Ashio Yoshimura

RETRACTED: Combination treatment of angiotensin-II receptor blocker and angiotensin-converting-enzyme inhibitor in non-diabetic renal disease (COOPERATE): a randomised controlled trial

Summary Background Present angiotensin-converting-enzyme inhibitor treatment fails to prevent progression of non-diabetic renal disease. We aimed to assess the efficacy and safety of combined treatment of angiotensin-converting-enzyme inhibitor and angiotensin-II receptor blocker, and monotherapy of each drug at its maximumdose, in patients with non-diabetic renal disease. Methods 336 patients with non-diabetic renal disease were enrolled from one renal outpatient department in Japan. After screening and an 18-week run-in period, 263 patients were randomly assigned angiotensin-II receptor blocker (losartan, 100 mg daily), angiotensin-converting-enzyme inhibitor (trandolapril, 3 mg daily), or a combination of both drugs at equivalent doses. Survival analysis was done to compare the effects of each regimen on the combined primary endpoint of time to doubling of serum creatinine concentration or end-stage renal disease. Analysis was by intention to treat. Findings Seven patients discontinued or were otherwise lost to follow-up. Ten (11%) of 85 patients on combination treatment reached the combined primary endpoint compared with 20 (23%) of 85 on trandolapril alone (hazard ratio 0·38, 95% CI 0·18–0·63, p=0·018) and 20 (23%) of 86 on losartan alone (0·40, 0·17–0·69, p=0·016). Covariates affecting renal survival were combination treatment (hazard ratio 0·38, 95% CI 0·18–0·63, p=0·011), age (1·30, 1·03–2·29, p=0·009), baseline renal function (1·80, 1·02–2·99, p=0·021), change in daily urinary protein excretion rate (0·58, 0·24–0·88, p=0·022), use of diuretics (0·80, 0·30–0·94, p=0·043), and antiproteinuric response to trandolapril (0·81, 0·21–0·91, p=0·039). Frequency of side-effects with combination treatment was the same as with trandolapril alone. Interpretation Combination treatment safely retards progression of non-diabetic renal disease compared with monotherapy. However, since some patients reached the combined primary endpoint on combined treatment, further strategies for complete management of progressive nondiabetic renal disease need to be researched.

Heparan sulfate of perlecan is involved in glomerular filtration.

Perlecan is a heparan sulfate proteoglycan and a major component of the glomerular basement membrane. To understand the role of heparan sulfate chains of perlecan in glomerular filtration, detailed analyses were performed of the kidneys of Hspg2(Delta)(3/)(Delta)(3) mice, whose perlecan lacks heparan sulfate attachment sites in N-terminal domain I. Macroscopic, histologic, and electron microscopic observations, as well as immunohistochemical and immunoelectron microscopic analyses using specific antibodies against perlecan and agrin core proteins, revealed no significant abnormalities in these mice under physiologic conditions. Polyethyleneimine staining demonstrated no significant changes in charge density in the glomerular basement membrane. Transcripts of other heparan sulfate proteoglycans, agrin, and collagen type XVIII, as well as perlecan, were expressed at similar levels to those in the wild-type littermates. Approximately 40% of the perlecan synthesized by Hspg2(Delta)(3/)(Delta)(3) fibroblasts was substituted with heparin sulfate and 60% was substituted with chondroitin sulfate. All of the perlecan synthesized by wild-type fibroblasts contained heparin sulfate, indicating an altered substitution of glycosaminoglycans on Hspg2(Delta)(3/)(Delta)(3) perlecan. Immunostaining indicated that the level of chondroitin sulfate was actually increased in the Hspg2(Delta)(3/)(Delta)(3) glomerular basement membrane. When administered intraperitoneally with BSA, Hspg2(Delta)(3/)(Delta)(3) mice exhibited remarkable proteinuria. These findings suggest that heparan sulfate chains of perlecan play an important role in glomerular filtration, especially of a large amount of protein.

Endothelial von Willebrand Factor Release Due to eNOS Deficiency Predisposes to Thrombotic Microangiopathy in Mouse Aging Kidney

