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Background The Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study was launched in 2013 to evaluate the complex interactions of genetic and environmental factors in multifactorial diseases. The present study describes the maternal baseline profile and perinatal data of participating mothers and infants. Methods Expectant mothers living in Miyagi prefecture were recruited from obstetric facilities or affiliated centers between 2013 and 2017. Three sets of self-administered questionnaires were collected, and the medical records were reviewed to obtain precise information about each antenatal visit and each delivery. Biospecimens, including blood, urine, umbilical cord blood, and breast milk, were collected for the study biobank. The baseline maternal sociodemographic characteristics, results of screening tests, and obstetric outcomes were analyzed according to the maternal age group. Results A total of 23 406 pregnancies involving 23 730 fetuses resulted in 23 143 live births. Younger maternal participants had a tendency toward a higher incidence of threatened abortion and threatened premature labor, while older age groups exhibited a significantly higher rate of low lying placenta, placenta previa, gestational diabetes and hypertensive disorders of pregnancy. Conclusions The present study clearly shows the distribution of maternal baseline characteristics and the range of perinatal outcomes according to maternal age group. This cohort study can provide strategic information for creating breakthroughs in the pathophysiology of perinatal, developmental, and noncommunicable diseases by collaborative data visiting or sharing.

We present a report of a 29-year-old woman with non-dipper type refractory hypertension due to the vascular compression of the medulla oblongata. The patient was diagnosed with hypertension at 17 years of age and underwent emergency Caesarean section at 26 weeks of gestation during 2 pregnancies due to severe high blood pressure. We suspected medullary compression by the curved posterior inferior cerebellar artery as the cause of her intractable hypertension, and she underwent Jannetta's decompression surgery. After the surgery, her blood pressure swiftly decreased to almost within the normal range, and her blood pressure pattern normalized to dipper type.

A screening of coronavirus disease 2019 (COVID-19) polymerase chain reaction (PCR) tests using saliva for pregnant women and their partners was performed at all 12 maternity facilities located in Himeji city between May 29 and September 5, 2020. Pregnant women at 37 or more weeks of gestation or who experienced threatened labor and their partners who cared for an infant underwent a saliva PCR test with informed consent. As a result, all of 1475 pregnant women and 1343 partners tested negative for COVID-19 PCR. There were no cases of false positive or false negative PCR tests. This cohort study revealed for the first time that a screening of COVID-19 PCR tests using saliva may be useful to sustain perinatal medical care during the pandemic period in Japan.

Background Theoretically, atrial natriuretic peptide (ANP), especially low-dose ANP, is beneficial in acute kidney injury (AKI). In this study, we examined whether low-dose ANP is effective in preventing or treating AKI by conducting an updated systematic review for randomized controlled trials (RCTs). Method We searched the Excerpta Medica database (EMBASE), PubMed, and Cochrane CENTRAL databases for RCTs that compare the effects of low-dose ANP (≤ 50 ng/kg/min) with a placebo or conventional therapy in at-risk patients or patients with AKI. The primary outcome was the incidence of new AKI (in prevention RCTs), while the secondary outcomes were in-hospital mortality rate, renal replacement therapy (RRT) requirement, length of hospital and intensive care unit (ICU) stay, incidence of hypotension, and peak serum creatinine levels. The risk-of-bias was evaluated using the Cochrane Collaboration risk-of-bias tool. Trial sequential analysis (TSA) was used for each outcome of interest. Results A total of 18 RCTs (16 prevention and two treatment trials) fulfilled our inclusion criteria. In prevention RCTs, the incidence of new AKI was significantly low in the low-dose ANP group (relative risk [RR] = 0.51; 95% confidence interval [CI] = 0.36–0.72; P = 0.0001) compared to the control group. In addition, the low-dose ANP group showed a significantly reduced RRT requirement in both prevention (RR = 0.17; 95% CI = 0.04–0.64; P = 0.009) and treatment (RR = 0.43; 95% CI = 0.20–0.93; P = 0.03) RCTs. Among secondary outcomes, in some cases, low-dose ANP was associated with a reduction in ICU and in-hospital stay. The risk-of-bias assessment and TSA results indicated that the sample sizes and qualities of the RCTs were insufficient to conclude the efficacy of low-dose ANP. Conclusion Low-dose ANP might be effective in preventing or treating AKI. However, the evidence accumulated so far is not strong enough to demonstrate ANP’s beneficial effects. The next step is to elucidate the effects of low-dose ANP by conducting multicenter, high-quality, large-sample RCTs. Trial registration PROSPERO registry CRD42017068568. Registered 20 June 2017. Electronic supplementary material The online version of this article (10.1186/s13054-019-2330-z) contains supplementary material, which is available to authorized users.

Clarifying allele frequencies of disease-related genetic variants in a population is important in genomic medicine; however, such data is not yet available for the Japanese population. To estimate frequencies of actionable pathogenic variants in the Japanese population, we examined the reported pathological variants in genes recommended by the American College of Medical Genetics and Genomics (ACMG) in our reference panel of genomic variations, 2KJPN, which was created by whole-genome sequencing of 2049 individuals of the resident cohort of the Tohoku Medical Megabank Project. We searched for pathogenic variants in 2KJPN for 57 autosomal ACMG-recommended genes responsible for 26 diseases and then examined their frequencies. By referring to public databases of pathogenic variations, we identified 143 reported pathogenic variants in 2KJPN for the 57 ACMG recommended genes based on a classification system. At the individual level, 21% of the individuals were found to have at least one reported pathogenic allele. We then conducted a literature survey to review the variants and to check for evidence of pathogenicity. Our results suggest that a substantial number of people have reported pathogenic alleles for the ACMG genes, and reviewing variants is indispensable for constructing the information infrastructure of genomic medicine for the Japanese population.

Personalized healthcare (PHC) based on an individual's genetic make-up is one of the most advanced, yet feasible, forms of medical care. The Tohoku Medical Megabank (TMM) Project aims to combine population genomics, medical genetics and prospective cohort studies to develop a critical infrastructure for the establishment of PHC. To date, a TMM CommCohort (adult general population) and a TMM BirThree Cohort (birth+three-generation families) have conducted recruitments and baseline surveys. Genome analyses as part of the TMM Project will aid in the development of a high-fidelity whole-genome Japanese reference panel, in designing custom single-nucleotide polymorphism (SNP) arrays specific to Japanese, and in estimation of the biological significance of genetic variations through linked investigations of the cohorts. Whole-genome sequencing from >3,500 unrelated Japanese and establishment of a Japanese reference genome sequence from long-read data have been done. We next aim to obtain genotype data for all TMM cohort participants (>150,000) using our custom SNP arrays. These data will help identify disease-associated genomic signatures in the Japanese population, while genomic data from TMM BirThree Cohort participants will be used to improve the reference genome panel. Follow-up of the cohort participants will allow us to test the genetic markers and, consequently, contribute to the realization of PHC.

Acute kidney injury (AKI) is a syndrome which has a broad range of etiologic factors depending on different clinical settings. Because AKI has significant impacts on prognosis in any clinical settings, early detection and intervention are necessary to improve the outcomes of AKI patients. This clinical guideline for AKI was developed by a multidisciplinary approach with nephrology, intensive care medicine, blood purification, and pediatrics. Of note, clinical practice for AKI management which was widely performed in Japan was also evaluated with comprehensive literature search.

Juxtaglomerular apparatus (JGA) hyperplasia rarely happened in renal biopsy and has been controversial clinically, because synthesis and secretion of renin were susceptible to the effect of clinical condition and medication. Here we present the case of a 39-year-old who got JGA hyperplasia of IgA nephropathy (IgAN) after long-term inhibition of the renin-angiotensin system (RAS) with an angiotensin receptor blocker (ARB), and a direct renin inhibitor (DRI) in combination with a diuretic. He was diagnosed with IgAN in his first renal biopsy, and was treated with supra-maximal dosages of ARB, DRI and a diuretic. In the second biopsy, because of the massive proteinuria and occurrence of steroid-induced diabetes, it was revealed that the area and the number of JGA cells were strikingly increased in observed glomeruli. Immunohistopathologically, the both specimens were stained by human renin antibody. The hyperplastic JG cells contained a large amount of renin granules. Putative renin granules were observed in some interstitial cells adjacent to an afferent arteriole by electron microscopy. The increasing response of renin granules co-localized in prominent JGA hyperplasia should be worried while physicians treat hypertensive patients with potent RAS inhibitors and diuretics even though they have diabetes. This is the first report showing a clinical course of forming prominent JGA hyperplasia directly after a full combination of RAS inhibitors and diuretics in adult IgA nephropathy.

