Filippo Aucella

Occult hepatitis B virus infection in dialysis patients: a multicentre survey

Background : The epidemiology and clinical significance of occult hepatitis B virus infection (serum hepatitis B surface antigen‐negative patients with detectable hepatitis B virus viraemia in serum) remains controversial with only limited information about its prevalence in patients on long‐term dialysis.

Calcitriol Increases Burst-Forming Unit-Erythroid Proliferation in Chronic Renal Failure

Background: Calcitriol (C) improves anemia in chronic renal failure. This effect may be related to the suppression of iPTH release, or to a direct effect on erythropoiesis. Methods: Thirty-three patients with chronic renal failure were enrolled; among them, 24 were on chronic hemodialysis and 9 on conservative management. None had other chronic or hematological disease, aluminum levels were below 20 µg/l and DFO testing was negative. The iPTH range was 250–480 pg/l. None were treated with C or r-HuEpo. In vitro study: Samples were drawn for a basal erythroid precursor (burst forming unit-erythroid BFU-E) study: Mononuclear cells were incubated for 14 days with r-HuEpo 3U/ml (A), r-HuEpo 3U/l + C 30 pg (B), r-HuEpo 3U/ml + C 300 pg (C), or r-HuEpo 30 U/ml + C 300 pg (D). In vivo study: After the basal evaluation, 10 patients on chronic dialysis were treated with C (Calcijex-Abbott) 1 µg three times a week, and 4 patients served as controls. BFU-E studies were performed after 1, 2 and 4 months. Results: In vitro, culture B showed increased BFU-E proliferation vs. A (41 ± 23 vs. 27 ± 15, p < 0.02); in cultures C and D, proliferation was 61 ± 31 and 78 ± 42, respectively, p < 0.01 vs. A. There was no difference among patients with predialysis renal failure and those on dialysis. BFU-E proliferation was inversely related to basal Hb (p < 0.04) and CRP levels (p < 0.05). During the in vivo study, all cultures showed a progressive increase in proliferation without a plateau level (basal, after 1, 2 and 4 months, respectively) In A: 17 ± 8, 22 ± 13, 30.9 ± 14.9, 41.4 ± 20; in B: 27.3 ± 15, 35.6 ± 20, 45.5 ± 21, 57 ± 26; in C: 48.2 ± 20.6, 63.7 ± 32, 75.7 ± 37, 83 ± 40; in D: 72 ± 24, 91 ± 42, 106 ± 42, 110 ± 42.3 (all p < 0.001). Hb and Hct showed a significant increase (p < 0.03) in the treatment group. The decrease in iPTH was not related to BFU-E proliferation. Conclusions: In chronic uremia, C has a direct effect on erythroid precursors proliferation, as demonstrated both in vitro and in vivo, with a synergistic effect with r-HuEpo. C may be a useful adjuvant therapy to r-HuEpo treatment.

Methylarginines and mortality in patients with end stage renal disease: A prospective cohort study

OBJECTIVES AND METHODS Methylarginines like asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are formed by post-translation methylation of arginine residues in proteins. ADMA inhibits nitric-oxide synthase and predicts clinical outcomes in various diseases including end stage renal disease (ESRD). SDMA competes with L-arginine for cell entry and is associated with organ failure in patients with severe illness. We investigated the inter-relationships between methylargines, L-arginine and other risk factors and tested the prediction power of methylargines for mortality in a prospective cohort study including 288 ESRD patients. RESULTS ADMA and SDMA exceeded the upper limit of the corresponding normal range in almost all cases (98% and 100%, respectively) and were inter-related (r=0.30, P<0.001) but only ADMA was associated with L-arginine. SDMA, was inversely related with haemoglobin (r=-0.23, P<0.001) and this association was independent of other risk factors. During the follow-up, 140 patients died. In unadjusted analysis, ADMA was strongly related to death [hazard ratio (HR) (1micromol/L): 2.07, 95% CI: 1.31-3.26] while plasma SDMA and L-arginine were largely unrelated to survival. In a multiple Cox regression model adjusting for potential confounders, ADMA maintained an independent relationship with death [HR: 1.92, 95% CI: 1.16-3.16]. CONCLUSIONS Methylarginines ADMA and SDMA are inter-related. ADMA is associated with L-arginine while SDMA correlates inversely with haemoglobin. ADMA but not SDMA is a death predictor in ESRD. Given the exceedingly high risk for death of this population, establishing whether or not ADMA is causally implicated in the high death risk of ESRD is a research priority.

