Kayo Kaneko

Relationship between Abdominal Fat Accumulation and Insulin Resistance in Hemodialysis Patients

It is well known that obesity and insulin resistance are closely related to the development of type 2 diabetes. However, the exact pathogenic mechanism underlying the insulin resistance in renal disease has not been clarified. The purpose of the present study was to clarify the contribution of abdominal (visceral and subcutaneous) fat accumulation to insulin resistance and various clinical parameters, including C-reactive protein (CRP), in hemodialysis (HD) patients. Visceral and subcutaneous fat areas (VFA and SFA) were evaluated at the umbilical level by CT. Insulin resistance was estimated by the homeostasis model assessment−insulin resistance index (HOMA-IR) in 80 HD patients. Insulin resistance and CRP seemed to be closely correlated with fat-related parameters such as body mass index (BMI), VFA and SFA. HOMA-IR was positively correlated with BMI, VFA, SFA, triglycerides (TG), remnant-like particle (RLP)-cholesterol and CRP in simple regression analysis. In multiple stepwise regression analysis, SFA and RLP-cholesterol were predominant determinants of HOMA-IR in HD patients. Furthermore, CRP was positively correlated with BMI, VFA, SFA, TG, high-density lipoprotein (HDL)-cholesterol, atherosclerosis index (AI), immunoreactive insulin (IRI) and HOMA-IR in simple regression analysis. In multiple stepwise regression analysis, VFA and HDL-cholesterol were predominant determinants of CRP in HD patients. In conclusion, insulin resistance and CRP were related to fat-related parameters such as BMI, VFA and SFA in HD patients. Furthermore, the contribution of SFA to insulin resistance was much higher than that of VFA, while the opposite relation was recognized for CRP. (Hypertens Res 2008; 31: 83−88)

Quantitative evaluation and assessment of peritoneal morphologic changes in peritoneal dialysis patients

BACKGROUND Morphologic changes of the peritoneum such as peritoneal fibrosis and vasculopathy develop during peritoneal dialysis (PD). In 2002, Williams et al. reported microscopic characteristics of peritoneal changes in PD patients. These studies pointed out the importance of establishing a global standard for qualitative and quantitative histological evaluations. The objectives of the present study are (i) to verify the methods for assessing peritoneal thickness and classifying vasculopathy in peritoneal specimens using the assessment of Williams et al. and (ii) to propose a simple assessment that reflects clinical features such as PD duration and peritoneal function. METHODS Parietal peritoneal samples were obtained from 35 patients that included 27 patients with PD and 8 uraemic patients without PD. In all samples, the maximum and average thicknesses of the submesothelial compact (SMC) zone were measured to assess peritoneal interstitial fibrosis using KS400 imaging analysis. Vasculopathy was also assessed by calculation of patency rates of the vascular lumens using the diameter and area, and by measurement of dimensions of vascular wall hyalinization in each vessel specimen. RESULTS The median values of maximum and average thicknesses of the SMC zone exceeded 200 μm in uraemic patients without PD treatment. There was a significant relationship between the maximum and average thicknesses of the SMC zone (P < 0.0001). Four to 30 vessels were examined in each participant. Various grades of vasculopathy were observed in each specimen. According to the predominant vasculopathy found in each vessel, the prevalence of serious vasculopathy increased with increasing PD duration. Vascular patency calculated from wall thickness was significantly related to that calculated by the area and to the thickness of hyalinization. Average vascular patency assessed from 5 to 10 vessels in each patient having diameters ranging from 10 to 40 μm was related to PD duration and to peritoneal function (D4/P). CONCLUSIONS A random-points measurement of average SMC thickness provides a descriptive evaluation of the severity of peritoneal fibrosis that minimizes artefacts during processing and avoids human error. In addition, the average patency in post-capillary venules appears to accurately reflect clinical features such as PD duration and peritoneal permeability.

