Kar N. Lai

Smad7 Inhibits Fibrotic Effect of TGF-β on Renal Tubular Epithelial Cells by Blocking Smad2 Activation

It has been shown that transforming growth factor-beta (TGF-beta) is a potent mediator in renal fibrosis and that Smad proteins are critical intracellular mediators in TGF-beta signaling. It is here reported that TGF-beta mediates renal fibrogenesis in tubular epithelial cells (TEC) in association with the activation of Smad2 and that overexpression of Smad7 blocks this fibrotic process. Using a normal rat kidney tubular epithelial cell line (NRK52E), it was determined that TGF-beta1 induces Smad2 phosphorylation and nuclear localization in both a dose- and time-dependent manner. The activation of Smad2 was evident at 5 min (20%), peaked at 15 to 30 min (85%), and declined to baseline levels by 2 h (5 to 10%). This was associated with de novo expression of collagens I, III, and IV and the transformation of TEC into a myofibroblast phenotype with de novo expression of alpha-smooth muscle actin (alpha-SMA) and with the loss of E-cadherin (>50%). To investigate a negative regulatory role of Smad7 in renal fibrosis, the Smad 7 gene was stably transfected and its expression was tightly controlled by doxycycline into NRK52E cells. Overexpression of Smad7 induced by doxycycline results in marked inhibition of TGF-beta-induced Smad2 activation (90% downward arrow) with the prevention of collagen synthesis and myofibroblast transformation. Thus, Smad2 activation occurs in the fibrogenic response of TEC to TGF-beta, and this process is blocked by overexpression of Smad7. This indicates that Smad signaling is a key pathway of TGF-beta-mediated renal fibrosis and suggests that treatments targeting the inactivation of Smad2 by overexpression of Smad7 may provide a new therapeutic strategy for renal fibrosis.

Family-Based Association Study Showing that Immunoglobulin A Nephropathy Is Associated with the Polymorphisms 2093C and 2180T in the 3′ Untranslated Region of the Megsin Gene

Immunoglobulin A nephropathy (IgAN) is considered to be a multifactorial disease with genetic and environmental factors contributing to its pathogenesis. The genes involved in susceptibility and progression of the disease have not yet been clearly elucidated. Megsin (SERPINB7) is an important candidate gene, predominantly expressed in glomerular mesangium and upregulated in IgAN. To investigate the potential role of this and other genes in IgAN, patients with biopsy-proven IgAN were recruited, as were family members, for a family-based association study. The genotypes of the polymorphisms C2093T and C2180T within the 3 untranslated region of the gene were determined by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. The results were analyzed by transmission disequilibrium test (TDT) and haplotype relative risk (HRR). TDT analyses revealed that Megsin 2093C and 2180T alleles were significantly more transmitted from heterozygous parents to patients than expected (C2093T: 127 trios, P = 0.034, C2180T: 100 trios, P = 0.002). Extended TDT showed increased cotransmission of the 2093C and 2180T alleles (232 families, P < 0.001). HRR revealed that the 2093C and 2180T alleles were more often transmitted to patients (P = 0.014, <0.001, respectively). Genetic variation in Megsin confers susceptibility to IgAN.

Differential effects of advanced glycation end‐products on renal tubular cell inflammation

Aim:u2003 The authors recently showed that advanced glycation end‐products (AGE) in the form of glycated albumin (GA) upregulated renal tubular expression of interleukin (IL)‐8 and soluble intercellular adhesion molecule‐1 (sICAM‐1), but not other important cytokines known to mediate diabetic nephropathy. This implies that other molecules such as the carbonyl intermediates of AGE or other modified protein lysine‐albumin may participate in diabetic tubular injury.

Rare inborn errors associated with chronic hepatitis B virus infection

Chronic hepatitis B (CHB) is a major global health issue. The role of rare genetic variants in CHB has not been elucidated. We aimed to identify rare allelic variants predisposing to CHB. We performed exome sequencing in 50 CHB patients who had no identifiable risk factors for CHB and 40 controls who were healthy and hepatitis B surface antibody‐positive, but had never received hepatitis B vaccination. We selected six rare variant alleles and followed up their association with disease status by Sanger sequencing in a case‐control study comprising 1,728 CHB patients and 1,636 healthy controls. The latter had either not been immunized with hepatitis B vaccine or had uncertain vaccination status. Our results showed that transmembrane protein 2 p.Ser1254Asn, interferon alpha 2 p.Ala120Thr, its regulator NLR family member X1 p.Arg707Cys, and complement component 2 p.Glu318Asp were associated with CHB, with P values of <1.0 × 10−7, 2.76 × 10−5, 5.08 × 10−5, 2.78 × 10−4 and odds ratios (ORs) of 2.45, 4.08, 2.34, and 1.97, respectively. The combined P value was <2.0 × 10−16. As there has been no indication of immunological functions for the associated gene, transmembrane protein 2, we further studied its expression by immunohistochemistry, real‐time polymerase chain reaction, and western blotting. Our results showed that it was strongly expressed by healthy hepatocytes, but its expression was reduced in liver tissues with CHB, hepatitis B viral (HBV) genome‐containing HepG2.2.15 cells, as compared with healthy liver tissues and non‐HBV genome‐containing HepG2 cells (P = 0.022 and 0.0036, respectively). Conclusion: We identified four missense mutations associated with CHB, our results providing evidence for rare inborn genetic defects that contribute to increased host susceptibility to CHB. (HEPATOLOGY 2012;56:1661–1670)

