Yoshihiko Kanno

Connective Tissue Growth Factor Expressed in Tubular Epithelium Plays a Pivotal Role in Renal Fibrogenesis

Connective tissue growth factor (CTGF) is one of the candidate factors that are thought to mediate the downstream profibrotic action of TGF-beta. However, its precise role in renal interstitial fibrogenesis has not yet been clarified. It was demonstrated previously that CTGF was expressed in tubular epithelial cells that had been engulfed by interstitial fibrosis in the remnant kidney of the subtotal nephrectomy (SNx) model. In the present study, co-cultures of tubular epithelial cells (mProx24) and tubulointerstitial fibroblasts (TFB) that mimic the subepithelial mesenchyme in the kidney were used to study the profibrotic effects of TGF-beta1-induced CTGF. In these co-cultures, TGF-beta1 treatment resulted in significantly increased mRNA levels of type I collagen and fibronectin in the TFB. These effects were both direct and indirect, with the latter being mediated by CTGF derived from the co-cultured mProx24. Then TGF-beta1 transgenic mice were subtotally nephrectomized and treated with CTGF antisense oligodeoxynucleotide, and their kidneys were analyzed for fibrosis. Intravenous administration of CTGF antisense oligodeoxynucleotide significantly blocked CTGF expression in the proximal tubular epithelial cells in the remnant kidney of these animals despite the sustained level of TGF-beta1 mRNA. This reduction in CTGF mRNA level paralleled a reduction in mRNA levels of matrix molecules as well as proteinase inhibitors plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase-1, suppressing renal interstitial fibrogenesis. In conclusion, tubular CTGF acts as a downstream mediator of the profibrotic effects of TGF-beta1 in the remnant kidney, which is a promising target for antifibrotic drugs designed to treat TGF-beta1-dependent interstitial fibrosis.

Effect of Angiotensin Receptor Blockers on Cardiovascular Events in Patients Undergoing Hemodialysis: An Open-Label Randomized Controlled Trial

BACKGROUND Cardiovascular disease is the leading cause of mortality in patients with kidney failure treated with hemodialysis (HD). Although angiotensin receptor blockers (ARBs) reduce cardiovascular disease (CVD) events in patients with diabetes and chronic kidney disease, their effect in patients with kidney failure on HD therapy is not known. STUDY DESIGN Open-labeled randomized trial. SETTING & PARTICIPANTS Patients aged 30 to 80 years receiving HD 2 to 3 times weekly for 1 to 5 years at 5 university-affiliated dialysis centers. INTERVENTIONS Treatment with ARBs (valsartan, candesartan, and losartan) versus without ARBs after stratification by sex, age, systolic blood pressure, and diabetes. OUTCOMES The primary end point is the development of fatal and nonfatal CVD events, defined as the composite of CVD death, myocardial infarction, stroke, congestive heart failure, coronary artery bypass grafting, or percutaneous coronary intervention. The secondary end point is all-cause death. RESULTS 366 subjects initially were randomly assigned to an ARB or no ARB (control), but after a run-in phase, 180 were retained in each group. Mean age was 60 years, 59% were men, 51% had diabetes, and mean predialysis systolic blood pressure was 154 mm Hg. There were 93 fatal or nonfatal CVD events (52%); 34 (19%) in the ARB group and 59 (33%) in the non-ARB group. After adjustment for age, sex, diabetes, systolic blood pressure, and center, treatment with an ARB was independently associated with reduced fatal and nonfatal CVD events (hazard ratio, 0.51; 95% confidence interval, 0.33 to 0.79; P = 0.002). There were 63 deaths (35%); 25 (14%) in the ARB group and 38 (21%) in the non-ARB group. After adjustment, all-cause mortality differed between the 2 groups (hazard ratio, 0.64; 95% confidence interval, 0.39 to 1.06; P = 0.1). LIMITATIONS Because of the small sample size of this trial, the large effect may be a spurious finding. Use of an open-label design and 3 different agents in the ARB group might have influenced results. CONCLUSION Use of an ARB may be effective in reducing nonfatal CVD events in patients undergoing long-term HD. A larger study is required to confirm these results.