Effect of AST-120 on Endothelial Dysfunction in Adenine-Induced Uremic Rats

Aim. Chronic kidney disease (CKD) represents endothelial dysfunction. Monocyte adhesion is recognized as the initial step of arteriosclerosis. Indoxyl sulfate (IS) is considered to be a risk factor for arteriosclerosis in CKD. Oral adsorbent AST-120 retards deterioration of renal function, reducing accumulation of IS. In the present study, we determined the monocyte adhesion in the adenine-induced uremic rats in vivo and effects of AST-120 on the adhesion molecules. Methods. Twenty-four rats were divided into control, control+AST-120, adenine, and adenine+AST-120 groups. The number of monocytes adherent to the endothelium of thoracic aorta by imaging the entire endothelial surface and the mRNA expressions of adhesion and atherosclerosis-related molecules were examined on day 49. The mRNA expressions of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells were also examined. Results. Adenine increased the number of adherent monocytes, and AST-120 suppressed the increase. The monocyte adhesion was related to serum creatinine and IS in sera. Overexpression of VCAM-1 and TGF-β1 mRNA in the arterial walls was observed in uremic rats. IS induced increase of the ICAM-1 and VCAM-1 mRNA expressions in vitro. Conclusion. It appears that uremic condition introduces the monocyte adhesion to arterial wall and AST-120 might inhibit increasing of the monocyte adherence with CKD progression.

Differentiation of bone marrow-derived cells into regenerated mesothelial cells in peritoneal remodeling using a peritoneal fibrosis mouse model

Marked thickening of the peritoneum and vasculopathy in the submesothelial compact zone have been reported in long-term peritoneal dialysis patients. Bone marrow (BM)-derived cell lines are considered to be useful tools for therapy of various diseases. To clarify the role of BM-derived cells in the peritoneal fibrosis (PF) model, we analyzed several lineages of cells in the peritoneum. BM cells from green fluorescent protein (GFP) transgenic mice were transplanted into naïve C57Bl/6 mice. Chlorhexidine gluconate (CG) was injected intraperitoneally to induce PF. Immunohistochemical analysis was performed with parietal peritoneum using anti-Sca-1 or -c-Kit and -GFP antibodies. Isolated BM cells were also transplanted into the CG-stimulated peritoneum. BM-derived cells from GFP transgenic mice appeared in the submesothelium from days 14 to 42. Both GFP- and stem cell marker-positive cells were observed in the submesothelium and on the surface. Isolated c-Kit-positive cells, transplanted into the peritoneal cavity, differentiated into mesothelial cells. In this study, we investigated whether or not BM-derived cells play a role in the repair of PF and immature cells have the potential of inducing repair of the peritoneum. The findings of this study suggest a new concept for therapy of PF.

CARDIAC FUNCTION AND STRUCTURE IN LONGITUDINAL ANALYSIS OF ECHOCARDIOGRAPHY IN PERITONEAL DIALYSIS PATIENTS

♦ Background: Echocardiography is widely used for the evaluation of cardiac structures and function. The prognostic value of assessment of left cardiac atrium (LA) size in peritoneal dialysis (PD) patients is still unclear. The objective of the present study is to investigate prospectively a longitudinal monitoring of echocardiography parameters after start of PD. We also investigated a correlation study among plasma atrial natriuretic peptide (ANP) level, LA size, and cardiac function undergoing aggressive treatment. ♦ Methods: Correlation among plasma ANP, LA size, and cardiac function was prospectively analyzed by Doppler echocardiography in 32 PD patients in Juntendo University Hospital, Tokyo. Measurement of these parameters was performed at 0, 6, 12, 18, and 24 months after start of PD. All patients were treated with an angiotensin type 1 receptor blocker to control blood pressure to less than 140/90 mmHg. Other antihypertensive drugs such as diuretics and/or calcium channel blockers were added if blood pressure rose to over 140/90 mmHg. Hemoglobin and hematocrit levels were targeted at 10.0 g/dL and 30.0% respectively with recombinant human erythropoietin treatment. A diuretic was added or patients decreased their water intake if ANP was more than 43.0 pg/mL or LA diameter (LAD) more than 39 mm, and for other basic markers of volume status. Cardiac function was measured before and after drainage of PD fluid to evaluate the influence of cardiac function. ♦ Results: LAD at start of dialysis (36 ± 4.6 mm) decreased significantly to 33 ± 3.3 mm (p < 0.05), 33 ± 3.2 mm (p < 0.05), and 33 ± 3.6 mm (p < 0.05) after 6, 12, and 24 months, respectively. Ejection fraction after 6 months was significantly increased compared with that at start of dialysis (p < 0.05). Left ventricular mass index (LVMI) after 6, 12, and 24 months was significantly decreased compared with that at start of dialysis (p < 0.05). ANP was 56 ± 39 pg/mL at start of dialysis and decreased significantly to 33 ± 19 pg/mL after 24 months (p < 0.05). ANP was significantly correlated with LAD (r = 0.412, p < 0.01), transmitral A wave flow velocity (r = 0.429, p < 0.01), and LVMI (r = 0.426, p < 0.01). Instillation of the dialysis fluid did not affect any parameters except inferior vena cava dimension. ♦ Conclusion: This study demonstrates a reduction in LA size and LVMI in PD patients followed over 24 months. Left ventricular structure, contraction, and compliance were well preserved in PD patients undergoing aggressive treatment based on measurements of plasma ANP and LAD.