Leptospirosis is a zoonotic and disaster-related infectious disease. It is mainly endemic in subtropical or tropical countries and has not been reported since 2009 in the Tohoku region (northern Japan), including the Yamagata and Miyagi Prefectures. However, we experienced four patients with leptospirosis in the Tohoku region from 2012 to 2014; three patients (#1-3) live in the agricultural areas of the Yamagata Prefecture and one patient (#4) was a visitor to the Miyagi Prefecture. Patient 1 (81-year-old female) is a villager, with a rat bite, while Patient 2 (77-year-old male) and Patient 3 (84-year-old female) are farmers and were infected probably during agriculture work. Patient 4 (40-year-old male US citizen) was infected while traveling in Thailand. They had chief complaint of fever, headache, and myalgia and showed manifestations of hyperbilirubinemia (mean, 4.35 mg/dL), thrombocytopenia and acute kidney injury (AKI). All patients were diagnosed by polymerase chain reaction using blood and/or urine samples and a microscopic agglutination test for the anti-Leptospira antibody. All the patients were treated with infused antibiotics, including minocycline. The patients underwent hemodialysis due to severe AKI (mean serum creatinine, 4.44 mg/dL), except for Patient 2 with the normal serum creatinine level (1.12 mg/dL). All the patients recovered and were discharged. The presence of the three patients in the Yamagata Prefecture implies that leptospirosis does re-emerge in the Tohoku region. Therefore, careful survey of the pathogen is necessary for febrile patients with AKI who engage in agriculture or have a recent history of travelling in subtropical or tropical countries.

Residents of areas affected by the Great East Japan Earthquake may suffer from diseases or health problems. We are conducting a cross-sectional study from 2012 to 2015 to investigate and address the health needs of schoolchildren affected by this disaster. In this paper, we describe the protocol and research perspectives of our long-term child health study, and present the results obtained immediately after the disaster. The parent-administered questionnaire includes the International Study of Asthma and Allergies in Childhood questionnaire for asthma and eczema symptoms, the Strengths and Difficulties Questionnaire (SDQ), and a questionnaire on influenza infection and vaccination status. In 2012, we distributed the questionnaire to 3,505 (2nd, 4th, 6th, and 8th graders) in three municipalities located in southern coastal area among the 28 municipalities, and 1,277 (36.4%) returned the completed questionnaire. Mean age was 11.1 ± 2.2 years old. The number of children with symptoms of wheeze and eczema in the past 12 months was 146 (11.4%) and 199 (15.6%), respectively. The SDQ total difficulties score revealed 174 (13.6%) children with some form of difficulty in their daily lives. From May 2011 to April 2012, 195 (15.3%) and 649 (50.8%) children received the influenza vaccination once and twice, respectively, and 532 (41.7%) had suffered from influenza. The prevalence of eczema symptoms or some form of difficulty was higher than the Japanese average. However, careful interpretation was required because of potential self-selection bias from the low response rate. We will continue this study of schoolchildren to provide aggregate findings.

In recent times, therapy for renal anemia has changed dramatically in that iron administration has increased and doses of erythropoiesis-stimulating agents (ESAs) have decreased. Here we used a prospective, observational, multicenter design and measured the serum ferritin and hemoglobin levels every 3 months for 2 years in 1086 patients on maintenance hemodialysis therapy. The associations of adverse events with fluctuations in ferritin and hemoglobin levels and ESA and iron doses were measured using a Cox proportional hazards model for time-dependent variables. The risks of cerebrovascular and cardiovascular disease (CCVD), infection, and hospitalization were higher among patients who failed to maintain a target-range hemoglobin level and who exhibited high-amplitude fluctuations in hemoglobin compared with patients who maintained a target-range hemoglobin level. Patients with a higher compared with a lower ferritin level had an elevated risk of CCVD and infectious disease. Moreover, the risk of death was significantly higher among patients with high-amplitude ferritin fluctuations compared with those with a low ferritin level. The risks of CCVD, infection, and hospitalization were significantly higher among patients who were treated with high weekly doses of intravenous iron compared with no intravenous iron. Thus, there is a high risk of death and/or adverse events in patients with hemoglobin levels outside the target range, in those with high-amplitude hemoglobin fluctuations, in those with consistently high serum ferritin levels, and in those with high-amplitude ferritin fluctuations.

Excess fluid distribution is a common disorder in peritoneal dialysis (PD) patients. Tacrolimus malabsorption may also occur in PD patients, and may lead to acute allograft rejection after transplantation. The purpose of this study was to evaluate the relationship between tacrolimus pharmacokinetics and excess fluid distribution according to pre-transplant dialysis modality.We retrospectively analyzed 41 adult living-donor kidney transplantations, including nine PD patients and 32 hemodialysis (HD) patients. We examined tacrolimus pharmacokinetics in the peri-operative period and determined the association between the tacrolimus absorption rate and body weight reduction. The absorption efficacy of tacrolimus was evaluated as the dose-normalized tacrolimus absorption rate. Tacrolimus concentrations in PD effluent were measured by high-performance liquid chromatography.The tacrolimus absorption rate on the day before kidney transplantation tended to be lower in PD patients than in HD patients; however, the rate improved after kidney transplantation and was similar in both groups of patients. The peak tacrolimus concentration time was later in PD patients than in HD patients. The body weight reduction after kidney transplantation was greater in PD patients than in HD patients, and was significantly associated with the change in tacrolimus absorption rate (p=0.04, r=0.32). Only 0.002% of the oral tacrolimus dose was removed by PD itself.Excess fluid distribution in PD patients appears to contribute to tacrolimus malabsorption rather than PD itself. We should consider the risk of tacrolimus malabsorption in patients with possible excess fluid distribution, particularly in PD patients.

Data regarding renal disease in the elderly (age ≥65 years old) and very elderly (age ≥80 years old) Japanese are extremely limited. The aim of this study was to examine the causes of renal disease and their clinical presentations in elderly patients who underwent renal biopsy.From July 2007 to November 2011, all of the elderly native renal biopsy patients who had been registered in the Japan Renal Biopsy Registry (J-RBR; 2802 including 1596 males and 1206 females) were identified. Their data were compared with a control group of 7416 patients who ranged in age from 20 to 64 years old and were registered on the J-RBR over the same period. In addition, the clinical and pathological classifications of 276 very elderly patients were also analyzed.The indications for biopsy were nephrotic syndrome (NS) in 36.2 and 50.7 % of the elderly and the very elderly patients, chronic nephritic syndrome in 31.8 and 17.4 %, and acute kidney injury including rapidly progressive glomerulonephritis in 18.6 and 22.5 %, respectively. Primary glomerular disease was the most frequent diagnosis, followed by MPO-ANCA-positive nephritis, IgA nephropathy (IgAN), and diabetic nephropathy. In primary GN including IgAN, membranous nephropathy (MN) was the most frequent histological type, followed by IgAN and minor glomerular abnormalities. A comparison with the control group showed that MN, MPO-ANCA-positive nephritis, and amyloid nephropathy were more common in the elderly (P 0.001), and IgAN was less common (P 0.001). As for nephrotic syndrome in the elderly, MN was the most common histological type, followed by minimal change NS, diabetic nephropathy, amyloid nephropathy, and focal segmental glomerulosclerosis. There was a significant discrepancy between the urinary protein/creatinine ratio and daily proteinuria after the 7th decade of life.Renal biopsy is a valuable diagnostic tool, even in elderly and very elderly Japanese patients. In the future, modified clinical guidelines for elderly renal disease should be developed.

