Yee-Yung Ng

Interleukin-1 induces tubular epithelial-myofibroblast transdifferentiation through a transforming growth factor-β1-dependent mechanism in vitro

Interleukin-1 (IL-1) has been shown to exert profibrotic activity in a number of disease models, including crescentic glomerulonephritis and pulmonary fibrosis, but the mechanisms by which this operates are poorly understood. Recent studies have identified a novel mechanism promoting renal fibrosis: tubular epithelial-myofibroblast transdifferentiation (TEMT). The present study examined whether IL-1 can stimulate TEMT in vitro. Cells of the normal rat kidney tubular epithelial cell line (NRK52E) were grown to confluence on collagen-coated plates and cultured for 5 days in the presence 1 to 20 ng/mL of IL-1alpha. Doses of 10 to 20 ng/mL of IL-1 caused transdifferentiation of NRK52E cells into myofibroblast-like cells. Scanning electron microscopy identified IL-1-induced morphological changes as a loss of apical-basal polarity and microvilli, cell hypertrophy, and the development of an elongated and invasive appearance. Phenotypically, IL-1-induced TEMT was characterized by de novo messenger RNA and protein expression of the mesenchymal marker alpha-smooth muscle actin, shown by Northern blotting, immunohistochemistry, and Western blotting. This was accompanied by loss of the epithelial marker E-cadherin. The addition of an excess of IL-1-receptor antagonist completely inhibited IL-1-induced TEMT. IL-1 was shown to stimulate the secretion of active transforming growth factor-beta1 (TGF-beta1) by NRK52E cells. Furthermore, the addition of a neutralizing anti-TGF-beta1 antibody inhibited IL-1-induced TEMT. In conclusion, IL-1 is a profibrogenic cytokine capable of inducing TEMT through a TGF-beta1-dependent mechanism. This may represent a novel mechanism by which IL-1 induces renal fibrosis in vivo.

A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology.

The digestion of fixed tissue sections is a critical step in the optimization of any in situ hybridization protocol. We describe a novel application of microwave oven heating to optimize mRNA detection in paraformaldehyde-fixed tissues by in situ hybridization using digoxigenin-labeled probes. This technique replaces protease digestion of fixed tissue sections with 10 min of microwave pretreatment, followed by either conventional hybridization or hybridization involving microwave incubation. This new technique has several advantages over the standard protease treatment-based methods presently in use. (a) Microwave oven heating is a simple, rapid, and highly reproducible technique. (b) Microwave pretreatment significantly increased the hybridization signal and reduced the background compared to conventional protease digestion. Consequently, the hybridization time required to obtain optimal mRNA detection was reduced to 30 min. (c) Ten minutes of microwave pretreatment produced an optimal hybridization signal in six different tissues using a variety of probes, demonstrating the general applicability of this technique. (d) Microwave heating of the probe during the hybridization step itself further reduced the hybridization time and substantially enhanced the hybridization signal obtained from proteinase K-digested tissue. (e) Microwave pretreatment caused no discernible loss of fine cell structure and tissue morphology compared to untreated tissue sections. In conclusion, microwave oven heating can replace the complicated strategies and poor reproducibility of protease treatment of tissue sections, resulting in a simple, rapid, more reliable and sensitive method that has general applicability for in situ hybridization.

Pentoxifylline inhibits transforming growth factor-beta signaling and renal fibrosis in experimental crescentic glomerulonephritis in rats.

Background/Aims: Pentoxifylline (PTX) has been shown to inhibit renal inflammation in a rat model of crescentic glomerulonephritis. The present study investigated the role of PTX in renal fibrosis in rats with crescentic glomerulonephritis. Methods: A rat model of accelerated anti-glomerular basement membrane glomerulonephritis was induced and treated with PTX or vehicle control for 3, 7, 14 and 28 days. The therapeutic effect and mechanism of PTX on renal fibrosis were examined by Northern blot and immunohistochemistry. Results: Diseased rats treated with vehicle control developed a severe crescentic glomerulonephritis with progressive renal fibrosis identified by a marked accumulation of α-SMA+ myofibroblasts and collagen matrix. This was associated with tubular epithelial-myofibroblast transition as evident by de novo expression of α-SMA and a loss of E-cadherin on damaged tubular epithelial cells. Further studies revealed that severe renal fibrosis was associated with upregulation of renal TGF-β1 and activation of TGF-β/Smad signaling, which was blocked by treatment with PTX. Conclusions: PTX may be an anti-fibrosis agent capable of inhibiting renal fibrosis in a rat model of crescentic glomerulonephritis. Blockade of TGF-β1 expression and Smad2/3 activation may be a mechanism by which PTX inhibits renal fibrosis.