Endothelial dysfunction is critical in the decline of renal function with. By using endothelial nitric oxide synthase knockout (eNOSKO) mice, we tested the hypothesis that a lack of endothelial nitric oxide synthase accelerates renal injury in the aging kidney. In contrast to control mice and young eNOSKO mice, aging eNOSKO mice showed greater renal injury and in particular developed a thrombotic microangiopathy, with mesangiolysis, endothelial swelling, endothelial cell loss, double-contour appearance of glomerular basement membrane (GBM), and thrombus formation. Thrombi, which were composed of fibrin, platelets, and von Willebrand factor (vWF), were identified predominantly in glomerular capillaries and rarely in arterioles, but not in larger vessels. In the tubulointerstitium, tubular degeneration and macrophage infiltration were also prominent in aging eNOSKO mice. Intraluminal vWF deposition was accompanied with thrombus formation, whereas mesangial deposition of vWF was associated with mesangial matrix expansion. Furthermore, the mesangial vWF deposition was detectable in young eNOSKO mice in which severe glomerular injury had not yet developed. Finally, a higher level of serum P-selectin in eNOSKO mice was consistent with the vWF behavior and suggested exocytosis of the Weibel-Palade body by the endothelium. In conclusion, a lack of endothelial nitric oxide synthase resulted in the development of glomerular thrombotic microangiopathy. A lack of nitric oxide likely contributed to the release of vWF, leading to thrombus formation in this model.

Autoregulation of Renal Blood Flow in Streptozocin-Induced Diabetic Rats

Autoregulation of renal blood flow (RBF) was studied in male Wistar rats. We studied 11 control rats, 11 rats with severe streptozocin (STZ)-induced hyperglycemia (diabetic group), and 10 moderately hyperglycemie rats made diabetic by injection of STZ but given 2–8 U s.c. insulin daily (insulin-treated group). RBF was measured by an electromagnetic flowmeter during stepwise reduction of renal perfusion pressure 4–8 wk after injection of STZ (older group). RBF autoregulation of the diabetic group was impaired compared with the control group. In the insulin-treated group, autoregulatory capability was less attenuated than in the diabetic group. The average autoregulatory index (ARI) of the diabetic group (0.61 ± 0.05) was greater than that of the control (0.24 ± 0.02, P < .01) and the insulin-treated (0.33 ± 0.07, P < .05) groups. To study the relationship between autoregulation and the duration of diabetes, an autoregulatory study was also made in a group of 22 rats (11 diabetic and 11 control) that were tested 2–3 wk after injection of STZ (younger group). The ARI in the younger diabetic group was smaller than that in the older diabetic group (P < .05). The results suggest that in uncontrolled diabetes RBF fluctuates with blood pressure change, and protection against hypertensive injury of glomerular capillaries may be diminished. Autoregulatory disability develops with time, and insulin treatment diminishes impairment of autoregulation. These findings may also explain the adverse consequences of hypertension on the progression of diabetic nephropathy in poorly controlled diabetes.

Elimination study of silver in a hemodialyzed burn patient treated with silver sulfadiazine cream

Silver sulfadiazine (SSD) cream is a potent agent for the treatment of burns. In a patient with end-stage renal disease, we observed a marked elevation in serum silver concentration in the course of 2 weeks of SSD cream therapy (200 g/d). Serum concentration of silver reached a maximum of 291 ng/mL in association with a rapid deterioration of mental status. SSD therapy was discontinued, and hemodialysis, hemofiltration, or plasma exchange was continually performed. Four months later, the patient died. At autopsy, profoundly elevated levels of silver were found in brain tissues of this patient (617.3, 823.7 ng/g wet tissue weight in the cerebrum and cerebellum, respectively). To determine the most efficient therapy to remove silver from serum, we compared hemodialysis (HD), hemofiltration (HF), and plasma exchange (PE). Both plasma exchange and hemofiltration were effective in decreasing serum silver, and their effects were additive. By contrast, HD was ineffective in reducing serum silver. This case illustrates that, on SSD cream therapy, burn patients with disturbed renal function are at risk of accumulating silver in serum and tissue to the level that may cause neuralgic decompensation. Removal of serum silver can best be effected by PE, particularly when combined with HF. In contrast, HD per se does not appear efficacious. None of these blood purification modalities improves deterioration of neurological status potentially attributable to silver deposition in brain tissues.

Reversible posterior leukoencephalopathy syndrome after blood transfusion in a patient with end-stage renal disease

A 42-year-old female end-stage renal disease (ESRD) patient with reversible posterior leukoencephalopathy syndrome (RPLS) post-transfusion during initiation of hemodialysis is reported. Eleven days after the onset of illness, we diagnosed encephalopathy as a grand mal seizure resulting from diffuse cerebral edema. One reason for the delayed diagnosis was that her symptom, a throbbing headache that occurred during her first dialysis, indicated dialysis disequilibrium syndrome. We must bear in mind that a small amount of transfusion could cause RPLS even during the first dialysis. To our knowledge, this is the first case report on RPLS after blood transfusion in an ESRD patient.