Background: Donor shortage is a serious problem worldwide and it is now debated whether kidneys from marginal donors are suitable for renal transplantation. Recent studies have shown that the findings of preimplantation kidney biopsy are useful to evaluate vasculopathy in the donated kidney, and may predict transplant outcomes in deceased- donor kidney transplantation. However, few studies have focused on the pathological findings of preimplantation biopsy in living-donor kidney transplantation. Therefore, we investigated whether arteriosclerotic vasculopathy in living-donor kidneys at the time of transplantation predicts the recipient’s kidney function (allograft function) later in life. Methods: We retrospectively analyzed 75 consecutive adult living-donor kidney transplants performed at Kagawa University Hospital. Renal arteriosclerotic vasculopathy was defined according to the presence of fibrous intimal thickening in the interlobular artery. Results: Forty-one kidneys exhibited mild arteriosclerotic vasculopathy on preimplantation kidney biopsies. The decreases in estimated glomerular filtration rate after donation were similar in donors with or without renal arteriosclerotic vasculopathy. Pre-existing arteriosclerotic vasculopathy did not affect graft survival rate, patient survival rate or the incidence of complications. Recipients of kidneys with arteriosclerotic vasculopathy had lower allograft function at 1 and 3 years after transplantation than the recipients of arteriosclerosis-free kidneys with or without donor hypertension. In multivariate analysis, fibrous intimal thickening on preimplantation biopsy was predictive of reduced allograft function at 1 year after transplantation. Conclusions: The present study demonstrated that mild arteriosclerotic vasculopathy in the donated kidney is an important pathological factor that reflects future impaired function of renal allografts from marginal donors.

We demonstrated previously that the blood pressure of patients with IgA nephropathy becomes salt sensitive as renal damage progresses. We also showed that increased urinary angiotensinogen levels in such patients closely correlate with augmented renal tissue angiotensinogen gene expression and angiotensin II levels. Here, we investigated the relationship between urinary angiotensinogen and salt sensitivity of blood pressure in patients with IgA nephropathy. Forty-one patients with IgA nephropathy consumed an ordinary salt diet (12 g/d of NaCl) for 1 week and a low-salt diet (5 g/d of NaCl) for 1 week in random order. The salt-sensitivity index was calculated as the reciprocal of the slope of the pressure-natriuresis curve drawn by linking 2 data points obtained during consumption of each diet. The urinary angiotensinogen:creatinine ratio was significantly higher in patients who consumed the ordinary salt diet compared with the low-salt diet (17.5 μg/g [range: 7.3 to 35.6 μg/g] versus 7.9 μg/g [range: 3.1 to 14.2 μg/g] of creatinine, respectively; P r =0.43; P =0.008) and changes in logarithmic urinary angiotensinogen:creatinine ratio ( r =0.37; P =0.017) but not with changes in urinary protein excretion ( r =0.18; P =0.49). In contrast, changes in sodium intake did not alter the urinary angiotensinogen:creatinine ratio in patients with Ménière disease and normal renal function (n=9). These data suggest that the inappropriate augmentation of intrarenal angiotensinogen induced by salt and associated renal damage contribute to the development of salt-sensitive hypertension in patients with IgA nephropathy.

Drug discovery and development is a lengthy and expensive process. Testing new agents in humans at an early stage could reduce the time and costs involved in identifying drugs that are likely to succeed in clinical studies. New guidance has outlined the concept of exploratory clinical trials, which provide important information on a drug's distribution as well as its physiological and pharmacological effects in humans. This strategy reduces the need for preclinical testing by limiting the dose and duration of exposure to a new drug in humans to below those required by the traditional testing of investigational new drugs. Exploratory, first-in-man studies should provide insights into human physiology and pharmacology, identify therapeutic targets relevant to disease and increase our knowledge of a drug's characteristics. Implementation of a new drug also requires the development of useful biomarkers of disease and of the drug's efficacy, as well as sensitive molecular imaging techniques. In this Review, we outline the benefits of exploratory clinical trials, especially in academia, and provide an overview of the experimental tools necessary for rational drug discovery and development.

H. Ohsaki, E. Hirakawa, M. Nakamura, Y. Norimatsu, H. Kiyomoto and R. Haba Expression of vimentin and high-molecular-weight cytokeratin (clone 34sE12) in differentiating reactive renal tubular cells from low-grade urothelial carcinoma cells in voided urine Objective: Reactive renal tubular cells show features of an atypical repair reaction. Differentiation between reactive renal tubular cells and low-grade urothelial carcinoma (LG-UC) cells can therefore be a diagnostic challenge based on morphology alone. In this study, we evaluated the diagnostic utility of vimentin and a high-molecular-weight cytokeratin antibody (clone 34sE12) in differentiating reactive renal tubular cells from LG-UC. Methods: We evaluated voided urine cytology and surgical specimens from 40 patients with renal disease, and 17 patients with LG-UC. All slides were stained with vimentin and 34sE12. Results: In the reactive renal tubular cells in voided urine cytology, vimentin showed strong cytoplasmic staining in 39/40 (97.5%) cases, but all were negative for 34sE12. LG-UC cells showed positive staining for 34sE12 in 3/17 (17.6%) cases, whereas none were positivity for vimentin. The reactive renal tubular cells of histological specimens in the renal disease group demonstrated positive for vimentin in all 40 cases and all were negative for 34sE12. The LG-UC group showed abnormal staining for 34sE12 in 4/17 (23.5%) cases, whereas none were positive for vimentin. Conclusions: Vimentin expression in urine cytology can help to distinguish reactive renal tubular cells from LG-UC. However, 34sE12 does not appear to be a useful adjunct to distinguish these two groups in voided urine cytology.

Although left ventricular (LV) hypertrophy and diastolic function assessed by echocardiography and chronic kidney disease (CKD) have been established as predictors of cardiovascular events in hypertensive patients, the relationships between the echocardiographic parameters and renal function have not been fully examined. We examined which echocardiographic parameter correlates best with estimated glomerular filtration rate (eGFR) in patients with cardiovascular risk factors. Enrolled in the study were 309 patients (mean age 67 +/- 13 y) with cardiovascular risk factors. Echocardiography was performed to measure left ventricular mass index (LVMI) as an index of LV hypertrophy. Transmitral early to atrial velocity (E/A) ratio and peak early diastolic mitral annular velocity (E') were measured as indexes of LV diastolic function. E/E' was calculated as a parameter of LV preload. eGFR was measured using the equation proposed by the Japanese Society of Nephrology. The correlations of LVMI (r = -0.333, p0.001) and hypertension (r = -0.326, p0.001) to eGFR were closer than those of E' (r = 0.276, p0.001) and E/A (r = 0.224, p0.001) to eGFR. Stepwise regression analysis showed that hypertension (beta coefficient = -0.211, p0.001) and LVMI (beta coefficient = -0.206, p0.001) were independently associated with eGFR. The E/E' increased with a decrease in eGFR, and E/E' in CKD stage 5 (16.0 +/- 6.8) was significantly higher than that in patients in whom eGFRor = 90 mL/min/1.73 m(2) (10.5 +/- 4.5) (p0.001). Left ventricular diastolic function may be influenced by the increase in LV preload due to progression of CKD stage. Therefore, LV hypertrophy may be superior to LV diastolic dysfunction in predicting low eGFR in patients with CKD using echocardiography.

H. Ohsaki, E. Hirakawa, K. Kagawa, M. Nakamura, H. Kiyomoto and R. Haba Value of computer-assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low-grade urothelial carcinoma Objective: To assess whether computer-assisted quantitative morphological parameters can be an effective tool for objectively distinguishing reactive renal tubular cells from low-grade urothelial carcinoma cells (LG-UCs) in voided urine. Methods: Nuclear morphometry was performed by a computer-assisted image analyser system on Papanicolaou-stained cytological specimens. The circumference of reactive renal tubular cells (n = 40) or LG-UC (n = 20) nuclei were manually traced, and the following nuclear morphometric parameters were analysed: (i) area, (ii) perimeter, (iii) roundness factor, (iv) maximum length, and (v) linear factor. For each nuclear measurement, we calculated the maximum, minimum, mean and standard deviation. Results: The mean nuclear area and nuclear perimeter were higher in reactive renal tubular cells compared to the LG-UCs. The mean of roundness and linear factors (reflecting a tendency for the nuclear outline to be regular and oval, respectively) were higher in LG-UCs compared with reactive renal tubular cells. Among nuclear areas, the nuclear perimeter, roundness factors and maximum length did not show any significant differences between reactive renal tubular cells and LG-UCs. On the other hand, the linear factor showed a mean higher value among LG-UCs than reactive renal tubular cells (P = 0.023). Conclusions: Of five quantitative nuclear morphological parameters, only linear factor was statistically significant in differentiating reactive renal tubular cells in renal disease from LG-UCs.