A case–control study of calciphylaxis in Japanese end-stage renal disease patients

BACKGROUND Calciphylaxis, also called calcific uremic arteriolopathy, is a rare and often fatal complication of end-stage renal disease and is characterized by painful skin ulceration, necrosis, medial calcification and intimal proliferation of small arteries. Studies in western countries have reported incidences ranging from 1 to 4% in chronic hemodialysis patients. Since no systematic studies of calciphylaxis have ever been performed in Japan, we conducted a nationwide survey and a case-control study to identify the characteristics of calciphylaxis in the Japanese dialysis population. METHODS Firstly, we sent a questionnaire to 3760 hemodialysis centers in Japan, asking whether calciphylaxis cases had been encountered in the past, and detailed clinical data regarding each case were then collected from the centers. In addition, two control dialysis patients matched for age and duration of hemodialysis to each calciphylaxis case were identified at the participating centers, and their data were analyzed to identify risk factors for calciphylaxis. RESULTS Responses to the questionnaire were obtained from 1838 centers (48.3%), and 151 centers reported that a total of 249 cases had been encountered. Sixty-four centers agreed to participate in the case-control study, and detailed clinical data in regard to 67 cases were obtained. In 28 of the 67 cases, a definite diagnosis of calciphylaxis was made by our study group based on the clinical characteristics and skin biopsy findings. A univariate logistic regression model comparing them with 56-matched controls identified warfarin therapy [odds ratio (OR) 11.4, 95% confidence interval (CI)] 2.7-48.1, P=0.0009], each 1 g/dL decline in serum albumin level (OR 19.8, 95% CI 4.4-89.5, P=0.0001), each 100 mg/dL increment in plasma glucose level (OR 3.74, 95% CI 1.08-12.9, P=0.037) and each 1 mg/dL increment in adjusted serum calcium level (OR 3.2, 95% CI 1.63-6.30, P=0.0008) at the time of diagnosis as significantly associated with calciphylaxis, but no significant associations were found with female gender, vitamin D analog therapy, serum phosphate level, adjusted calcium-phosphate products or serum alkaline-phosphatase level. Warfarin therapy and lower serum albumin levels were still significant risk factors after a multivariate logistic regression model analysis. CONCLUSION The results of this study showed that warfarin therapy and lower serum albumin levels are significant and strong risk factors for the development of calciphylaxis in chronic hemodialysis patients in Japan.

Newly Developed Immobilized Polymyxin B Fibers Improve the Survival of Patients with Sepsis

Background: Sepsis and septic shock are still major causes of morbidity and mortality in spite of the availability of powerful and broadly active antibiotics. Methods: A prospective, open and randomized trial of the effect of immobilized polymyxin fibers (PMX-F) on the survival of patients with sepsis throughout a follow-up period of 28 days or until discharge, if earlier, was carried out. Ninety-eight patients were included who met at least 4 of the criteria for systemic inflammatory response syndrome due to infection. The patients were classified into three groups based on their Acute Physiology and Chronic Health Evaluation (APACHE) II score. Results: The overall survival rate was significantly improved by using PMX-F compared to the control group (41 vs. 11%) (p = 0.002). In patients with an APACHE II score less than 20, treatment with PMX-F was shown to improve outcome (65 vs. 19%) (p = 0.01). In cases of more severe sepsis with an APACHE II score of 20–29, PMX-F still maintained efficacy in improving outcome (40 vs. 11%) (p = 0.04). However, PMX-F treatment did not improve the survival rate in patients with an APACHE II score of greater than 30 (survival rate 7 vs. 0%) (p = 0.59). Conclusion: From these results, it is concluded that treatment with PMX-F in patients with sepsis is effective and prolongs the survival rate when applied at an early stage of sepsis. However, in severe sepsis, this therapy does not improve the survival rate.

Effects of Candesartan on Cardiovascular Outcomes in Japanese Hypertensive Patients

In recent years, angiotensin receptor blockers (ARBs) have begun to represent a markedly larger percentage of the antihypertensive agents used in Japan. However, it remains uncertain whether ARBs are effective for protecting against hypertension-related organ damage in the general Japanese population. In the present report, we describe the results of a single blind, randomized, prospective study conducted in 1999–2002 and employing a total of 2,048 essential hypertensive subjects (sitting blood pressure 140–180/90–110 mmHg) aged 35–79 years. Subjects were randomly assigned to receive the ARB candesartan, 2 to 12 mg daily, or conventional antihypertensive drugs other than angiotensin converting enzyme inhibitors or ARBs. We used Cox regression analysis to compare the two regimens. The primary outcome was assessed by hospitalization due to stroke, myocardial infarction, and congestive heart failure. Blood pressure was reduced from 162.1/91.1 to 140.1/78.9 mmHg in the candesartan group and from 165.9/95.9 to 138.4/81.1 mmHg in the conventional therapy group. The main outcomes were as follows: there was a 39% reduction in hospitalization for stroke (5.8 vs. 9.4 cases: relative risk [RR]: 0.61; 95% confidence interval [CI]: 0.41–0.84; p<0.05) and a 57% reduction in hospitalization for myocardial infarction (RR: 0.44; CI: 0.21–0.84; p<0.05) with the candesartan-based treatment compared with the conventional treatment. In spite of a significant difference in the total incidence of both stroke and myocardial infarction, there was no significant reduction in the incidence of congestive heart failure (15% reduction: 4.3 vs. 5.0; RR: 0.85; CI: 0.57–1.26). Further analysis in stratifying the subjects with or without a past history of cardiovascular diseases including stroke and myocardial infarction revealed that candesartan reduced the incidence of stroke (61% reduction; RR: 0.39; CI: 0.15–0.43; p<0.01) and congestive heart failure (49% reduction; RR: 0.51; CI: 0.23–0.92; p<0.05) but not myocardial infarction (RR: 0.74; CI: 0.36–1.48; p=0.1) in hypertensive patients with a past history. However, conventional treatment was superior to candesartan-based treatment in reducing the incidence of stroke in the patients without a past history of cardiovascular diseases (66% reduction; RR: 0.34; CI: 0.16–0.69; p<0.05). This is the first demonstration that an ARB-based antihypertensive treatment was superior to the conventional treatment for reducing the risk of stroke and myocardial infarction in Japanese hypertensive patients, especially in the patients with a past history of cardiovascular diseases.

