Rafael T. Krmar

Hypertension-Linked Mutation in the Adducin α-Subunit Leads to Higher AP2-μ2 Phosphorylation and Impaired Na+,K+-ATPase Trafficking in Response to GPCR Signals and Intracellular Sodium

&agr;-Adducin polymorphism in humans is associated with abnormal renal sodium handling and high blood pressure. The mechanisms by which mutations in adducin affect the renal set point for sodium excretion are not known. Decreases in Na+,K+-ATPase activity attributable to endocytosis of active units in renal tubule cells by dopamine regulates sodium excretion during high-salt diet. Milan rats carrying the hypertensive adducin phenotype have a higher renal tubule Na+,K+-ATPase activity, and their Na+,K+-ATPase molecules do not undergo endocytosis in response to dopamine as do those of the normotensive strain. Dopamine fails to promote the interaction between adaptins and the Na+,K+-ATPase because of adaptin-&mgr;2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na+,K+-ATPase activity, &mgr;2-subunit hyperphosphorylation, and impaired Na+,K+-ATPase endocytosis. Thus, increased renal Na+,K+-ATPase activity and altered sodium reabsorption in certain forms of hypertension could be attributed to a mutant form of adducin that impairs the dynamic regulation of renal Na+,K+-ATPase endocytosis in response to natriuretic signals.

Carotid Artery Intima-Media Thickness and Distensibility in Children and Adolescents Reference Values and Role of Body Dimensions

Carotid intima-media thickness (cIMT) and carotid artery distensibility are reliable screening methods for vascular alterations and the assessment of cardiovascular risk in adult and pediatric cohorts. We sought to establish an international reference data set for the childhood and adolescence period and explore the impact of developmental changes in body dimensions and blood pressure (BP) on carotid wall thickness and elasticity. cIMT, the distensibility coefficient, the incremental modulus of elasticity, and the stiffness index &bgr; were assessed in 1155 children aged 6 to 18 years and sex-specific reference charts normalized to age or height were constructed from 1051 nonobese and nonhypertensive children. The role of body dimensions, BP, and family history, as well as the association between cIMT and distensibility, was investigated. cIMT increased and distensibility decreased with age, height, body mass index, and BP. A significant sex difference was apparent from the age of 15 years. Age- and height-normalized cIMT and distensibility values differed in children who are short or tall for their age. By stepwise multivariate analysis, standardized systolic BP and body mass index were independently positively associated with cIMT SD scores (SDS). Systolic BP SDS independently predicted all distensibility measures. Distensibility coefficient SDS was negatively and &bgr; SDS positively associated with cIMT SDS, whereas incremental modulus of elasticity was independent of cIMT. Morphological and functional aspects of the common carotid artery are particularly influenced by age, body dimensions, and BP. The reference charts established in this study allow to accurately compare vascular phenotypes of children with chronic conditions with those of healthy children.

Long-term follow-up after rituximab for steroid-dependent idiopathic nephrotic syndrome

BACKGROUND In patients with refractory steroid-sensitive nephrotic syndrome (SSNS), treatment with rituximab has shown encouraging results; however, long-term follow-up data are not available. METHODS We performed a retrospective analysis of 37 patients (25 boys) with steroid-dependent nephrotic syndrome who were treated with rituximab (375 mg/m(2) given weekly for one to four courses). Long-term follow-up data (>2 years, median 36, range 24-92.8 months) are available for 29 patients (12 boys). RESULTS Twenty-six of 37 (70.3%) patients remained in remission after 12 months. Relapses occurred in 24 (64.8%) patients after a median of 9.6 (range 5.2-64.1) months. Time to first relapse was significantly shorter in patients receiving one or two compared to three or four initial infusions. In the 29 patients with long-term follow-up for >2 years, 12 (41%) patients remained in remission after the initial rituximab course for >24 months, 7 (24.1%) patients without further maintenance immunosuppression. Nineteen children received two to four repeated courses of rituximab increasing the total number of patients with long-term remission to 20 (69%), remission including 14 (48%) patients off immunosuppression. The proportion of patients with long-term remission was not related to the number of initial rituximab applications. No serious side effects were noted. CONCLUSION Rituximab is an effective treatment option in the short- and long-term control of treatment refractory SSNS. Further controlled studies are needed to address optimal patient selection, dose and safety of rituximab infusions.