H. Ohsaki, E. Hirakawa, Y. Kushida, S. Yokoshita, M. Nakamura, H. Kiyomoto and R. Haba Can cytological features differentiate reactive renal tubular cells from low-grade urothelial carcinoma cells? Objective: To compare the cytomorphological and immunocytochemical features of reactive renal tubular cells and low-grade urothelial carcinoma cells (LG-UCs). Methods: We examined 15 cytological parameters in 38 cases with reactive renal tubular cells in renal disease and 20 cases of LG-UCs from bladder cancer that had been diagnosed by histological examination. Voided urine cytological parameters evaluated were as follows: (i) maximum cell numbers of clusters, (ii) cannibalism, (iii) rosette-like arrangement, (iv) hobnail-shaped cells, (v) vacuolated cytoplasm, (vi) intracytoplasmic haemosiderin, (vii) irregular nuclear contours, (viii) chromatin pattern, (ix) prominent nucleoli, (x) cast encasement, (xi) casts, (xii) dysmorphic erythrocytes, (xiii) isomorphic erythrocytes, (xiv) necrosis, and (xv) vimentin reactivity. The above parameters were determined using Mann–Whitney U-test and chi-square test, with differences considered significant at P

Tonsillectomy and steroid pulse (TSP) therapy was proposed as a curative treatment for IgA nephropathy by Hotta et al. (Am J Kidney Dis 38:736-742, 2001) based on data that about 50% of patients achieved clinical remission (CR) of urinary abnormalities.As a primary survey, we sent a questionnaire and letter to 848 hospitals in Japan, each of which employed a Fellow of the Japanese Society of Nephrology between October and December of 2006, in order to gather information about the prevalence and efficacy of TSP therapy for patients with IgA nephropathy. As a secondary survey, we collected data from both low- and high-CR-rate groups to determine which factors predicted resistance to TSP therapy.A total of 2,746 patients received TSP therapy between 2000 and 2006. The CR rates, calculated by measuring urinary criteria 6 and 12 months after TSP therapy, were 32.0% (347/1,085) and 45.6% (452/991), respectively. Analysis of the 30 hospitals in which TSP therapy had been performed on at least ten patients revealed that the CR rates varied from below 10% to 100%. A secondary survey of ten hospitals revealed that, after correction of the CR rate from each hospital, patients could be categorized into three groups: those with a low CR rate (122 patients in four hospitals), a middle CR rate (78 patients in four hospitals), and a high CR rate (103 patients in two hospitals). The CR rate of all patients (N = 303) was 54.1%. A comparison of patient data between the low- and high-CR-rate groups showed a significant difference in age at onset (years; P = 0.05), amount of proteinuria (g/day; P = 0.02), total protein (g/dl; P = 0.02), pathological grade (P = 0.009), and prognostic score as described by Wakai et al. [Nephrol Dial Transplant 21:2800-2808, 2006, (P = 0.04)]. Univariate analysis revealed that there was a significant difference between non-CR and CR subgroups in duration from diagnosis until TSP therapy (6.9 +/- 6.8 versus 5.3 +/- 5.2 years; P = 0.02), amount of proteinuria (1.5 +/- 1.6 versus 0.8 +/- 0.8 g/day; P0.0001), serum creatinine (0.99 +/- 0.40 versus 0.87 +/- 0.34 mg/dl; P = 0.006), pathological grade (P = 0.0006), and Wakai et al.'s prognostic score (37.4 +/- 17.8 versus 28.1 +/- 15.1; P0.0001). A multivariate logistic analysis demonstrated that resistance to TSP therapy depends on age at onset, amount of proteinuria, hematuria grade, and pathological grade, and a score predicting resistance to TSP therapy could be derived by the formula: [(-0.0330) x (age) + (0.4772) x log (amount of proteinuria) - (0.0273) x (hematuria grade: 0, 1, 2, and 3) + (0.7604) x (pathological grade: 1, 2, 3, and 4) - 0.1894]. A receiver operating characteristic (ROC) curve showed that patients with a resistance score of greater than -0.02 easily resist TSP therapy (sensitivity 69%, specificity 75%, positive likelihood ratio 2.76).TSP therapy shows promise as a treatment that can bring about CR of urinary abnormalities, but unfortunately the average CR rate is about 50% at 1 year after treatment. Predictive factors for resistance to TSP therapy are age at onset, amount of proteinuria, hematuria grade, and pathological grade. The present study suggests that patients with either early-stage or mild to moderate IgA nephropathy easily achieve CR following TSP therapy, whereas patients with late-stage or severe disease are prone to TSP therapy resistance.

Recent studies have shown that blocking non-proteolytically activated prorenin with a decoy peptide for the handle region of the prorenin prosegment (HRP) inhibits the development of microvascular complications in diabetic animals. In the present study, we investigated whether non-proteolytic activation of prorenin contributes to the development of fructose-induced insulin resistance. Rats were fed a standard diet (n = 10), a 60% high fructose diet (n = 16), or a high fructose diet + HRP (0.1 mg kg(-1) day(-1), n = 16) for 10 weeks. Fructose-fed rats showed higher systolic blood pressure (SBP), fasting plasma triglycerides, total cholesterol and insulin levels; which, except for SBP, were suppressed by HRP. The responses of plasma glucose and insulin levels to oral glucose loading were significantly greater in fructose-fed rats than in standard diet-fed rats. The HRP normalized the enhanced responses of plasma glucose and insulin levels that were observed in fructose-fed rats. Moreover, HRP suppressed the enhanced prorenin activation and angiotensin II formation in the soleus muscle of fructose-fed rats. These data suggest that local angiotensin II generation in skeletal muscle, induced by non-proteolytic activation of prorenin, contributes to the development of insulin resistance induced by a high fructose diet.

Background Peripheral arterial disease (PAD) is a major complication of haemodialysis (HD), especially in patients with diabetes mellitus. Although previous reports have indicated that low-density lipoprotein apheresis (LDL-A) improves arteriosclerosis in PAD patients, the mechanism by which LDL-A affects PAD is still unclear. In this study, we tested the hypothesis that LDL-A attenuates reactive oxygen species (ROS) production in HD patients with PAD. Methods Twenty HD patients with PAD were investigated in this study. Clinical effects were evaluated by thermography and angiography. Oxidative stress in serum was evaluated by thiobarbituric acid reactive substances (TBARS) and expression of p22phox mRNA. Results Ischaemic symptoms due to PAD were gradually improved in 13 patients (65%) after LDL-A. One session of LDL-A removed approximately 75% of LDL from serum. Some patients exhibited dramatic improvement of severe symptoms of PAD such as skin ulcers after serial performance of LDL-A. The levels of LDL cholesterol, malondialdehyde-modified LDL, high-sensitivity C-reactive protein, vascular endothelial growth factor, international normalized ratio of pro-thrombin time and bradykinin were decreased after a single session of LDL-A, although there were no additional changes after 10 sessions of LDL-A. The levels of fibrinogen and p22phox mRNA were decreased by a single session of LDL-A, and these decreases continued over the entire period of treatment. TBARS was decreased after a course of LDL-A. Conclusions LDL-A improved ischaemic symptoms in HD patients with PAD by reducing ROS production in leucocytes. We conclude that LDL-A is an effective therapy for patients with HD complicated by PAD.