Qualification of arterial stiffness as a risk factor to the progression of chronic kidney diseases

Background: Reflection pressure may influence the clinical course of chronic kidney diseases (CKDs). The relationship between the augmentation index (AI) and progression of non-diabetic CKDs was characterized. Methods: Ninety-nine patients were enrolled into the study. Pulse wave form analysis was performed to determine AI that assesses arterial stiffness. Results: In a cross-sectional study, a multiple regression analysis found that AI correlated positively to age and weight, and negatively to height and heart rate (R2 = 0.50). Furthermore, echocardiography was performed in 51 patients who gave their consent. In male patients under angiotensin inhibition, left ventricular mass index increased as AI was elevated (r = 0.33, slope = 0.85 ± 0.30 g/m2/%, p < 0.05, n = 23). A prospective study was performed in 41 patients who consented to having their creatinine clearance measured repeatedly. In the patients with angiotensin inhibition a higher basal AI resulted in a greater annual decrease in creatinine clearance (r = –0.52, slope = –0.43 ± 0.14 ml/min/year/%, p < 0.01, n = 27). Conclusion: The present data indicate that AI as well as angiotensin contribute to the development of left ventricular hypertrophy. Furthermore, our results suggest that in addition to angiotensin, AI is a risk factor of progression of non-diabetic CKDs.

Cellular mechanisms mediating rat renal microvascular constriction by angiotensin II.

To assess cellular mechanisms mediating afferent (AA) and efferent arteriolar (EA) constriction by angiotensin II (AngII), experiments were performed using isolated perfused hydronephrotic kidneys. In the first series of studies, AngII (0.3 nM) constricted AAs and EAs by 29+/-3 (n = 8, P < 0.01) and 27+/-3% (n = 8, P < 0.01), respectively. Subsequent addition of nifedipine restored AA but not EA diameter. Manganese (8 mM) reversed EA constriction by 65+/-9% (P < 0.01). In the second group, the addition of N-ethylmaleimide (10 microM), a Gi/Go protein antagonist, abolished AngII- induced EA (n = 6) but not AA constriction (n = 6). In the third series of experiments, treatment with 2-nitro-4-carboxyphenyl-N, N-diphenyl-carbamate (200 microM), a phospholipase C inhibitor, blocked both AA and EA constriction by AngII (n = 6 for each). In the fourth group, thapsigargin (1 microM) prevented AngII-induced AA constriction (n = 8) and attenuated EA constriction (8+/-2% decrease in EA diameter at 0.3 nM AngII, n = 8, P < 0.05). Subsequent addition of manganese (8 mM) reversed EA constriction. Our data provide evidence that in AAs, AngII stimulates phospholipase C with subsequent calcium mobilization that is required to activate voltage-dependent calcium channels. Our results suggest that AngII constricts EAs by activating phospholipase C via the Gi protein family, thereby eliciting both calcium mobilization and calcium entry.

Add-On Angiotensin Receptor Blocker in Patients Who Have Proteinuric Chronic Kidney Diseases and Are Treated with Angiotensin-Converting Enzyme Inhibitors