Spectrum of Steroid-Resistant and Congenital Nephrotic Syndrome in Children: The PodoNet Registry Cohort

BACKGROUND AND OBJECTIVES Steroid-resistant nephrotic syndrome is a rare kidney disease involving either immune-mediated or genetic alterations of podocyte structure and function. The rare nature, heterogeneity, and slow evolution of the disorder are major obstacles to systematic genotype-phenotype, intervention, and outcome studies, hampering the development of evidence-based diagnostic and therapeutic concepts. To overcome these limitations, the PodoNet Consortium has created an international registry for congenital nephrotic syndrome and childhood-onset steroid-resistant nephrotic syndrome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Since August of 2009, clinical, biochemical, genetic, and histopathologic information was collected both retrospectively and prospectively from 1655 patients with childhood-onset steroid-resistant nephrotic syndrome, congenital nephrotic syndrome, or persistent subnephrotic proteinuria of likely genetic origin at 67 centers in 21 countries through an online portal. RESULTS Steroid-resistant nephrotic syndrome manifested in the first 5 years of life in 64% of the patients. Congenital nephrotic syndrome accounted for 6% of all patients. Extrarenal abnormalities were reported in 17% of patients. The most common histopathologic diagnoses were FSGS (56%), minimal change nephropathy (21%), and mesangioproliferative GN (12%). Mutation screening was performed in 1174 patients, and a genetic disease cause was identified in 23.6% of the screened patients. Among 14 genes with reported mutations, abnormalities in NPHS2 (n=138), WT1 (n=48), and NPHS1 (n=41) were most commonly identified. The proportion of patients with a genetic disease cause decreased with increasing manifestation age: from 66% in congenital nephrotic syndrome to 15%-16% in schoolchildren and adolescents. Among various intensified immunosuppressive therapy protocols, calcineurin inhibitors and rituximab yielded consistently high response rates, with 40%-45% of patients achieving complete remission. Confirmation of a genetic diagnosis but not the histopathologic disease type was strongly predictive of intensified immunosuppressive therapy responsiveness. Post-transplant disease recurrence was noted in 25.8% of patients without compared with 4.5% (n=4) of patients with a genetic diagnosis. CONCLUSIONS The PodoNet cohort may serve as a source of reference for future clinical and genetic research in this rare but significant kidney disease.

The 14-3-3 Protein Translates the NA+,K+-ATPase α1-Subunit Phosphorylation Signal into Binding and Activation of Phosphoinositide 3-Kinase during Endocytosis

Clathrin-dependent endocytosis of Na+,K+-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the α-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na+,K+-ATPase α-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na+,K+-ATPase α-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na+,K+-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the α-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na+,K+-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the α-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na+,K+-ATPase endocytosis.

Genetic screening in adolescents with steroid-resistant nephrotic syndrome

Genetic screening paradigms for congenital and infantile nephrotic syndrome are well established; however, screening in adolescents has received only minor attention. To help rectify this, we analyzed an unselected adolescent cohort of the international PodoNet registry to develop a rational screening approach based on 227 patients with nonsyndromic steroid-resistant nephrotic syndrome aged 10-20 years. Of these, 21% had a positive family history. Autosomal dominant cases were screened for WT1, TRPC6, ACTN4, and INF2 mutations. All other patients had the NPHS2 gene screened, and WT1 was tested in sporadic cases. In addition, 40 sporadic cases had the entire coding region of INF2 tested. Of the autosomal recessive and the sporadic cases, 13 and 6%, respectively, were found to have podocin-associated nephrotic syndrome, and 56% of them were compound heterozygous for the nonneutral p.R229Q polymorphism. Four percent of the sporadic and 10% of the autosomal dominant cases had a mutation in WT1. Pathogenic INF2 mutations were found in 20% of the dominant but none of the sporadic cases. In a large cohort of adolescents including both familial and sporadic disease, NPHS2 mutations explained about 7% and WT1 4% of cases, whereas INF2 proved relevant only in autosomal dominant familial disease. Thus, screening of the entire coding sequence of NPHS2 and exons 8-9 of WT1 appears to be the most rational and cost-effective screening approach in sporadic juvenile steroid-resistant nephrotic syndrome.