Recent studies indicate a role of chymase in the regulation of angiotensin II (AngII) formation in cardiovascular and renal tissues. We investigated a possible contribution of chymase to AngII formation and to renal fibrosis in unilateral ureteral obstruction (UUO). Eight-week-old Syrian hamsters were subjected to UUO and treated with vehicle, the specific chymase inhibitor (CI) 4-[1-(4-methyl-benzo[b]thiophen-3-ylmethyl)-1H-benzimidazol-2-ylsulfanyl]-butyric acid (50 mg/kg, twice a day, p.o.), or the selective AT1-receptor blocker olmesartan (10 mg/kg per day, p.o.) for 14 days. UUO-induced renal interstitial fibrosis was associated with increases in renal mRNA levels of α-smooth muscle actin (SMA), type I collagen, and transforming growth factor (TGF)-β. The UUO hamsters showed markedly higher AngII contents and increased AT1-receptor mRNA level in the obstructed kidney than sham-operated ones. In contrast, angiotensin-converting enzyme (ACE) protein expression was significantly lower in UUO hamsters. In UUO hamsters, treatment with CI or olmesartan significantly decreased AngII levels in renal tissue and mRNA levels of α-SMA, type I collagen, and TGF-β and ameliorated tubulointerstitial injury. On the other hand, neither CI nor olmesartan changed systolic blood pressure, renal ACE, and AT1-receptor protein levels. These data suggest that chymase-dependent intrarenal AngII formation contributes to the pathogenesis of interstitial fibrosis in obstructed kidneys of hamsters. Keywords:: chymase, angiotensin II, angiotensin-converting enzyme, unilateral ureteral obstruction

Background Peritoneal mesothelial cells play an important role in peritoneal dialysis and are often exposed to dialysis fluid containing high glucose levels. Loss of peritoneal function is a major complication associated with long-term peritoneal dialysis. In this study, we hypothesized that high glucose levels induce apoptosis, and that insulin attenuates this apoptosis in peritoneal mesothelial cells. To clarify this hypothesis, we examined the effects of insulin on the phosphatidylinositol 3-kinase/Akt signaling pathway and apoptosis in rat peritoneal mesothelial cells. Methods Phosphorylated insulin receptor and Akt were detected by western blot analysis. Apoptosis was evaluated by measuring caspase 3 activity and by TUNNEL staining. Results Insulin (100 nmol/L) increased tyrosine phosphorylation of insulin receptor in peritoneal mesothelial cells. Furthermore, insulin (1-100 nmol/L) dose-dependently stimulated Akt phosphorylation. Treatment with the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (10 micromol/L) attenuated insulin-induced Akt phosphorylation, indicating that insulin phosphorylates Akt via a phosphatidylinositol 3-kinase-dependent pathway. Insulin attenuated caspase 3 activity and decreased the number of TUNNEL-positive cells. The phosphatidylinositol 3-kinase inhibitors and overexpression of a dominant-negative mutant of Akt inhibited the effect of insulin on apoptosis. Conclusions The present data indicate that insulin attenuates high glucose-induced apoptosis via the phosphatidylinositol 3-kinase/Akt signaling pathway in peritoneal mesothelial cells. Therefore, the insulin signaling pathway may play a protective role in peritoneal function.

OBJECTIVES Beneficial effects of angiotensin II type 1 receptor blockers have been indicated for patients with diabetic nephropathy. We investigated the effects of an angiotensin II type 1 receptor blocker, telmisartan, on intrarenal angiotensin II levels and the progression of albuminuria or glomerular injury in type 2 diabetic Otsuka Long-Evans Tokushima Fatty rats with microalbuminuria. METHODS AND RESULTS Otsuka Long-Evans Tokushima Fatty rats were randomly treated with telmisartan (10 mg/kg/day, orally), hydralazine (25 mg/kg/day in drinking water) or vehicle from the initiation of albuminuria (13 weeks old). At this age, Otsuka Long-Evans Tokushima Fatty rats showed low but detectable albuminuria (1.0 +/- 0.1 mg/day) and higher systolic blood pressure, postprandial blood glucose and kidney angiotensin II levels than age-matched nondiabetic Long-Evans Tokushima Otsuka rats. At 35 weeks of age, vehicle-treated Otsuka Long-Evans Tokushima Fatty rats did not show apparent glomerular injury or tubulointerstitial fibrosis but did exhibit severe albuminuria (72.6 +/- 5.9 mg/day) and accumulation of cytoplasmic granules containing albumin in podocytes. Otsuka Long-Evans Tokushima Fatty rats also showed higher systolic blood pressure, postprandial blood glucose, collagen gene expression, desmin staining (a marker of podocyte injury) and angiotensin II levels than Long-Evans Tokushima Otsuka rats. Treatment with telmisartan did not affect postprandial blood glucose but decreased systolic blood pressure, collagen gene expression, desmin staining and angiotensin II levels. Telmisartan also prevented the development of albuminuria (0.6 +/- 0.1 mg/day at 35 weeks old) and accumulation of cytoplasmic granules. Hydralazine treatment resulted in a similar reduction in systolic blood pressure and partially attenuated the albuminuria (35.4 +/- 1.8 mg/day at 35 weeks old) but did not affect the other parameters. CONCLUSION The present results suggest the contribution of augmented intrarenal angiotensin II levels to the initiation and progression of albuminuria as well as podocyte abnormalities in type 2 diabetic rats. Angiotensin II blockade may inhibit the transition from microalbuminuria to overt nephropathy through prevention of intrarenal angiotensin II augmentation, independently of changes in blood pressure and glucose levels.

There is increasing evidence indicating the roles of aldosterone and mineralocorticoid receptor (MR) in the pathogenesis of renal injury. In rats, chronic treatment with aldosterone and salt results in severe proteinuria and renal tissue injury, characterized by glomerulosclerosis and tubulointerstitial fibrosis. Aldosterone-induced renal tissue injury is associated with increases in reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs) or Rho-kinase. Treatment with a selective MR antagonist, eplerenone, prevents aldosterone-induced increases in ROS levels and MAPK activity and ameliorates renal injury. In vitro studies have revealed that MR is highly expressed in glomerular mesangial cells (RMCs), podocytes, and renal interstitial fibroblasts. In these renal cells, aldosterone induces cellular injury through NADPH oxidase–dependent ROS production and activation of MAPKs or Rhokinase. Such aldosterone-induced renal cellular injury is markedly attenuated by treatment with eplerenone. These data suggest that aldosterone induces renal injury via activation of MR through mechanisms that cannot be simply explained by changes in blood pressure. In this review, we summarized recent findings on the roles of aldosterone and MR in the pathogenesis of renal injury with particular emphasis on potential underlying mechanisms. Keywords:: aldosterone, mineralocorticoid receptor, reactive oxygen species (ROS), extracellular signal–regulated kinases (ERK) 1/2, eplerenone, kidney

Objective: Aerobic exercise is known to have multiple beneficial effects including renal protection in type 2 diabetes mellitus and obesity. However, the mechanisms regulating these actions of aerobic exercise remain unclear. The present study evaluated the effects of chronic aerobic exercise on early stage of diabetic nephropathy focusing on renal oxidative stress and nitric oxide (NO), using Zucker diabetic fatty (ZDF) rats as an animal model of obese type 2 diabetes. Methods: Male ZDF rats (6 weeks old) underwent forced treadmill exercise for 8 weeks (Ex-ZDF). Sedentary ZDF (Sed-ZDF) and Zucker lean (Sed-ZL) rats served as controls. After the last exercise session, all rats were subjected to an intra-peritoneal glucose tolerance test. Result: Exercise ameliorated hyperglycemia with increased insulin secretion, and raised the values of homeostasis model assessment for β-cell function (HOMA-β) in ZDF rats. Exercise also reduced albumin excretion and normalized creatinine clearance in ZDF rats. Endothelial (e) and neuronal (n) NO synthase (NOS) protein expression, and NOS activity in the kidneys of Sed-ZDF rats were lower compared with Sed-ZL rats, while both NOS expression and NOS activity were upregulated by exercise in ZDF rats. Although there was no significant difference in plasma levels of adiponectin between the groups, plasma levels of leptin were significantly higher in the Ex-ZDF group compared with the Sed-ZL and Sed-ZDF groups. Exercise decreased plasma and urinary thiobarbituric acid-reactive substances (TBARS) as an index of lipid peroxidation in ZDF rats. Additionally, renal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and expression of p47phox protein were significantly higher in the kidneys of the Sed-ZDF group compared with the Sed-ZL group, and there was no significant difference between the Ex-ZDF and Sed-ZL groups. Immunoblots for Nox4, Nox2, namely gp91phox and p22phox protein were undetected in the kidneys of the Sed-ZDF and Ex-ZDF group as well as the Sed-ZL group. In addition, expression of nitrotyrosine as an index of peroxynitrite (ONOO–) formation was significantly lower in the kidneys of the Sed-ZDF group compared with the Sed-ZL group, and was significantly higher in the Ex-ZDF group compared with the Sed-ZDF group. Further, morphometric evidence of renal damage was alleviated in response to exercise. Conclusion: Upregulated NOS activity and NOS protein, and ameliorated NADPH oxidase activity and p47phox protein in the kidneys may be potential mechanisms by which chronic aerobic exercise can reduced early diabetic nephropathy in ZDF rats. Chronic aerobic exercise does have beneficial effects and may be a novel therapeutic approach for preventing the development of renal dysfunction in patients with type 2 diabetes mellitus and obesity.