The benefit of the add-on angiotensin II receptor blocker candesartan to angiotensin-converting enzyme (ACE) inhibitors in inhibition of progression of nephropathy in hypertensive patient with nondiabetic renal disease compared with monotherapy with ACE inhibitors remains controversial. All patients were previously treated with ACE inhibitors. Urinary protein excretion of patients exceeded 1.0 g/d despite treatment with ACE inhibitors. Ninety hypertensive patients with chronic renal insufficiency were randomly assigned to one of two groups. One group received ACE inhibitor plus candesartan (2 to 12 mg/d), and a control group received only ACE inhibitor. The target BP was < or = 130/80 mmHg. The primary outcome was the changes in serum creatinine and the reduction of proteinuria. The mean duration of follow-up was 3.1 +/- 0.4 yr. At years 2 and 3, systolic and diastolic BP were reduced from 140 +/- 3/84 +/- 2 to 129 +/- 1/78 +/- 2 mmHg (candesartan group) and from 135 +/- 2/85 +/- 2 to 130 +/- 2/80 +/- 2 mmHg (ACE inhibitors group). In both groups, both systolic and diastolic BP decreased significantly from the beginning to the end of the study (P < 0.01). The serum creatinine concentration increased from 3.02 +/- 0.27 to 3.38 +/- 0.49 mg/dl (candesartan plus ACE inhibitor group) versus 3.00 +/- 0.37 to 4.48 +/- 0.57 mg/dl (ACE inhibitor group; P < 0.01) at year 3. Although the level of proteinuria significantly declined in each group (P < 0.05), the degree of reductions in proteinuria was greater in the candesartan group than in the ACE inhibitors group (P < 0.01). In the patients who were treated with candesartan and ACE inhibitor or ACE inhibitor alone, pretreatment proteinuria correlated significantly with decline of renal function, whereas reduction of proteinuria negatively correlated with decline in renal function in the patients who were treated with candesartan. Candesartan with an ACE inhibitor is effective in slowing the progression of renal insufficiency in hypertensive patients with nondiabetic renal disease through reduction of proteinuria.

Bradykinin Decreases Plasminogen Activator Inhibitor-1 Expression and Facilitates Matrix Degradation in the Renal Tubulointerstitium under Angiotensin-Converting Enzyme Blockade

A number of experimental and clinical investigations support the notion that angiotensin-converting enzyme inhibitor (ACEi) and angiotensin II type 1 receptor blocker (ARB) compounds attenuate renal fibrosis. Fibrosis can be attenuated by either suppressing matrix formation or facilitating matrix degradation. In this study, drugs of ACEi and ARB classes were tested for their ability to facilitate matrix degradation in the kidney. A murine model system in which cyclosporin A (CsA) treatment for a specified period caused interstitial matrix deposition in the kidney was used. CsA was then discontinued, and experimental procedures were initiated to investigate matrix degradation. Benazepril, an ACEi, facilitated matrix degradation via the bradykinin (BK) B2 receptor on tubular epithelial cells in the kidney, whereas CGP-48933, an ARB, did not. In this murine model of CsA nephropathy under ACE blockade, plasminogen activator inhibitor-1 (PAI-1) expression was decreased in tubular epithelial cells, possibly leading to conversion of plasminogen to plasmin by plasminogen activator and subsequent activation of matrix metalloproteinases. These findings were confirmed in this study by measurements of plasmin activity, collagenolytic activity, and matrix metalloproteinase activities in the kidneys. In tubular epithelial cells stimulated in vitro, BK suppressed PAI-1 gene expression. All of these results suggest that ACEi can decrease PAI-1 expression via BK, thereby facilitating matrix degradation via activation of degradative enzymes to reduce interstitial matrix deposition.

SELF-MEASURED SYSTOLIC BLOOD PRESSURE IN THE MORNING IS A STRONG INDICATOR OF DECLINE OF RENAL FUNCTION IN HYPERTENSIVE PATIENTS WITH NON-DIABETIC CHRONIC RENAL INSUFFICIENCY

While blood pressure is a recognized major determinant of renal function deterioration, the role of self blood pressure measurement (BPM) in predicting the loss of renal function in hypertensive patients with chronic renal insufficiency (CRI) has not been adequately addressed. One hundred and thirteen patients (F/M: 46/67; 56±1 years) with CRI (mean serum creatinine: 1.87±0.08; range: 1.4 to 3.5 mg/dl; average urinary protein excretion: 1.2±0.2 g/24 hrs.) were followed for 3 years. The record of renal biopsy revealed that 74 patients had IgA nephropathy, 16 had chronic glomerulonephritis, and 6 had membranous nephropathy, while 17, unbiopsied patients had underlying renal disease of unknown origin. Self BPM were made at regular intervals throughout the course of the study. All recorded blood pressures were included in a stepwise multiple regression analysis in which the decline in GFR per year was the dependent variable. Patients were primarily treated with a combination of amlodipine (5 to 20 mg daily), a calcium antagonist, and benazepril(2.5 to 5 mg daily), an ACE inhibitor in an effort to reduce their blood pressure at the office to <130/85 mmHg. The simple correlation between blood pressures (i.e., office, home morning and home evening) and the decline in GFR were all statistically significant. The correlation coefficients of determination for this model were as follows: r=0.64 for home morning SBP; 0.43 for office SBP; 0.39 for office DBP; and 0.38 for home morning DBP. The level of urinary protein excretion did not correlate with the decline in GFR. These data suggest that self BPM improves prognostic ability in hypertensive patients with CRI.