Essential trace element status and clinical outcomes in long-term dialysis patients: A two-year prospective observational cohort study

BACKGROUND & AIMSnEssential trace elements are involved in many biological processes for normal cell function including immunological defense against oxidation and infection. Deficiency of these elements generally leads to illness or even death in the general population. Therefore, we investigated the predictive values of trace element status on clinical outcomes in dialysis patients, who are more prone to trace element deficiency.nnnMETHODSnWe enrolled 111 prevalent patients on maintenance dialysis from a Taipei tertiary-care referral hospital and measured serum levels of selenium, copper, and zinc. Patients were followed for 2 years or until death or withdrawal.nnnRESULTSnMultivariate Cox regression analysis indicated that patients with diabetes mellitus (HR, 2.162 [95% CI, 1.105-4.232], p=0.024), prior stroke (HR, 3.876 [95% CI, 1.136-13.221], p=0.030), and zinc deficiency (HR, 0.979 [95% CI, 0.966-0.992], p=0.002) were more likely to be hospitalized for infectious diseases. Furthermore, beyond traditional risk factors, such as old age and hypoalbuminemia, multivariate Cox regression also indicated that lower serum level of zinc independently predicts overall mortality (HR, 0.973 [95% CI, 0.948-0.999], p=0.046).nnnCONCLUSIONSnIn long-term dialysis patients, the serum level of zinc was an independent predictor of future hospitalization due to infectious diseases and of overall mortality.

Spiral Computed Tomographic Angiography – A New Technique for Evaluation of Vascular Access in Hemodialysis Patients

Spiral computed tomographic angiography (CTA), a new noninvasive imaging technique, was used to study 10 arteriovenous fistulas (AVF) in 9 hemodialysis patients. Digital subtraction angiography (DSA) was also performed as a gold standard for comparison. AVF stenosis was graded by a four-point scale: grade 0, well patency of supplying artery, anastomosis and drainage vein; grade 1, <50% stenosis; grade 2, 50–70% stenosis; grade 3, 70–99% stenosis, and grade 4, total occlusion. We found CTA correlated closely to DSA in detecting both stenosis and dilatation of AVF and it spared all the shortcomings of DSA. CTA has the potential to be alternative for imaging of dialysis fistulas. Further studies will be performed to specify the role of CTA images in the assessment of the hemodialysis vascular access.

Comparison of peptic ulcer disease risk between peritoneal and hemodialysis patients.

Background: Compared to the general population, patients with end-stage renal disease (ESRD) have increased peptic ulcer and upper GI bleeding complication rates. However, the risk factors for peptic ulcer among ESRD patients are unknown. Methods: In this retrospective study, we enrolled 827 incident dialysis patients and diagnosed peptic ulcer on the basis of endoscopic findings; information on the morbidities and medical prescription were obtained directly from medical records. A Cox regression hazard model was used to identify risk factors for peptic ulcer. Results: During the 10-year study period, 481 patients underwent an endoscopic exam. Peptic ulcers were detected in 153 patients. Age (p = 0.025), peritoneal dialysis (p = 0.022), diabetes mellitus (p = 0.020), congestive heart failure (p = 0.015), low serum albumin (p = 0.008) and high gamma-glutamyl transpeptidase (γ-GT) levels (p = 0.002) are risk factors for peptic ulcers among ESRD patients. Ulcer severity (p = 0.004) and aspirin prescription (p = 0.043), but not Helicobacter pylori infection, influenced the ulcer recurrence rate. Conclusion: The risk factors for peptic ulcer have some differences between ESRD patients and general population. In patients with high risk of upper GI bleeding, peritoneal dialysis and aspirin should be prescribed with caution.

Ankle-Brachial Index Is a Powerful Predictor of Renal Outcome and Cardiovascular Events in Patients with Chronic Kidney Disease

Ankle-brachial index (ABI) is an accurate tool to diagnose peripheral arterial disease. The aim of this study was to evaluate whether ABI is also a good predictor of renal outcome and cardiovascular events in patients with chronic kidney disease (CKD). We enrolled 436 patients with stage 3–5u2009CKD who had not been undergoing dialysis. Patients were stratified into two groups according to the ABI value with a cut point of 0.9. The composite renal outcome, including doubling of serum creatinine level and commencement of dialysis, and the incidence of cardiovascular events were compared between the two groups. After a median follow-up period of 13 months, the lower ABI group had a poorer composite renal outcome (OR = 2.719, P = 0.015) and a higher incidence of cardiovascular events (OR = 3.260, P = 0.001). Our findings illustrated that ABI is a powerful predictor of cardiovascular events and renal outcome in patients with CKD.