Carmine Stallone

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.

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.

Systematic Monitor Disinfection Is Effective in Limiting HCV Spread in Hemodialysis

Background: Patients on chronic hemodialysis are at high risk of HCV infection due to nosocomial transmission. The strict adhesion to universal precautions is the first step in prevention, but other simple tools such as systematic monitor disinfection and the use of separate machines for anti-HCV-positive patients need to be evaluated. Methods: A 5-year prospective study was carried out in 4 dialysis centers enrolling 135 patients. General precautions were adopted, but anti-HCV-positive patients were not isolated. In period A, lasting 24 months, monitor disinfection was performed after each dialysis session with sodium hypochlorite; peracetic acid was also used 3 times a week. In period B, lasting 36 months, 3 dialysis units (77 patients) prolonged the same preventive protocol of period A, while another unit (58 patients) also adopted the use of separate machines for anti-HCV-positive subjects. A third-generation ELISA anti-HCV test was performed every 2 months throughout the study. Results: Anti-HCV antibodies were initially detected in 43 patients (31.8%), prevalence rate ranging from 25 to 39.4%. One seroconversion occurred in period A, with an overall seroconversion rate of 0.54%/year. Also in period B one seroconversion occurred (unit 2), seroconversion rate of 0.36%/year. Therefore the mean seroconversion rate throughout the 5 years was 0.43%/year. Conclusion: Systematic monitor disinfection may be a simple and quite effective tool to avoid nosocomial transmission of HCV infection in the hemodialysis setting. In our opinion its use is mandatory. The use of separate machines for anti-HCV-positive patients seems unnecessary.

Polymorphism of the Angiotensin-Converting Enzyme Gene in End-Stage Renal Failure Patients

The plasma levels of angiotensin-converting enzyme (ACE) are modulated by the insertion (I)/deletion (D) polymorphism within the ACE gene locus. An association between progressive renal disease, raised cardiovascular risk, and ACE plasma levels has been shown. To evaluate the genotype frequencies of the I/D polymorphism in terminal renal failure, we have enrolled 341 dialysis patients (321 on hemodialysis and 20 on peritoneal dialysis) in a district of southern Italy (Foggia). As controls, 1,307 subjects from the same area have been enrolled. Genomic DNA was obtained from leukocytes, and the ACE I/D polymorphism was determined by polymerase chain reaction. Among uremics, 151 subjects (44.3%) carried the DD genotype, 149 (43.7%) the ID, and 41 (12.0%) the II genotype. In controls, 560 subjects (42.8%) had the DD genotype, 577 (44.1%) the ID, and 170 (13.1%) the II genotype (p = n.s.). Among patients, the frequency of DD subjects was higher in men (48.3%) than in women (39.7%, p < 0.01). A slight different frequency of the DD genotype was found according to the duration of dialysis treatment: 47.5% in patients on dialysis up to 60 months and 41.7 and 40.6% in those with a dialytic age of 60–120 and >120 months, respectively (p for trend: 0.53). Patients with or without cardiovascular diseases, such as hypertension, left ventricular hypertrophy, coronary artery disease, and chronic cardiac failure, did not exhibit any difference in ACE I/D allele and genotype frequencies (p always >0.05). In conclusion, frequencies of the ACE DD genotype were similar in uremics and in controls and did not differ between patients with and without cardiovascular diseases. A nonsignificant inverse relationship with the time spent on dialysis was observed, suggesting that ACE I/D polymorphism may influence the cardiovascular death rate.

Effect of a plasma sodium biofeedback system applied to HFR on the intradialytic cardiovascular stability. Results from a randomized controlled study

Background Intradialytic hypotension (IDH) is still a major clinical problem for haemodialysis (HD) patients. Haemodiafiltration (HDF) has been shown to be able to reduce the incidence of IDH. Methods Fifty patients were enrolled in a prospective, randomized, crossover international study focussed on a variant of traditional HDF, haemofiltration with endogenous reinfusion (HFR). After a 1-month run-in period on HFR, the patients were randomized to two treatments of 2 months duration: HFR (Period A) or HFR-Aequilibrium (Period B), followed by a 1-month HFR wash-out period and then switched to the other treatment. HFR-Aequilibrium (HFR-Aeq) is an evolution of the haemofiltration with endogenous reinfusion (HFR) dialysis therapy, with dialysate sodium concentration and ultrafiltration rate profiles elaborated by an automated procedure. The primary end point was the frequency of IDH. Results Symptomatic hypotension episodes were significantly lower on HFR-Aeq versus HFR (23 ± 3 versus 31 ± 4% of sessions, respectively, P l= l0.03), as was the per cent of clinical interventions (17 ± 3% of sessions with almost one intervention on HFR-Aeq versus 22 ± 2% on HFR, P <0.01). In a post-hoc analysis, the effect of HFR-Aeq was greater on more unstable patients (35 ± 3% of sessions with hypotension on HFR-Aeq versus 71 ± 3% on HFR, P <0.001). No clinical or biochemical signs of Na/water overload were registered during the treatment with HFR-Aeq. Conclusions HFR-Aeq, a profiled dialysis supported by the Natrium sensor for the pre-dialysis Na+ measure, can significantly reduce the burden of IDH. This could have an important impact in every day dialysis practice.

Reduction of mononuclear cytokine production in hemodialysis patients treated with steam-sterilized low-flux polysulphone membranes.