Objective It has been reported that hyporesponsiveness to erythropoiesis-stimulating agent (ESA) is associated with adverse events in patients on maintenance hemodialysis (MHD). However, it has not been determined whether higher iron storage is associated with an improved response, including better survival, to ESA. Design and Method We measured serum ferritin, hemoglobin (Hb), and transferrin saturation (TSAT) levels every three months for two years in 1,095 MHD patients. The weekly dose of ESA to Hb ratio was also calculated as an index of ESA responsiveness (ERI). Results A significant correlation (p280); however, serum ferritin and TSAT levels did not predict a higher ERI. In the time-dependent Cox hazard model, the risk for a composite event in the patients with a high ERI (≥280) and a high ferritin level (≥100 ng/mL) was significantly greater (hazard ratio [HR], 2.09, P = 0.033) than that for patients with a high ERI and a low ferritin (

Angiotensin II regulates vasoconstriction, homeostasis of salt and water, and cardiovascular hypertrophy and remodeling. Angiotensin II is a potent activator of NAD(P)H oxidase in the cardiovascular system, and augments production of reactive oxygen species. Numerous signaling pathways in response to angiotensin II are mediated by reactive oxygen species and oxidative stress is deeply associated with the progression of cardiovascular disease. The purpose of this review is to discuss the mechanism of reactive oxygen species formation and the pathophysiological effects of angiotensin II in the cardiovascular system.Recent studies have demonstrated novel molecular mechanisms of reactive oxygen species generation by angiotensin II and signaling pathways including cell proliferation, hypertrophy and apoptosis. In spite of these findings that strongly suggest the benefits of angiotensin II inhibition for cardiovascular disease, the clinical effects of angiotensin II-induced reactive oxygen species on the cardiovascular system are still controversial.We focus on the effects of angiotensin II-induced oxidative stress on cardiovascular function and remodeling after discussing the source of reactive oxygen species and novel signaling pathways in response to reactive oxygen species.

After the Great East Japan Earthquake of 2011, there has been a concern about health problems among children. Therefore, we investigated the prevalence of wheeze and eczema symptoms and associated factors among children in areas primarily affected by the disaster. From 2012 to 2014, we distributed the parent-administered questionnaire to 25,198 children in all 233 public schools in the 13 municipalities of Miyagi Prefecture in northeast Japan. A total of 7,155 responses (mean age 10.5 ± 2.2 years) were received (response rate: 28.4%). The prevalence of allergic symptoms according to the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire in 2nd, 4th, 6th, and 8th graders was 12.4%, 9.9%, 9.3%, and 5.6% for wheeze, and 20.1%, 18.0%, 14.0%, and 12.4% for eczema. In multivariate logistic analysis, younger age, history of hospitalization, and difficulties in children's daily lives as assessed by the Strengths and Difficulties Questionnaire (SDQ), were significantly and consistently associated with both allergic symptoms (both P < 0.05). Living in a coastal municipality was also associated with eczema symptoms (P = 0.0278). The prevalence of eczema symptoms in the 2nd (20.1%) and 8th (12.4%) grades was significantly higher than previously reported in Japan. Living in a coastal municipality was independently associated with eczema symptoms, and psychometric properties were also closely linked to allergic symptoms. These findings are clinically important for understanding the risks of allergic disorders after natural disasters.

Transition of adolescent and young adult (AYA) patients with childhood-onset chronic kidney diseases (C-CKD) from pediatric to adult renal services has received increasing attention. However, information on transition of Japanese patients with C-CKD is limited. The Transition Medicine Working Group, in collaboration with the Japanese Society for Nephrology, the Japanese Society for Pediatric Nephrology and the Japanese Society of Pediatric Urology, conducted a retrospective cross-sectional study in 2014 on issues concerning the transition of Japanese patients with C-CKD. Few institutions in Japan had transition programs and/or transition coordinators for patients with C-CKD. Refusal to transfer by patients or their families, lack of concern about transition and inability to decide on transfer were common reasons for non-transfer of patients still followed by pediatric renal services. Around 25 % of patients who had ended or interrupted follow-up by pediatric renal services presented to adult renal services because of symptoms associated with C-CKD. Patients with various types of childhood-onset nephrourological diseases were transferred from pediatric to adult renal services. IgA nephropathy, minimal change nephrotic syndrome and congenital anomalies of the kidney and urinary tract were the most frequent primary kidney diseases in adult patients with C-CKD. These survey results indicate the need for introduction of transitional care for Japanese AYA patients with C-CKD. Consensus guidelines for the optimal clinical management of AYA patients with C-CKD are required to ensure the continuity of care from child to adult renal services.

Background Periodontal disease is a less common but important cause of septic pulmonary embolism (SPE). However, the pathogens causing periodontal disease-associated SPE (PD-SPE) have been poorly understood. Actinomyces species are resident microbiota in the oral cavity. Here we report a case of PD-SPE caused by Actinomyces species, which was identified by anaerobic culture of bronchoalveolar lavage fluid (BAL). Case presentation A 64-year-old Asian man, complicated with severe chronic periodontitis, was admitted with chest pain and fever. Chest CT revealed multiple bilateral pulmonary nodules located subpleurally. We diagnosed the case as SPE associated with periodontitis. Although blood cultures were negative for the usual 5-day incubation, anaerobic culture of the BAL fluid sample yielded Actinomyces species. Antibacterial therapy alone did not ameliorate the symptoms; however, additional dental treatment, including tooth extraction, promptly did. The patient was discharged 23 days after admission. The 3-month follow-up revealed no recurrence of the symptoms and complete resolution of the lung lesions. Conclusion This case demonstrated that Actinomyces species can cause PD-SPE. Additionally, clinicians should consider performing appropriate anaerobic culture of BAL fluid to identify the pathogen of SPE, and to ordering dental treatment, if necessary, in addition to antibiotics for the initial management of PD-SPE.

Objective Mechanical forces and angiotensin II influence the structure and function of vascular cells, and play an important role in reactive oxygen species production. In this study, we examined the effects of mechanical stretch and angiotensin II on the expression of p22-phox and Nox-1, essential membrane components of NADPH oxidase, and superoxide production in rat vascular smooth muscle cells (VSMCs). Methods and results Neither a stretch force nor angiotensin II alone altered p22-phox and Nox-1 expression in VSMCs. Combined stimulation markedly increased p22-phox and Nox-1 mRNA, however, which was associated with increased NADPH oxidase activity, superoxide production and total 8-iso-prostaglandin F2α concentration. The increases in p22-phox mRNA levels induced by a stretch force in combination with angiotensin II were prevented by treatment with an angiotensin type I (AT1) receptor antagonist, RNH-6270 (100 nmol/l). Protein expression of the AT1 receptor was upregulated by a stretch force. Conclusions These data indicate that mechanical stretch and angiotensin II synergistically increase NADPH oxidase expression in VSMCs, and suggest that part of this mechanism is mediated through an upregulation of the AT1 receptor induced by mechanical stretch. The combined effects of mechanical strain and angiotensin II might promote vascular damage through the production of superoxide in a hypertensive state.

Recent studies have indicated that derangement of peritubular capillary (PTC) circulation with consequent tubulointerstitial hypoxia plays a pivotal role in the pathogenesis of renal injury. The present study was performed to determine whether azelnidipine, a new dihydropyridine calcium channel blocker, attenuates angiotensin II (AngII)-induced peritubular ischemia in anesthetized rats. The superficial PTCs were visualized directly using an intravital fluorescence videomicroscope system, and the PTC blood flow was evaluated by analyzing the velocity of fluorescein isothiocyanate-labeled erythrocytes. Intravenous infusion of AngII (50 ng/kg/min, 10 min) significantly increased mean arterial pressure (MAP) and renal vascular resistance (RVR) (by 35 +/- 3% and 110 +/- 32%, respectively), and decreased total renal blood flow (RBF) and PTC erythrocyte velocity (by -34 +/- 4 and -37 +/- 1%, respectively). Treatment with azelnidipine (5 microg/kg/min i.v., 10 min) had no effect on basal MAP, RBF, RVR, or PTC erythrocyte velocity. However, azelnidipine markedly attenuated the AngII-induced increases in MAP (7 +/- 3%) and RVR (40 +/- 4%) and decreases in RBF (-24 +/- 1%) and PTC erythrocyte velocity (-22 +/- 1%). Similar attenuation in the AngII-induced responses of MAP, RBF, RVR, and PTC erythrocyte velocity were observed in rats treated with a higher dose of azelnidipine (20 microg/kg/min i.v., 10 min), which significantly decreased basal MAP and RVR and increased RBF and PTC erythrocyte velocity. These data suggest that calcium channel blockade attenuates AngII-induced peritubular ischemia, which may be involved in its beneficial effects on renal injury.