Eplerenone-Mediated Aldosterone Blockade Prevents Renal Fibrosis by Reducing Renal Inflammation, Interstitial Cell Proliferation and Oxidative Stress

Background/Aims: Prolonged elevation of serum aldosterone leads to renal fibrosis. Inflammation also plays a role in the pathogenesis of renal disease. We used a rat model of interstitial renal fibrosis to test the hypothesis that eplerenone-mediated aldosterone blockade prevents renal fibrosis due to its anti-inflammatory and anti-proliferative effects. Methods: Eplerenone (a selective aldosterone blocker) or vehicle (control), was given to male Wistar rats (50 mg/kg, twice daily) for 7 days before unilateral ureteral obstruction (UUO) and for an additional 28 days after surgery. Body weight, blood pressure, renal histo-morphology, immune-staining for macrophages, monocyte chemotactic protein-1, proliferating cell nuclear antigen, α-smooth muscle actin, and serum and urine markers of renal function and oxidative stress were determined for both groups on 7, 14, and 28 days after surgery. Results: Epleronone had no effect on body weight or blood pressure. However, eplerenone inhibited the development of renal fibrosis, inflammation (macrophage and monocyte infiltration), interstitial cell proliferation, and activation of interstitial cells (α-SMA expression). Epleronone also reduced oxidative stress. Conclusion: The anti-fibrotic effect of eplerenone appears to be unrelated to its effect on blood pressure. Eplerenone inhibits renal inflammation, interstitial cell proliferation, phenotypic changes of interstitial cells, and reduces oxidative stress.

Expression of epidermal growth factor and its receptor in rabbits with ischaemic acute renal failure

Urinary immunoreactive epidermal growth factor (EGF) levels decrease, and renal immunoreactive EGF levels increase in rats with ischaemic acute renal failure (ARF). We investigated the immunohistochemical localization of EGF and EGF receptor in rabbits with ischaemic ARF to clarify the significance of renal EGF. Male New Zealand White rabbits underwent right nephrectomy prior to a 60 min renal artery clamp. At 3, 6, 24, 48, 72 and 96 h after ischaemia, serum urea nitrogen and serum creatinine were determined. Guinea pig anti-rabbit EGF antibody and monoclonal anti-EGF receptor antibody were used for the primary incubation. EGF was immunolocalized to the ascending limb of Henle and the distal convoluted tubule in the normal right kidneys. However, in the post ischaemic left kidneys at 6, 24, 48 and 72 h, immunoreactivity of EGF was associated with proximal tubules. In the normal kidneys, antibody to EGF receptor reacted with distal tubules and collecting ducts. In the ischaemic kidneys, EGF receptor was localized in the basolateral membrane in the proximal tubules. The expression of EGF and EGF receptor in renal tubules may play an important role in repair following ischaemic renal damage.

Mesangial Proliferative Nephritis in Man Is Associated with Increased Expression of the Cell Survival Factor, Bcl-2

Although most studies suggest that the hypercellularity in mesangial proliferative nephritis is due to increased cell proliferation, we hypothesized that it may also be due to increased expression of survival factors that may block their removal (apoptosis). We therefore studied the expression of apoptosis preventing/delaying the bcl-2 gene product in the glomerulus with various human glomerulonephritides. Immunohistochemistry for Bcl-2, proliferating cell associated protein (Ki-67) and α-smooth muscle actin (α-SMA) was performed on 55 biopsied kidney tissues: 6 cases of orthostatic proteinuria as a control (OP); 6 cases of diffuse proliferative lupus nephritis (WHO type IV, LN-MPGN); 24 cases of IgA nephropathy (IgA); 9 cases of minimal change nephrosis and 10 cases of idiopathic membranous nephropathy. The number of Ki-67-positive cells and the expression of α-SMA in the glomerulus were significantly higher in LN-MPGN and IgA. There was a significant positive correlation between glomerular Bcl-2 expression and glomerular cell proliferation evaluated by the number of Ki-67-positive cells (r = 0.605, p < 0.01) or glomerular α-SMA expression (r = 0.674, p < 0.01). Glomerular expression of Bcl-2 in IgA or LN-MPGN was significantly higher than that in OP (p < 0.01 and p < 0.05 vs. OP, respectively). The Bcl-2-positive cells were present in mesangial locations and demonstrated a perinuclear pattern. These results suggest that maintenance of glomerular hypercellularity in human glomerular diseases is partly due to the prevention of mesangial cell death via Bcl-2 expression.

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

SummaryThe ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18–21 particles/1000 nm of LRE) with fairly regular interspacing (45–65 nm) as reported previously.This is the first report to identify the three-dimensional ultrastructure of anionic sites of GBM, and provides new information on the location and distribution of anionic sites in the glomerular capillary wall. In addition, these studies suggest that several chemical procedures used in conventional transmission electron microscopy to visualize PEI tracers, may produce structural changes and disarrangement of PEI particles that can be avoided with the quick-freezing and deep-etching method.