglobin (r = 0.32; P < .01), total protein (r = -0.33; P < .01), erythrocyte count (r = 0.26; P < .05), and fasting glucose (r = -0.25; P < .05). Zonulin was not affected by sex, type of immunosuppressive therapy, presence of diabetes, coronary artery disease, heart failure, hypertension, or cause of end-stage renal disease. Zonulin was not related to any of the iron parameters studied. In multiple regression analysis, predictors of zonulin were total protein and thyroglobulin-binding protein, explaining 46% of variation. CONCLUSIONS Zonulin, with its poorly defined function, does not seem to play a role in the anemia in kidney allograft recipients; however, it seems to be related to the absorption process in the gut.

Molecular Study of Proteinuria in Patients Treated with B12 Supplements: Do Not Forget Megaloblastic Anemia Type 1

Background/Aims: Current consensus supports the notion that proteinuria is a marker of renal disease with prognostic implications. Whereas most chronic kidney disease patients with proteinuria would often require antiproteinuric agents, there are some exceptions. Megaloblastic anemia type 1 (MGA1) is characterized by megaloblastic anemia due to congenital selective vitamin B12 malabsorption and proteinuria. In the present study, we describe 2 Israeli Jewish patients with MGA1 and isolated proteinuria. Methods: Because of their origin, the patients were screened for the presence of the already studied Tunisian AMN mutation, by direct sequencing the corresponding region from genomic DNA. PCR products were purified and sequenced. Results: Genomic DNA sequencing of the AMN gene of both patients confirmed that the acceptor splice site in intron 3 was changed from CAG to CGG (208-2A→G). Conclusion: We determined the molecular basis of MGA1 in both patients and discuss the involvement of the cubilin/AMN complex in this pathology and its role in the development of the proteinuria. We also discuss the questionable significance of antiproteinuric treatment for these patients.

Iron metabolism in kidney allograft recipients: still a mystery?

INTRODUCTION All living organisms have evolved sophisticated mechanisms to maintain appropriate iron levels in their cells and within their body. Recently our understanding of iron metabolism has dramatically increased. Overt labile plasma iron (LPI) represents a component of non-transferrin-bound iron (NTBI) that is both redox active and chelatable, capable of permeating into organs and inducing tissue iron overload. LPI measures the iron-specific capacity of a given sample to produce reactive oxygen species. We studied NTBI correlations with markers of iron status and inflammation in prevalent kidney allograft recipients. METHODS Complete blood count, urea, creatinine, serum lipids, fasting glucose, ferritin, serum iron, and total iron-binding capacity (TIBC) were studied by standard laboratory method in the hospital central laboratory. NTBI was assessed by FeROS eLPI kit by Aferrix Ltd (Tel Aviv, Israel). A test result of 0.6 U of LPI or more indicated a potential for iron-mediated production of reactive oxygen species in the sample. RESULTS In kidney transplant recipients NTBI was correlated with TIBC (r=.46, P<.001) and ferritin (r=.31, P<.05), with tendencies to correlate with C-reactive protein. Patients with LPI units≥0.6 showed higher serum iron (P<.05), TIBC (P<.05), ferritin (P<.001) and mean corpuscular volume. High ferritin values together with elevated NTBI content were observed among patients undergoing multiple transfusions before and/or after transplantation. CONCLUSIONS Elevated NTBI as well as ferritin levels in kidney transplant patients may be due to disturbed iron metabolism, since the human body has no possibility to remove an iron excess. NTBI could be responsible for excessive synthesis of reactive oxygen species. Therefore, it may be linked to complications such as atherosclerosis, which is frequently encountered among this population.