Novel Assay Using Total Hepatitis C Virus (HCV) Core Antigen quantification for Diagnosis of HCV Infection in Dialysis Patients

ABSTRACT Dialysis patients remain a high-risk group for hepatitis C virus (HCV) infection. The current diagnosis of HCV infection among dialysis patients includes serological assays and nucleic acid amplification technology (NAT) for assessing serum anti-HCV antibody and HCV viremia, respectively. However, current NAT techniques are expensive and labor-intensive and often lack standardization. An assay prototype designed to detect and quantify total HCV core antigen (total HCV core Ag) protein in serum and plasma in the presence or absence of anti-HCV antibodies has been recently developed. A comparison between a total anti-HCV core Ag enzyme-linked immunosorbent assay (ELISA) and a quantitative HCV RNA assay based on reverse transcription-PCR (RT-PCR) (Amplicor HCV Monitor test) was performed using a large (n = 305) cohort of ELISA HCV 3.0 HCV-negative and -positive patients on maintenance dialysis. The concentrations of HCV core Ag and HCV RNA levels (measured by RT-PCR) were significantly correlated (r = 0.471, P = 0.0001) over a wide range of HCV RNA levels and were maintained among different HCV genotypes (HCV genotype 1, r = 0.862, P = 0.0001; HCV genotype 2, r = 0.691, P = 0.0001). We estimated that 1 pg of total HCV core Ag per ml is equivalent to approximately 19.952 IU of HCV RNA per ml, even if the wide range in the ratio of core Ag to HCV RNA (95% confidence intervals, 2.8 × 103 to 1.6 × 105 IU/ml) precluded definitive conclusions. In summary, total HCV core Ag proved to be useful for performing HCV RNA measurement among dialysis patients in routine laboratories without the need for special equipment or training. The present study supports the use of the total anti-HCV core Ag ELISA for assessing viral load among dialysis patients with HCV infection.

TRPC6 Mutations in Children with Steroid-Resistant Nephrotic Syndrome and Atypical Phenotype

BACKGROUND AND OBJECTIVES Mutations in the TRPC6 gene have been recently identified as the cause of late-onset autosomal-dominant focal segmental glomerulosclerosis (FSGS). To extend the screening, we analyzed TRPC6 in 33 Italian children with sporadic early-onset SRNS and three Italian families with adult-onset FSGS. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS TRPC6 mutation analysis was performed through PCR and sequencing. The effects of the detected amino acid substitutions were analyzed by bioinformatics tools and functional in vitro studies. The expression levels of TRPC6 and nephrin proteins were evaluated by confocal microscopy. RESULTS Three heterozygous missense mutations (c.374A>G_p.N125S, c.653A>T_p.H218L, c.2684G>T_p.R895L) were identified. The first new mutation, p.H218L, was found in a 18-year-old boy who presented a severe form of FSGS at the age of 8 years. The second, p.R895L, a new de novo mutation, was identified in a girl with collapsing glomerulosclerosis at the age of 2 years. The former mutation, p.N125S, was found in two siblings with early-onset steroid-resistant nephrotic syndrome (SRNS) at the ages of 4 and 14 years. Renal immunofluorescence revealed upregulated expression of TRPC6 and loss of nephrin in glomeruli. The intracellular calcium concentrations were significantly higher in the cells expressing all mutant TRPC6 channels compared with cells expressing wild-type TRPC6. CONCLUSIONS Our findings suggest that TRPC6 variants can also be detected in children with early-onset and sporadic SRNS (4 of 33 patients). Moreover, in one patient a new de novo TRPC6 mutation was associated with a rare severe form of childhood collapsing glomerulosclerosis with rapid progression to uremia.