CLINICAL COURSE AND OUTCOMES OF SINGLE-ORGANISM ENTEROCOCCUS PERITONITIS IN PERITONEAL DIALYSIS PATIENTS

♦ Background and Objectives: Enterococci are part of the normal flora of the gastrointestinal tract. They can cause enteric peritonitis, which is a serious complication of peritoneal dialysis (PD). However, the clinical course and outcome of PD-related Enterococcus peritonitis remains unclear. ♦ Methods: We reviewed all Enterococcus peritonitis episodes occurring in our dialysis unit from 1995 to 2009. ♦ Results: During the study period, 1421 episodes of peritonitis were recorded. Of 29 episodes (2.0%) that were attributable to single-organism Enterococcus, 12 episodes were caused by E. faecalis; 9, by E. faecium; and the remaining 8, by other Enterococcus species. The overall rate of ampicillin resistance was 41.4%. Recent use of antibiotics was associated with the development of ampicillin-resistant Enterococcus (ARE) peritonitis (hazard ratio: 12.53; p = 0.04). The primary response rate of Enterococcus peritonitis was significantly higher than that of Escherichia coli peritonitis (89.7% vs. 69.9%, p = 0.038), but the primary response rate was not significantly lower for ARE peritonitis than for ampicillin-susceptible Enterococcus (ASE) peritonitis (83.3% vs. 94.1%, p = 0.553). However, significantly more patients with ARE had received vancomycin (83.3% vs. 23.5%, p = 0.003), with a longer mean duration of vancomycin treatment (11.8 ± 6.9 days vs. 3.7 ± 6.8 days, p = 0.005). ♦ Conclusions: Recent use of antibiotics was a risk factor for the development of ARE peritonitis. Outcomes in ASE and ARE peritonitis were similar, but vancomycin was required during treatment for ARE peritonitis, in turn possibly predisposing the patients to infections caused by vancomycin-resistant organisms.

Interleukin-5 messenger RNA expression in peripheral blood CD4+ cells in asthma

BACKGROUNDnIL-5 has been implicated in the pathogenesis of asthma through its regulatory role on eosinophil survival, proliferation, and effector function.nnnOBJECTIVEnThe study was designed to investigate the relationships between IL-5 messenger RNA expression in circulating CD4+ cells and serum concentrations of eosinophil cationic protein (ECP), a marker of eosinophil activation, and disease activity in asthma.nnnMETHODSnIL-5 gene expression was assessed semiquantitatively in ex vivo stimulated CD4+ cells by reverse transcription-polymerase chain reaction and serum ECP concentration measured from venous blood samples collected from patients with acute severe asthma before the commencement of systemic steroid therapy (day 1) and on day 7 and from patients with stable asthma and healthy volunteers.nnnRESULTSnIL-5 gene expression was significantly higher in patients with acute asthma before steroid treatment than in those with stable disease and healthy subjects (p < 0.0001). Similar results were obtained with serum ECP levels: levels in patients with acute asthma were highest (20.30 +/- 5.31 micrograms/L), followed by levels in patients with stable asthma (2.76 +/- 0.65 micrograms/L) and levels in normal control subjects (1.37 +/- 0.06 micrograms/L; p < 0.01 for all comparisons). Significant falls in both IL-5 expression and serum ECP level were seen on day 7 (p < 0.001) and coincided with a significant improvement in peak expiratory flow (p < 0.0001). Significant correlations were observed between IL-5 expression and ECP level (rho = 0.39, p < 0.01), IL-5 expression and peak expiratory flow (rho = -0.55, p < 0.0002), and peak expiratory flow and ECP level (rho = -0.32, p < 0.04).nnnCONCLUSIONnOur data therefore support an important regulatory role of IL-5 on eosinophil function in human asthma in vivo.