Copeptin and Its Relation to Arteriovenous Fistula (AVF) Type and NYHA Class in Hemodialysis Patients

Copeptin is cosynthesized with vasopressin, also known as anti-diuretic hormone, with similar plasma levels. In the past 2 years, copeptin has been studied as a diagnostic and prognostic marker in infections and other diseases. In patients with decompensated heart failure, copeptin was an accurate prognostic marker for mortality. Cardiovascular disease is a major contributor to the mortality and morbidity in chronic kidney disease. Creation of an arteriovenous fistula (AVF) might contribute to the development or worsening of congestive heart failure (CHF). The aim of the study was to assess associations between copeptin, New York Heart Association (NYHA) class, and the location of the AVF in hemodialysis (HD) patients. The cross-sectional study was performed on a cohort of 93 clinically stable HD patients. Patients with proximal AVF tend to be older, with decreased renal residual function and increased NYHA functional class. These patients were also highly anemic, had more acidosis, and had increased high-sensitivity C-reactive protein along with increased copeptin and NT-proBNP levels. These changes were also associated with significant changes in all intra-cardiac dimensions, including right ventricle, both atria, and intraventricular septum and increase in end-systolic and end-diastolic left ventricular intra-cardiac dimensions. In multiple logistic regression analysis, the only associate of copeptin was NYHA functional class. Copeptin level in HD patients depends on cardiac function and it might be involved in the pathophysiology of cardiovascular disease in these patients. Proximal AVF creation might contribute to the development or worsening of CHF in HD patients.

Renal disease with OCRL1 mutations: Dent-2 or Lowe syndrome?

Dent disease is an X-linked tubulopathy, characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis that may progress to advanced renal failure [1,2]. During the last decade, loss-of-function mutations of the CLCN5 gene, which is located in chromosome Xp11.22 and encodes the renal chloride/proton antiporter ClC-5, have been consistently reported in patients with Dent disease [3]. However, in about 40% of patients with a Dent-like phenotype, no CLCN5 mutations are found [4]. In 2005, Hoopes et al. [4] showed for the first time that the Dent phenotype may also be caused by mutations in the OCRL1 gene, which encodes a phophatidylinositol 4,5-phosphate (PIP2) 5-phosphatase, located in the trans-Golgi network. These findings were confirmed by other reports [4–6] and led to the definition of two types of Dent disease: Dent-1, caused by CLCN5 mutations (OMIM #300009) and Dent-2, caused by OCRL1 mutations (OMIM #300555), which accounts for about 15–20% of Dent cases [7]. PIP2 5-phosphatase, which is encoded by the OCRL1 gene, is expressed ubiquitously in human tissues, including the eyes, kidneys and brain, the main organs involved in Lowe syndrome. This protein is distributed predominantly in the Golgi complex, lysosomes and endosomes [8]. Both ClC-5 and PIP2 5-phosphatase are expressed in endosomes of the proximal tubular cells and thought to be related to the recycling of multi-ligand receptors, such as megalin and cubilin, which are commonly involved in both Dent-1 and Dent-2 disease. Further genetic heterogeneity is assumed to exist, since there are patients expressing the distinctive phenotype of Dent disease, in which no mutation was identified in either CLCN5 or OCRL1 genes [4]. The OCRL1 gene is also mutated in the oculocerebrorenal syndrome of Lowe (OMIM #309000), which is a rare X-linked disorder, characterized by bilateral congenital cataracts, mental retardation and a selective proximal tubular dysfunction very similar to the renal phenotype of patients with Dent disease [9]. However, progressive renal failure is common, and is typically more aggressive and occurs at an earlier age than in Dent-2 disease. Thus, loss of Ocrl1 function can cause a spectrum of renal, as well as systemic symptoms. In this issue of Journal of Pediatric Genetics, Bockenhauer et al. [10] report their study of 14CLCN5 negative patients from 12 families with a phenotype resembling Dent-2 disease, for defects in OCRL1. In six of these patients, they identified three novel mutations and another three known mutations in the OCRL1 gene. None of these mutations has been described in patients with the classic Lowe syndrome. The renal phenotype of these patients was similar to that of patients harboring the CLCN5 gene mutations, except for a lower prevalence of nephrocalcinosis. *Corresponding author: Dganit Dinour, Department of Nephrology and Hypertension, The Chaim Sheba Medical Center, Tel-Hashomer, Israel. E-mail: Dganit.Dinour@sheba.health.gov.il. Journal of Pediatric Genetics 1 (2012) 3–5 DOI 10.3233/PGE-2012-002 IOS Press 3