WT1 mutations in nephrotic syndrome revisited. High prevalence in young girls, associations and renal phenotypes

WT1 mutations have been considered a rare cause of nephrotic syndrome but recent reports challenge this assumption. Exclusion of inherited forms is a basic point in any therapeutic strategy to nephrotic syndrome since they do not respond to drugs. We screened for WT1 mutations in 200 patients with nephrotic syndrome: 114 with steroid resistance (SRNS) and 86 with steroid dependence (SDNS) for whom other inherited forms of nephrotic syndrome (NPHS2, CD2AP) had been previously excluded. Three girls out of 32 of the group with steroid resistance under 18 years presented classical WT1 splice mutations (IVS9+5G>A, IVS9+4C>T) of Frasier syndrome. Another one presented a mutation coding for an amino acid change (D396N) at exon 9 that is typical of Denys-Drash syndrome. All presented resistance to drugs and developed end stage renal failure within 15 years. Two girls of the Frasier group presented a 46 XY karyotype with streak gonads while one was XX and had normal gonad morphology. In the two cases with IVS9+5G>A renal pathology was characterized by capillary wall thickening with deposition of IgG and C3 in one that was interpreted as a membrane pathology. Foam cells were diffuse in tubule-interstitial areas. In conclusion, WT1 splice mutations are not rare in females under 18 years with SRNS. This occurs in absence of a clear renal pathology picture and frequently in absence of phenotype change typical of Frasier syndrome. In adults and children with SDNS, screening analysis is of no clinical value. WT1 hot spot mutation analysis should be routinely done in children with SRNS; if the molecular screening anticipates any further therapeutic approach it may modify the long term therapeutic strategy.

Molecular analysis of NPHS2 and ACTN4 genes in a series of 33 Italian patients affected by adult-onset nonfamilial focal segmental glomerulosclerosis.

Background: Mutations in the NPHS2 gene, encoding podocin, and in the ACTN4 gene, encoding α-actinin-4, have been identified in familial childhood-onset forms of focal and segmental glomerulosclerosis (FSGS). NPHS2 may be also responsible for some sporadic cases. The role of NPHS2 and ACTN4 in the adult sporadic form of the disease is being clarifying. Methods: Thirty-three adult subjects affected by sporadic FSGS were studied at molecular level. At biopsy, 12 patients had nephrotic syndrome, 5 patients had isolated proteinuria and 16 patients showed proteinuria and hematuria. Glomerular filtration rate (GFR) was in the normal range in 19 subjects and 14 patients had a variable degree of renal failure. Multiplex families presenting with a clear familial inheritance for proteinuria or other congenital nephrotic syndrome were excluded. The whole coding region, all intron/exon boundaries and flanking intronic regions of NPHS2 gene and the exon 8, i.e. hot-spot mutations of the ACTN4 gene, were analyzed in all patients by denaturing high-performance liquid chromatography (DHPLC) to search disease-causing defects. Results: The analysis identified four already described and two new polymorphisms, IVS3–21C>T and IVS3–46C>T, on the NPHS2 gene. Moreover, the R229Q allele was identified in 3/33 patients and in 7/124 controls, accounting for an allelic frequency of 0.045 and 0.028, respectively. The new intronic polymorphism IVS7–54C>T was also found in the exon 8 of the ACTN4 gene. Conclusions: In this study, we exhaustively analyzed the NPHS2 and the exon 8 of the ACTN4 genes in a series of sporadic ‘adult-onset’ FSGS patients. No causative mutations were found while the R229Q allele was identified in 3 patients confirming its possible role as a ‘disease-associated NPHS2 allele’ although its pathogenetic involvement needs to be further clarified. Moreover, the description of new intronic polymorphisms in both genes is reported.