Comparison of Plasma Clearance of Iohexol and Urinary Clearance of Inulin for Measurement of GFR in Children

BACKGROUND Very few studies have been published that compare plasma clearance of iohexol (Cio) with renal clearance of inulin (Cin). STUDY DESIGN Diagnostic test study. SETTING & PARTICIPANTS 60 children aged 11.6 ± 4.5 years with different kidney disorders were investigated. INDEX TEST Plasma Cio calculated from the slope and a single point. REFERENCE TEST Renal Cin with continuous infusion during water diuresis. Results were compared with the correlation coefficients, bias and precision, accuracy percentage, root mean square error, and intraclass correlation. OTHER MEASUREMENTS Measured creatinine clearance and estimated glomerular filtration rate based on serum creatinine level and height. RESULTS Mean Cin was 70.7 ± 41.3 (SD) mL/min/1.73 m². Mean differences between Cio and Cin were 2.65 and 2.00 mL/min/1.73 m² for the slope and single-point methods, respectively. Precision was ±16 mL/min/1.73 m² and intraclass correlation was 0.92 in both methods. Proportions of Cio within 30% of Cin were 83.3% and 86.7% for the slope and single-point methods, respectively. LIMITATIONS A limited number of patients; no adults were studied. CONCLUSIONS Plasma Cio shows good agreement with renal Cin.

Prospective analysis of carotid arterial wall structure in pediatric renal transplants with ambulatory normotension and in treated hypertensive recipients.

Abstract:  Increased carotid IMT was found to be associated with cardiovascular risk factors. As pediatric renal transplants are at high risk for cardiovascular disease, we examined whether there is a relationship between BP and IMT in normotensive and in treated hypertensive recipients after transplantation. Thirty‐one recipients aged 10 ± 3.5 yr (16 M, 15 F) underwent repeated carotid ultrasound examinations 5.4 ± 3.2 yr after transplantation with a 4.1 ± 1 yr interval and were followed with annual ambulatory BP monitoring. Baseline IMT was significantly higher in transplants compared with controls. When recipients were again investigated, follow‐up IMT measurements were similar compared with measurements obtained at baseline. The analysis of variance showed that baseline IMT both in recipients with strict normotension, i.e., ambulatory normotension without antihypertensive therapy at baseline and throughout the study period (n = 9), and in recipients with treated hypertension or newly diagnosed hypertension (n = 22) was significantly higher than in healthy controls (n = 21). Baseline IMT did not differ between these subgroups of recipients. Similarly, pairwise comparisons showed that baseline and follow‐up IMT within each subgroup of recipients were not significantly different. Overall and regardless of time‐point, no significant associations were found between systolic and diastolic 24‐h BP, daytime BP, night‐time BP, ambulatory BP standard deviation scores, BP loads and IMT. Our results suggest that increased IMT in pediatric renal transplants does not seem to be related to BP but more likely to other factor(s) not investigated in this study.

Use of annual ABPM, and repeated carotid scan and echocardiography to monitor cardiovascular health over nine yr in pediatric and young adult renal transplant recipients

Balzano R, Lindblad YT, Vavilis G, Jogestrand T, Berg UB, Krmar RT. Use of annual ABPM, and repeated carotid scan and echocardiography to monitor cardiovascular health over nine yr in pediatric and young adult renal transplant recipients.
Pediatr Transplantation 2011: 15: 635–641.

ABPM vs. office blood pressure to define blood pressure control in treated hypertensive paediatric renal transplant recipients

Abstract:  While 24‐h ambulatory blood pressure monitoring (ABPM) is an established tool for monitoring antihypertensive therapy in adults, data in children are scarce. We retrospectively analysed whether office blood pressure (BP) is reliable for the diagnosis of BP control in 26 treated hypertensive paediatric renal transplants. Controlled office BP was defined as the mean of three replicate systolic and diastolic BP recordings less than or equal to the 95th age‐, sex‐ and height‐matched percentile on the three‐outpatient visits closest to ABPM. Controlled ABPM was defined as systolic and diastolic daytime BP ≤95th distribution adjusted height‐ and sex‐related percentile of the adapted ABPM reference. Eight recipients (30%) with controlled office BP were in fact categorized as having non‐controlled BP by ABPM criteria. Overall, when office BP and ABPM were compared using the Bland and Altman method, the 95% limits of agreement between office and daytime values ranged from −12.6 to 34.1 mmHg for systolic and −23.9 to 31.7 mmHg for diastolic BP, and the mean difference was 10.7 and 3.9 mmHg respectively. Office readings miss a substantial number of recipients who are hypertensive by ABPM criteria. Undertreatment of hypertension could be avoided if ABPM is applied as an adjunct to office readings.