Improvement of peritoneal calcification after parathyroidectomy in a peritoneal dialysis patient.

Peritoneal calcification is one of the complications of peritoneal dialysis (PD). It can become serious, leading to severe abdominal pain and even death. Possible mediators of peritoneal calcification in PD patients are assumed to include acetate buffer, overdosage of vitamin D, repeated peritonitis, hypertonic dialysate, calciphylaxis and secondary hyperparathyroidism (SHPT). However, the mechanism and treatment of peritoneal calcification are controversial. Few reports have appeared on improvement of peritoneal calcification after parathyroidectomy (PTX) for SHPT of long duration. We report herein the case of a 48-year-old man on dialysis for 17 years including PD for 14 years. In 1989, he was admitted to hospital because of end-stage renal disease (ESRD), and started treatment with PD. Abdominal computed tomography (CT) first showed peritoneal calcification in August 2002. Peritoneal calcification did not improve despite conventional treatment including discontinuation of PD, control of calcium phosphate product to less than 55 mg2/dl2, removal of the peritoneal catheter and empirical prednisolone (PSL) usage. The intact parathyroid hormone (i-PTH) level was increased over 1,000 pg/ml and extra-osseous calcification occurred. Total PTX was performed in November 2004. Postoperatively, the i-PTH level decreased immediately and calcium phosphate product was maintained in the reference range. Abdominal CT after PTX showed improvement of peritoneal calcification in September 2005. It appeared that PTX could be used to treat patients with persistent peritoneal calcification not responding to conventional treatment. It was postulated that SHPT might play a crucial role in accelerating peritoneal calcification in PD patients.

Paracrine effects of transplanted mesothelial cells isolated from temperature-sensitive SV40 large T antigen gene transgenic rats during peritoneal repair

Background The prevention and restoration of peritoneal damage is a critical mission in peritoneal dialysis (PD). Transplantation of mesothelial cells has been suggested to suppress peritoneal injury during PD. Few studies have examined the efficacy and safety of cell transplantation. We evaluated the paracrine effects of mesothelial transplantation during peritoneal repair using immortalized temperature-sensitive mesothelial cells (TSMCs) in chlorhexidine gluconate (CG)-induced peritoneal fibrosis rats. Methods Continuous-infusion pumps containing 8% CG were placed into the abdominal cavity for 21 days. After the removal of the pumps, the TSMCs were injected into the peritoneal cavity at Day 22 (Tx-1 group) or 29 (Tx-2 group). Morphological findings and mRNA expressions of regeneration-related factors were examined at Days 22, 29 and 35. Results Peritoneal thickness was aggravated in the Tx-1 group. Levels of transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 mRNA in the Tx-1 group at Day 35 were comparable with those at Day 22. The levels of Snail, B-Raf and ERK-1, markers of epithelial to mesenchymal transition and of the RAS/MAPK pathway in the Tx-1 group, were significantly higher than those in the Tx-2 group. TGF-β and VEGF were produced from the transplanted mesothelial cells and the surrounding cells in the Tx-1 group. Conclusion It appears that the paracrine effect of transplanted mesothelial cells during peritoneal repair is associated with its surrounding condition. It is important to determine the most appropriate time for developing peritoneal repair through mesothelial transplantation.