Recently, we demonstrated that in rats treated chronically with aldosterone and salt, severe tubulointerstitial fibrosis is associated with the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK1/2). Here, we investigated whether aldosterone stimulates collagen synthesis via ERK1/2-dependent pathways in cultured rat renal fibroblasts. Gene expression of mineralocorticoid receptor (MR) and types I, II, III, and IV collagen was measured by real-time polymerase chain reaction (PCR). MR protein expression and ERK1/2 activity were evaluated by Western blotting analysis with anti-MR and anti–phospho-ERK1/2 antibodies, respectively. Collagen synthesis was determined by [ 3 H]-proline incorporation. Significant levels of MR mRNA and protein expression were observed in rat renal fibroblasts. Treatment with aldosterone (0.1 to 10 nmol/L) increased ERK1/2 phosphorylation in a concentration-dependent manner with a peak at 5 minutes. Aldosterone (10 nmol/L) also increased the mRNA levels of types I, III, and IV collagen at 36 hours but had no effect on the type II collagen mRNA level. [ 3 H]-proline incorporation was significantly increased by aldosterone in both the medium and cell layer at 48 hours. Aldosterone-induced ERK1/2 phosphorylation was markedly attenuated by pretreatment with eplerenone (10 μmol/L), a selective MR antagonist, or PD98059 (10 μmol/L), a specific inhibitor of MAPK kinase/ERK kinase, which is the upstream activator of ERK1/2. In addition, both eplerenone and PD98059 prevented the aldosterone-induced increases in types I, III, and IV collagen mRNA and [ 3 H]-proline incorporation. These results suggest that aldosterone stimulates collagen gene expression and synthesis via MR-mediated ERK1/2 activation in renal fibroblasts, which may contribute to the progression of aldosterone-induced tubulointerstitial fibrosis.

Objectives We sought to clarify the mechanism for neovascularization by ultrasonic microbubble destruction (US/MB) and its ability to improve the function of ischemic limbs. Background In tissue, US/MB can cause capillary rupture, leading to angiogenesis and arteriogenesis. Methods Seven days after removal of the femoral artery (day 0) in mice, microbubble/ultrasound treatment was performed by intermittent insonation (1.6 MHz, mechanical index 1.1) to the ischemic limbs after intravenous infusion of phospholipid-stabilized microbubbles BR14 (US/MB group). Effects were compared with those in untreated mice with ischemic limbs (control group). Results Immunostaining of the treated muscles revealed a greater leukocyte (CD45-positive cell) count in the US/MB group on days 3 and 7. These cells included F4/80-positive cells (macrophages) and CD3-positive cells (T-lymphocytes), both of which were immunoreactive to vascular endothelial growth factor (VEGF) antibody. Muscular VEGF content by Western blotting was elevated in the US/MB group on day 3, which declined but remained greater until day 21. The US/MB group showed a greater capillary density by alkaline phosphatase stain on day 7 without further increase at day 21. Surface vascularity of the muscles and blood flow were greater in the US/MB group on day 7, which further increased by day 21. Moreover, the US/MB group showed a two-fold longer treadmill time compared with the untreated control group on day 21. None of these favorable effects were observed in mice treated with ultrasound only or microbubbles only. Conclusions Ultrasonic destruction of microbubbles delivered to the ischemic limbs can recruit inflammatory cells producing VEGF, which is followed by neovascularization and functional improvement, and thus has a therapeutic potential.

We demonstrated recently that chronic administration of aldosterone to rats induces glomerular mesangial injury and activates mitogen-activated protein kinases including extracellular signal-regulated kinases 1/2 (ERK1/2). We also observed that the aldosterone-induced mesangial injury and ERK1/2 activation were prevented by treatment with a selective mineralocorticoid receptor (MR) antagonist, eplerenone, suggesting that the glomerular mesangium is a potential target for injuries induced by aldosterone via activation of MR. In the present study, we investigated whether MR is expressed in cultured rat mesangial cells (RMCs) and involved in aldosterone-induced RMC injury. MR expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. Cell proliferation and micromechanical properties were determined by [ 3 H]-thymidine uptake measurements and a nanoindentation technique using an atomic force microscope cantilever, respectively. ERK1/2 activity was measured by Western blotting analysis with an anti-phospho–ERK1/2 antibody. Protein expression and immunostaining revealed that MR was abundant in the cytoplasm of RMCs. Aldosterone (1 to 100 nmol/L) dose-dependently activated ERK1/2 in RMCs with a peak at 10 minutes. Pretreatment with eplerenone (10 μmol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [ 3 H]-thymidine incorporation and decreased the elastic modulus, indicating cellular proliferative and deforming effects of aldosterone, respectively. These aldosterone-induced changes in cellular characteristics were prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor, PD988059 (100 μmol/L). The results indicate that aldosterone directly induces RMC proliferation and deformability through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.

Whether temporary angiotensin II (AngII) blockade at the prediabetic stage attenuates renal injury in type 2 diabetic OLETF rats later in life was investigated. OLETF rats were treated with an AT(1) receptor antagonist (olmesartan, 0.01% in food), angiotensin-converting enzyme inhibitor (temocapril, 0.01% in food), a combination of the two, or hydralazine (25 mg/kg per d) at the prediabetic stage (4 to 11 wk of age) and then monitored without further treatment until 50 wk of age. At 11 wk of age, blood glucose levels and urinary protein excretion (U(protein)V) were similar between OLETF and control LETO rats. However, OLETF rats showed higher kidney AngII contents and type IV collagen mRNA expression than LETO rats at this age. These decreased with olmesartan, temocapril, and a combination of these but not with hydralazine. At 50 wk of age, diabetic OLETF rats showed higher BP, U(protein)V, and intrarenal AngII levels than LETO rats. Temporary AngII blockade did not affect glucose metabolism or the development of hypertension in OLETF rats but significantly suppressed proteinuria and ameliorated glomerular injury. However, no parameters were affected by temporary hydralazine treatment. The present study demonstrated that intrarenal AngII and type IV collagen expression are already augmented long before diabetes becomes apparent in OLETF rats. Furthermore, temporary AngII blockade at the prediabetic stage attenuates the progression of renal injury in these animals. These data suggest that early AngII blockade could be an effective strategy for preventing the development of type 2 diabetic renal injury later in life.

Antihypertensive treatment with dihydropyridine calcium channel blockers elicits sympathetic nerve activation, which may contribute to cardiovascular events. However, recent clinical studies showed that treatment with azelnidipine, a new dihydropyridine calcium channel blocker, significantly reduced blood pressure in hypertensive patients while either maintaining or actually decreasing heart rate (HR). In this study, we examined the effects of azelnidipine and amlodipine on systemic hemodynamics and renal sympathetic nerve activity (RSNA) in anesthetized spontaneously hypertensive rats (SHR). We also examined the effects of these agents on baroreflex functions by infusing phenylephrine (30 microg/kg/min, i.v.) and sodium nitroprusside (10 microg/kg/min, i.v.) into azelnidipine- or amlodipine-treated SHR. Fifty min after administration of azelnidipine (10 microg/kg/min for 10 min, i.v.), mean arterial pressure (MAP) significantly decreased from 153+/-5 to 122+/-5 mmHg; however, HR and integrated RSNA did not change significantly (from 352+/-9 to 353+/-10 beats/ min and 115+/-5% of baseline, respectively). Infusion of amlodipine (50 microg/kg/min for 10 min) elicited similar effects on MAP (from 152+/-5 to 120+/-4 mmHg). However, amlodipine significantly increased HR (from 351+/-9 to 375+/-11 beats/min) and integrated RSNA (165+/-5% of baseline). Analyses of baroreflex function curves revealed that azelnidipine-treated rats showed a smaller baroreflex function than amlodipine-treated rats (p