MRI visualization of rodent liver structure and peritoneal adhesion with dialyzate enhancement

This study investigated the use of peritoneal dialysis fluid (dialyzate) as a MR contrast agent to visualize the liver structure and peritoneal adhesion in rats at 7 T. Intraperitoneal injection of dialyzate (∼0.1 ml/g) yielded excellent and consistent intraperitoneal enhancement that delineated the liver lobular structure in all rats studied (N = 8). It also allowed the MR detection of peritoneal adhesions that were surgically induced. MR measurements of adhesion surface areas correlated well with the postmortem estimations (R = 0.99; N = 6). Dialyzate persisted in the intraperitoneal cavity for up to 2 days. T1 and T2 values of undiluted dialyzate were found to be 3017.5 ± 35.3 ms and 108.4 ± 2.0 ms, respectively. These findings demonstrated dialyzate‐enhanced MRI as a potentially valuable technique to localize certain activities within liver (such as local tumor metastasis), and to monitor therapeutic interventions (e.g., against peritoneal adhesion) in preclinical research using small animal models. Magn Reson Med 59:1170–1174, 2008.

Novel genes and variants associated with IgA nephropathy by co-segregating with the disease phenotypes in 10 IgAN families.

BACKGROUNDnPreviously, a large proportion of the genetic components predisposing individuals to IgA nephropathy (IgAN) have been unidentified. Familial IgAN is enriched with genetic variations predisposing individuals to the disease. Whole exome sequencing is an effective way to explore disease-causing genes and gene variants.nnnMETHODSnWe performed exome sequencing on the probands from each of ten IgAN families, and on one of the unaffected member from 7 of the families. Sanger sequencing, bioinformatics and co-segregation analysis were performed for all available family members to detect deleterious genetic variation. The relatedness of the families was tested by haplotype analyses.nnnRESULTSnSix deleterious variants in 4 genes were observed to be associated with IgA nephropathy by co-segregating with the disease phenotypes in study families. MYCT1 p.Asp22Glufs*34 was associated with IgAN by co-segregating with its phenotypes in families 2, 7, and 9; DEFA4 p.Ala8Pro, p.Ala8Val, c.172+1G>T co-segregated in families 1, 2, and 3; ZNF543 p.Pro226Ala co-segregated in families 3, 5, and 6 and CARD8 p.Val98Lysfs*26 co-segregated in families 7 and 8. Among these genes, MYCT1, CARD8 and ZNF543 are novel. Our haplotype analyses showed that families in which the same variation(s) were co-segregating with IgAN were unrelated, except for DEFA4. Of the families carrying DEFA4, families 2 and 3 were possibly related, but not family 1, indicating that common genes/variations in these families were not due to the same founder. Interfamilial sharing of different co-segregating genes was also observed, demonstrating the polygenic nature of this disease.nnnCONCLUSIONSnWe discovered 6 deleterious variants in 4 genes associated with familial IgAN. These genes are good candidate genes that appear to be causally related to IgAN and warrant further study.

TRAC Variants Associate with IgA Nephropathy

The T cell receptor alpha constant gene (TRAC) encodes the constant region of the alpha chain for the T cell receptor, and the association of its gene variants with IgA nephropathy remains controversial. The authors resequenced the gene in 100 patients with IgA nephropathy and 100 controls, tested its linkage disequilibrium pattern, constructed haplotypes, and performed association and functional studies. First, the association between TRAC variants and IgA nephropathy was tested in 704 patients and 704 controls. Next, these 704 patients were divided into two independent datasets--310 with family member(s) and 394 single patients--to test the association separately. Results showed that the gene is located in a recombination hot spot, with nine linkage disequilibrium blocks within a 6.9-kb region. There is a hypervariable region with six single-nucleotide polymorphisms (SNPs) in an 85-bp stretch in intron 1. We identified multiple SNPs and two haplotypes that associate with IgA nephropathy (P = 0.0000013-0.0096 by logistic regression for SNPs; P = 0.0003 and P = 0.0398 for haplotype associations). The family-based study replicated both haplotype findings, and the 394 single-patient case-control study replicated the association with haplotype 1 (P = 0.0033). The overtransmitted/observed haplotypes demonstrated reduced transcription activity compared with the undertransmitted/observed haplotypes. In conclusion, this study suggests an association between TRAC variants and susceptibility to IgA nephropathy.

An Unusual Organism for PD-Related Peritonitis: Hafnia Alvei

1. Kubota M, Kanazawa M, Takahashi Y, Io H, Ishiguro N, Tomino Y. Implantation of presternal catheter using Moncrief technique: aiming for fewer catheter-related complications. Perit Dial Int 2001; 21(Suppl 3):S205–8. 2. Danielsson A, Blohme L, Tranaeus A, Hylander B. A prospective randomized study of the effect of a subcutaneously “buried” peritoneal dialysis catheter technique versus standard technique on the incidence of peritonitis and exit-site infection. Perit Dial Int 2002; 22:211–19. 3. Kubota M, Ishiguro N, Tomino Y, Koide H. Campylobacter fetus subspecies fetus peritonitis in continuous ambulatory peritoneal dialysis. Nephron 1993; 65:487–8. doi: 10.3747/pdi.2009.00078