Establishment of a peritoneal mesothelial cell line from a transgenic rat harbouring the temperature-sensitive simian virus 40 large T-antigen gene

BACKGROUND Establishing a peritoneal mesothelial cell (MC) line in which the native characteristics of primary MCs are constantly maintained in vivo is of great significance for investigating their morphological and functional changes in peritoneal dialysis. We established transgenic (Tg) rats that expressed the temperature-sensitive tsA58 mutant of the simian virus 40 large T-antigen (tsSV40T), which served as a source of immortalized rat cell lines. The cells were immortalized at a permissive temperature of 33 degrees C, although they were differentiated at a non-permissive temperature of 38 degrees C. In this study, we established a novel MC line from tsSV40T Tg rats and evaluated its characteristics. METHODS MCs were isolated from 8-week-old tsSV40T Tg rats and cloned. MCs from 8-week-old Wistar rats were used as controls. These cells were immunohistochemically and phenotypically evaluated by immunofluorescence, phase contrast and electron microscopy. The production of plasminogen activator inhibitor 1 (PAI-1) from MCs stimulated by tumour necrosis factor-alpha (TNF-alpha) was measured. RESULTS The tsSV40T MCs showed a cobblestone-like appearance at 33 and 38 degrees C, which was similar to normal primary cultured MCs. Microvilli-like structures were observed on the cell surface by a scanning electron microscope at 33 and 38 degrees C. Wilms tumour-1 and pancytokeratin, as MC markers, were expressed at 33 and 38 degrees C. Following TNF-alpha stimulation, PAI-1 production of tsSV40T MCs was similar to that of normal primary cultured MCs. CONCLUSION We established a novel, conditionally immortalized MC line that continuously retained the characteristics of primary cultured peritoneal MCs. This cell line might be a useful tool for various types of in vitro biological research on peritoneal dialysis.

Scavenging of reactive oxygen species by astaxanthin inhibits epithelial–mesenchymal transition in high glucose-stimulated mesothelial cells

Background High glucose concentrations influence the functional and structural development of the peritoneal membrane. We previously reported that the oral administration of astaxanthin (AST) suppressed peritoneal fibrosis (PF) as well as inhibited oxidative stress, inflammation, and epithelial–mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) in a chlorhexidine-induced PF rat model. This suggests that oxidative stress induction of EMT is a key event during peritoneal damage. The present study evaluated the therapeutic effect of AST in suppressing EMT, in response to glucose-induced oxidative stress. Methods Temperature-sensitive mesothelial cells (TSMCs) were cultured in the presence or absence of AST and then treated with 140 mM glucose for 3 or 12 hours. Expression levels of TNF-α, TGF-β, and VEGF were determined at the mRNA and protein levels, and nuclear factor kappa B (NF-κB) activity was evaluated. We measured NO2−/NO3− concentrations in cellular supernatants and determined 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in mitochondrial and nuclear DNA. The expressions of E-cadherin and alpha-smooth muscle actin (α-SMA) were evaluated by double immunofluorescence and protein levels. Results High glucose concentrations induced overproduction of reactive oxidative species (ROS), increasing 8-OHdG mitochondrial DNA and cytokine levels. The NF-κB pathway was activated in response to high glucose concentrations, whereas de novo α-SMA expression was observed with decreased E-cadherin expression. AST treatment attenuated ROS production, inflammatory cytokine production, NF-κB activation, and EMT. Conclusion The findings of the present study indicate that AST may have an anti-EMT effect due to anti-oxidative and anti-inflammatory activities by scavenging glucose-induced ROS from mitochondria in PMCs. AST may be an efficacious treatment for PF.

Comparison Between the Fixation of Peritoneal Dialysis Catheters to the Peritoneal Wall and the Conventional Placement Technique: Clinical Experience and Follow-Up of a New Implant Technique for Peritoneal Dialysis Catheters

Peritoneal dialysis (PD) catheters often become severely dislocated, which may lead to malfunction. With the aim of preventing this complication, we have developed a simple method of fixing the catheter downwards in the peritoneal cavity (fixation technique), a technique that does not require a laparoscope. Sixteen patients were implanted using the conventional placement technique and 25 patients were implanted using the fixation technique. The location of the catheter tip was classified from grade 1 (downward, normal) to 5 (dislocated). The frequency of dislocation (defined as the extended time and/or decrease in volume when draining the PD solution) was measured for both the fixation technique and conventional placement technique. There was a significant difference in grade between the fixation technique (2.72 ± 1.01) and conventional technique (3.92 ± 1.31). The time until first dislocation was significantly different between the fixation technique (59.3 ± 48.1 days) and conventional technique (8.8 ± 14.6 days). The time until any dislocation was significantly different between the fixation technique (69.2 ± 41.9 days) and conventional technique (12.9 ± 13.7 days). Complications were not significantly different between the fixation technique and conventional technique. The fixation technique appears to be simple, safe, and useful for preventing severe dislocation and for lengthening the time until dislocation in PD patients.