SUMMARY: Background: Leptin has been shown to function as an inhibitor of appetite and energy expenditure accelerator. However, it was recently reported that leptin has other important functions as a fibrogenetic factor and a novel, independent risk factor for coronary heart disease. The present study aimed to assess the blood concentration of leptin in acute uraemic rats by using various peritoneal dialysis (PD) solutions. Methods: To induce acute renal failure, the bilateral renal arteries were ligated via a mid-abdominal incision 1 h before starting PD. Rats were divided into four groups: 13.6 g/L glucose-containing dialysate (group L); 38.6 g/L glucose-containing dialysate (group H); 13.6 g/L glucose- and 25 g/L mannitol-containing dialysate with equal osmotic pressure to the dialysate of group H (group M); and renal failure without PD (group F). The concentrations of glucose, urea nitrogen (UN), leptin and insulin were measured at 0, 2 and 4 h after starting PD. Results: We observed significant blood UN suppression in all dialysed groups. Blood glucose was significantly higher in rats treated with the high glucose solution than in those treated with the low glucose solution. Insulin and leptin significantly increased in the high glucose solution group. There was a strong correlation between the blood glucose and insulin levels. We also found a strong correlation between the percentage changes in blood glucose and leptin. The relationship between the percentage changes in insulin and leptin were weak but significant. Conclusion: The high glucose PD solution resulted in increased circulating levels of leptin, glucose, and insulin, suggesting that these changes are linked with PD performed with glucose-based dialysis fluid.

Recent studies have implicated a contribution of oxidative stress to the development of hypertension. Studies were performed to determine the effects of the superoxide dismutase (SOD) mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine- N -oxyl (Tempol) on vascular superoxide production and renal sympathetic nerve activity (RSNA) in anesthetized Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Compared with WKY rats (n=6), SHR showed a doubled vascular superoxide production, which was normalized by treatment with Tempol (3 mmol/L, n=7). In WKY rats (n=6), Tempol (30 mg/kg IV) significantly decreased mean arterial pressure (MAP) from 108±5 to 88±6 mm Hg and HR from 304±9 to 282±6 beats/min. In SHR (n=6), Tempol significantly decreased MAP from 166±4 to 123±9 mm Hg and HR from 380±7 to 329±12 beats/min. Furthermore, Tempol significantly decreased RSNA in both WKY rats and SHR. On the basis of group comparisons, the percentage decreases in MAP (−28±4%), HR (−16±3%) and integrated RSNA (−63±6%) in SHR were significantly greater than in WKY rats (−17±3%, −9±2%, and −30±4%, respectively). In SHR, changes in integrated RSNA were highly correlated with changes in MAP (r=0.85, P

Exercise training is known to exert multiple beneficial effects including renal protection in type 2 diabetes mellitus and obesity. However, the mechanisms regulating these actions remain unclear. The present study evaluated the effects of chronic running exercise on the early stage of diabetic nephropathy, focusing on nitric oxide synthase (NOS), oxidative stress and glycation in the kidneys of Zucker diabetic fatty (ZDF) rats. Male ZDF rats (6 weeks old) underwent forced treadmill exercise for 8 weeks (Ex-ZDF). Sedentary ZDF (Sed-ZDF) and Zucker lean (Sed-ZL) rats served as controls. Exercise attenuated hyperglycemia (plasma glucose; 242 ± 43 mg/dL in Sed-ZDF and 115 ± 5 mg/dL in Ex-ZDF) with increased insulin secretion (plasma insulin; 2.3 ± 0.7 and 5.3 ± 0.9 ng/mL), reduced albumin excretion (urine albumin; 492 ± 70 and 176 ± 11 mg/g creatinine) and normalized creatinine clearance (9.7 ± 1.4 and 4.5 ± 0.8 mL/min per body weight) in ZDF rats. Endothelial (e) and neuronal (n) NOS expression in kidneys of Sed-ZDF rats were lower compared with Sed-ZL rats (p

The recent development of contrast echography has made renal enhancement possible through an intravenous injection of microbubble-based contrast. In animal models, tissue perfusion can be quantified using contrast echography by measurement of the rate at which microbubbles replenish tissue after their ultrasound-induced destruction. Our purpose in this study was to evaluate renal blood flow with contrast echography in humans. To increase the sensitivity for microbubbles, we used a combination of power Doppler harmonic and intermittent imaging. The pulsing interval (PI) was changed from 10 cardiac cycles to 1 cardiac cycle during an intravenous infusion of the contrast agent, and alterations in the intensity of the renal cortex were represented as a decline ratio (DR). In 24 patients with various renal diseases, we were able to observe all 48 kidneys with adequate enhancement of the renal cortex. At PI of 10 cardiac cycles, the enhancement was homogeneous and strong, while, obviously, changing PI from 10 to 1 cardiac cycles caused a decline of enhancement. An excellent correlation was found between DR using contrast echography and renal plasma flow determined by clearance and radionuclide measurements. An excellent correlation was found between the DR values determined by contrast echography and the renal plasma flow values determined using clearance and radionuclide measurements. These results suggest that DR may be useful for evaluation of both total and split renal blood flow. Thus the contrast echographic method presented here could succeed in assessing renal cortical blood flow less invasively than conventional methods in humans.

Background —Considerable controversy exists regarding impairment of cardiac function in diabetes mellitus (DM). We investigated the serial changes in left ventricular (LV) histopathology and LV filling dynamics in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which have been established as an animal model of type II DM. Methods and Results —In 54 OLETF and 54 non-DM rats, body weight, blood pressure, heart rate, and transmitral pulsed Doppler examinations were performed from 5 to 47 weeks of age. An oral glucose tolerance test was performed at 10, 20, and 30 weeks of age. The hearts were excised for histopathology, including immunohistochemistry and histomorphometry of collagen, and measurement of hydroxyproline at baseline and each stage of developing DM. In the prediabetic stage (15 weeks of age), in which fast blood glucose remained normal, OLETF rats manifested mild obesity, postprandial hyperglycemia, and hyperinsulinemia, and early diastolic transmitral inflow exhibited prolonged deceleration time (OLETF, 59±10 ms versus non-DM, 49±8 ms, P P 1 receptor II in LV myocytes of OLETF rats. At 15 weeks of age, the ratio of collagen area/visual field of LV wall in OLETF rats (8.3±1.3%) was larger than that in non-DM rats (4.9±1.8%, P P Conclusions —A metabolic abnormality present in the prestage of type II DM may produce LV fibrosis and alteration in cardiac function.

SUMMARY 1. Pharmacological inhibition of nitric oxide (NO) synthesis is known to produce acute and chronic hypertension in many animal species, but the underlying mechanisms mediating the hypertension are not completely understood. In particular, the pathogenetic roles of sodium sensitivity and the sympathetic nervous system in this model of hypertension are controversial. The present study was designed to test the hypothesis that long-term administration of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) to male Sprague-Dawley rats would produce a sodium-sensitive hypertension and that the enhanced activity of the sympathetic nervous system in this type of hypertension contributes to the sodium sensitivity. 2. NG-Nitro-L-arginine methyl ester was added to drinking fluid for 8 weeks at a concentration of 16 mg/dL. Rats received tap water for the first 4 weeks of the study and were then divided into two groups and placed on either a normal or high sodium intake (ingestion of either tap water or 0.9% NaCl, respectively). Awake systolic blood pressure was measured by the tail-cuff method every week. Urinary excretion rates of the stable NO metabolites and catecholamines during NO synthesis inhibition were examined. 3. Long-term administration of L-NAME produced a marked and sustained elevation in arterial pressure without altering urine flow, or sodium excretion rate. NG-Nitro-L-arginine methyl ester-induced hypertension was accompanied by a decreased urinary excretion of the stable NO metabolites NO2– and NO3– and was aggravated when rats drank 0.9% NaCl in place of tap water. Urinary excretion of adrenaline and noradrenaline, but not dopamine, in L-NAME-treated rats increased significantly within the first week of the study compared with control rats. L-Arginine (2 g/dL in drinking fluid) completely reversed the elevation of arterial pressure as well as the decrease in urinary NO2– and NO3– excretion and the increased urinary excretion of catecholamines associated with L-NAME treatment by 3 weeks of concomitant administration. 4. These results suggest that long-term inhibition of NO synthesis produces a sodium-sensitive hypertension and that changes in sympathetic nerve activity may, at least in part, contribute to the sodium sensitivity in this type of hypertension.