The ENPP1 Q121 Variant Predicts Major Cardiovascular Events in High-Risk Individuals: Evidence for Interaction With Obesity in Diabetic Patients

OBJECTIVE Insulin resistance (IR) and cardiovascular disease may share a common genetic background. We investigated the role of IR-associated ENPP1 K121Q polymorphism (rs1044498) on cardiovascular disease in high-risk individuals. RESEARCH DESIGN AND METHODS A prospective study (average follow-up, 37 months) was conducted for major cardiovascular events (myocardial infarction [MI], stroke, cardiovascular death) from the Gargano Heart Study (GHS; n = 330 with type 2 diabetes and coronary artery disease), the Tor Vergata Atherosclerosis Study (TVAS; n = 141 who had MI), and the Cardiovascular Risk Extended Evaluation in Dialysis (CREED) database (n = 266 with end-stage renal disease). Age at MI was investigated in cross-sectional studies of 339 type 2 diabetic patients (n = 169 from Italy, n = 170 from the U.S.). RESULTS Incidence of cardiovascular events per 100 person--years was 4.2 in GHS, 10.8 in TVAS, and 11.7 in CREED. Hazard ratios (HRs) for KQ+QQ versus individuals carrying the K121/K121 genotype (KK) individuals were 1.47 (95% CI 0.80–2.70) in GHS, 2.31 (95% CI 1.22–4.34) in TVAS, and 1.36 (95% CI 0.88–2.10) in CREED, and 1.56 (95% CI 1.15–2.12) in the three cohorts combined. In the 395 diabetic patients, the Q121 variant predicted cardiovascular events among obese but not among nonobese individuals (HR 5.94 vs. 0.62, P = 0.003 for interaction). A similar synergism was observed in cross-sectional studies, with age at MI being 3 years younger in Q121 carriers than in KK homozygotes among obese but not among nonobese patients (P = 0.035 for interaction). CONCLUSIONS The ENPP1 K121Q polymorphism is an independent predictor of major cardiovascular events in high-risk individuals. In type 2 diabetes, this effect is exacerbated by obesity. Future larger studies are needed to confirm our finding.

Review article: hepatitis C virus infection and type-2 diabetes mellitus in renal diseases and transplantation

A link between hepatitis C virus infection and development of diabetes mellitus has been suggested by many investigators; however, this remains controversial.

Physical Performance and Clinical Outcomes in Dialysis Patients: A Secondary Analysis of the Excite Trial

Background/Aims: Scarce physical activity predicts shorter survival in dialysis patients. However, the relationship between physical (motor) fitness and clinical outcomes has never been tested in these patients. Methods: We tested the predictive power of an established metric of motor fitness, the Six-Minute Walking Test (6MWT), for death, cardiovascular events and hospitalization in 296 dialysis patients who took part in the trial EXCITE (ClinicalTrials.gov Identifier: NCT01255969). Results: During follow up 69 patients died, 90 had fatal and non-fatal cardiovascular events, 159 were hospitalized and 182 patients had the composite outcome. In multivariate Cox models - including the study allocation arm and classical and non-classical risk factors - an increase of 20 walked metres during the 6MWT was associated to a 6% reduction of the risk for the composite end-point (P=0.001) and a similar relationship existed between the 6MWT, mortality (P<0.001) and hospitalizations (P=0.03). A similar trend was observed for cardiovascular events but this relationship did not reach statistical significance (P=0.09). Conclusions: Poor physical performance predicts a high risk of mortality, cardiovascular events and hospitalizations in dialysis patients. Future studies, including phase-2 EXCITE, will assess whether improving motor fitness may translate into better clinical outcomes in this high risk population.