An increased cytokine production, correlated with long term complications of uremic disease, has been described during hemodialysis. To identify possible differences in the cytokine release of differently sterilized membranes, we enrolled six uremic patients on chronic hemodialysis. The patients underwent dialysis with ETO-sterilized low-flux polysulphone membranes (F6, Fresenius AG) for at least three months (At), they were then switched to steam-sterilized polysulphone membranes (F6-HPS Fresenius AG) and further evaluations after one (B1) and two months (B2) were carried out. A final evaluation (A2) was made one month after switching back to F6 dialyzers. At each time period, samples were drawn to measure IL-1B released by cultured mononuclear cells (MN). Moreover, dialysate samples were collected to test endotoxin levels. C3a and C5a levels were assessed at 0, 5, 15 and 60 min from starting hemodialysis. Anti-ETO IgE levels were also assayed at A1, B1 and A2. The LAL test revealed a good quality dialysate. The mean pre-dialysis IL-1B levels were 215 pg/million cells at A1; falling to 49 at B1, and 54 at B2 (p≤0.01); there was then a sharp rebound at A2:284, p≤0.01. Post-dialysis levels followed the same pattern. No correlation between the dialysate endotoxin level and cytokine release was found. Complement activation did not change and in all the phases of the study no anti-ETO IgE was detected in any of the subjects. Our data suggest that the steam sterilized polysulphone membrane induces a lower cytokine release than the ETO sterilized membrane, although the mechanism by which it does so remains to be clarified.

Effectiveness of universal precautions in limiting nosocomial transmission of hepatitis C virus in haemodialysis units.

In spite of the high prevalence of anti-HCV positive patients in haemodialysis units (1), there are conflicting results regarding the incidence rate and, consequently, the preventive strategies. Here we report our experience with a three-year follow-up: 88 patients on ROT for at least 6 months were enrolled in three dialysis units. Mean duration of follow-up was 37.2 months. All patients enrolled were tested every 4 months with a 2nd generation antiHCV test (Elisa, Ortho); confirmation of positive samples was performed with Riba test (Ortho), RT-nested PCR was used to detect HCV RNA sequence in serum. The prevention of nosocomial transmission was set up as follows. No segregation of anti-HCV positive patients was set up, no dedicated machines were used, and no dialyzer was reused. Utmost attention was paid in applying universal precautions for prevention of community acquired infection as in Center For Disease Control Recommendation (2). The disinfection protocol for dialysis monitors included a chemical disinfection with peracetic acid or hypoclorite solution after the last dialysis session of the day, while only a washing cycle was applied between dialysis sessions. Anti-HCV antibodies were initially detected in 32 subjects (35.2%); 56 patients were seronegative (Tab. I). The prevalence of anti-HCV positive patients ranged from 18.18% to 41.37%. During the follow-up five seroconversions were observed: 1 in the first, 2 in the second, 2 in the third year (3 in the hospital center, 1 for each outside centers). Thus, mean incidence of seroconversion was 2.79% / year. All seroconverted patients but one were PCR positive and all but one had a positive Riba 2° test (one was indeterminate); two patients received blood transfusion. None of the seroconverted subjects were dialyzing on single pass monitors; three were dialyzed on the same machines of an anti-HCV positive patients: one of these was also transfused 19 months before SC; one was dialyzed next to an anti-HCV positive patient, and the last had none of these risk conditions, but was transfused 15 months before SC (Tab. II). Up to now there is no agreement on applying a segregation protocol for anti-HCV positive and negative patients as has been done for hepatitis B. However, some authors have advocated the segregation of anti-HCV positive patients (3, 4). Should an isolation protocol be accepted, there will be many clinical and organizing problems: the delay between HCV infection and detection of anti-HCV antibodies; the great variability of HCV genomes (5); when isolation was performed SC rate decreased but did not disappear (6); on the other hand, there are organizing problems: we will need up to four different facilities to dialyze patients with or without HBV and HCV infection or coinfec-

HLA System in Uremics with Cystic Diseases

We evaluated the prevalence of class I HLA antigens (A, B, C) in patients (pts) on regular dialysis treatment (RDT) affected with adult polycystic kidney diseases (APKD) (12 pts) and acquired renal cystic diseases (ARCD) (12 pts). 96 voluntary HLA-typed blood donors, randomly selected, were enrolled as healty controls. In APKD group we found the greater occurence of Bw22-Ag (p/0.004).

Cystic Diseases in Uremia and HLA System

We evaluated the prevalence of class I HLA antigen (A, B, C) in patients (pts) on regular dialysis treatment (RDT) affected with adult polycystic kidney diseases (APKD) (12 pts) and acquired renal cystic diseases (ARCD) (12 pts). 96 voluntary HLA-typed blood donors, randomly selected, were enrolled as healty controls. In APKD group we found the greater occurence of Bw22-Ag (p/0.004).

Bone GLA Protein in Chronic Renal Failure

Osteocalcin, or Bone Gla Protein (BGP), is a biological marker of bone turn over. To evaluate the relationship between serum BGP and other biochemical parameters in chronic renal failure, we measured BGP, PTH-C, alkaline phosphatase (ALP), calcium and phosphorus in 73 hemodialyzed patients. We used linear regression as statistical analysis. BGP was elevated in 68 patients and correlated with PTH (p<0.00001), ALP (p<0.005) and dialytic age (p<0.04). No correlation was found between BGP, calcium and phosphorus. Our study confirms that BGP correlates strongly with PTH. BGP have a promising role as a non invasive tool for assessment of bone histopatology.