Effect of Nonsupplemented Low-Protein Diet on the Initiation of Renal Replacement Therapy in Stage 4 and 5 Chronic Kidney Disease: A Retrospective Multicenter Cohort Study in Japan.

OBJECTIVE: In subjects with chronic kidney disease (CKD), the effect of low-protein diet (LPD) is expected to alleviate uremic symptoms. However, whether LPD is effective in preventing loss of kidney function is controversial. The aim of this study was to evaluate the association between LPD and renal outcomes. METHODS: We conducted a multicenter cohort study of 325 patients who suffered CKD stage 4 and 5 with eGFR ≥10 mL/min/1.73 m,2 between January 2008 and December 2014. The primary diseases of the patients were chronic glomerulonephritis (47.7%), nephrosclerosis (16.9%), diabetic nephropathy (26.2%), and others (9.2%). The patients were divided into four groups, based on the mean protein intake (PI)/day, group 1 (n = 76): PI < 0.5 g/kg ideal body weight/day, group 2 (n = 56): 0.5 ≤ PI < 0.6 g/kg/day, group 3 (n = 110): 0.6 ≤ PI < 0.8 g/kg/day, group 4 (n = 83): PI ≥ 0.8 g/kg/day. Dietary supplementation with essential amino acids and ketoanalogues was not used. The outcome measure was occurrence of renal replacement therapy (RRT) (hemodialysis, peritoneal dialysis, renal transplantation (excluding preemptive transplantation)) and all-cause mortality until December 2018. Cox regression models were used to examine whether LPD was associated with the risk of outcomes. RESULTS: During a mean follow-up of 4.1 ± 2.2 years. Thirty-three patients (10.2%) died of all causes, 163 patients (50.2%) needed to start RRT, and 6 patients (1.8%) received a renal transplant. LPD therapy of 0.5 g/kg/day or less was significantly related to a lower risk of RRT and all-cause mortality [Hazard ratio = 0.656; 95% confidence interval, 0.438 to 0.984, P = .042]. CONCLUSIONS: These results suggest that non-supplemented LPD therapy of 0.5 g/kg/day or less may prolong the initiation of RRT in stage 4 and 5 CKD patients.

Nationwide questionnaire survey on the management of chronic kidney disease for general practitioners in Japan.

BACKGROUND: In 2019, a nationwide questionnaire survey on the management of chronic kidney disease (CKD) was circulated to general practitioners (GPs) throughout Japan by The Japan Physicians Association. The aim was to assess the current state of CKD medical care in the country and evaluate the utilization of CKD-specific guidelines in the treatment by GPs. METHODS: The voluntary survey targeted all members of Japan Physicians Association, a nationwide organization consisting primarily of 15,000 GPs in clinics throughout the country. GPs were divided into groups: 171 GPs using and 414 GPs not using the guidelines. Comparisons between the groups' responses were made using propensity score matching and component cluster analysis. RESULTS: Overall responses revealed that the estimated glomerular filtration rate's utilization rate was high (95.1%). However, evidence-practice gaps in urine protein quantification and anemia remedy were prominent. There were significantly favorable answers in terms of CKD management in the user group compared with those in the non-user group, except for the questions about a urine check at the first visit, stopping the use of renin-angiotensin system inhibitors, and the target blood pressure for elderly CKD patients. The differences suggest that utilization of the CKD guidelines has improved CKD management practices by GPs. CONCLUSIONS: Further promotion of CKD guidelines utilization (28% in this survey) is considered valid for CKD medical education.

Tonsillectomy with steroid pulse therapy has more effect on the relapse rate than steroid pulse monotherapy in IgA nephropathy patients.

AIMS: Both steroid pulse (SP) monotherapy and the combination of tonsillectomy and SP therapy (TSP) are effective for achieving clinical remission (CR), defined as negative hematuria and proteinuria, in patients with IgA nephropathy (IgAN). The role of tonsillectomy in the treatment of IgAN has been analyzed only from the aspect of CR or renal survival after TSP treatment, so there is no evidence of its effect on the relapse after CR. METHODS: We retrospectively investigated relapse (re-appearance of urinary abnormalities) from CR after TSP or SP monotherapy in 62 IgAN patients (mean follow-up, 70.1 ± 35.3 months). The SP therapy comprised 0.5 g methylprednisolone administered intravenously on 3 consecutive days followed by oral prednisolone (30 mg/day) on 4 consecutive days, with the course repeated 3 times. Oral prednisolone (30 mg/day) was then given on alternative days and gradually tapered and finished over 1 year. Tonsillectomy was performed either before or within 6 months of starting SP therapy. RESULTS: At baseline, the mean age was 34.6 years, the mean serum creatinine (Cr) level was 0.9 mg/dl, and the mean level of proteinuria was 876 mg/day. There were no differences between the TSP group (41 patients) and SP monotherapy group (21 patients). In total, 24 of the TSP and 10 of the SP patients achieved CR. Of the 34 patients who achieved CR, 13 relapsed after TSP or SP monotherapy. Using Kaplan-Meier analysis, tonsillectomy was associated with a lower incidence of relapse from CR after treatment (p = 0.045). Multivariate Cox regression analysis revealed that tonsillectomy reduced the rate of from CR after SP therapy. CONCLUSION: Tonsillectomy was associated with a reduction in the relapse rate from CR after SP therapy in IgAN patients.

Comparison of the 2013 and 2019 Nationwide Surveys on the Management of Chronic Kidney Disease by General Practitioners in Japan.

In 2019, the Japan Physicians Association conducted a second nationwide survey on the management of chronic kidney disease (CKD) among the Japanese general practitioners (GPs). We aimed to clarify the changes in the state of CKD medical care by GPs since the 2013 survey. The 2013 and 2019 surveys included 2214 and 601 GPs, respectively, who voluntarily participated. The two surveys were compared, using propensity score matching to balance the background of the responded GPs. For the medical care of CKD, the frequency of urine or blood examination, use of estimated glomerular filtration rate (eGFR) value for CKD management, and continuous use of renin-angiotensin system inhibitors for their reno-protective effects were significantly higher in 2019 than in 2013 (all: p < 0.001). The medical cooperation in CKD management, the utilization of the clinical path for CKD management and the measurement of the eGFR during the medical health checkup were significantly increased in 2019, compared to those in 2013. More GPs felt dissatisfied with the components of CKD treatment by nephrologists (p < 0.001). The two surveys confirmed improvements in the level of medical care for CKD and a strengthening in cooperation. However, the dissatisfaction with the consultation with nephrologists did not necessarily improve.

Vascular calcification estimated by aortic calcification area index is a significant predictive parameter of cardiovascular mortality in hemodialysis patients.

BACKGROUND: Vascular calcification is a feature of arteriosclerosis. In hemodialysis (HD) patients, vascular calcification progresses rapidly. This study used the aortic calcification area index (ACAI), an index of vascular calcification, to evaluate vascular calcification factors in HD patients, to investigate correlations between ACAI and long-term prognosis and to assess correlations between various factors and long-term prognosis. METHODS: Subjects comprised 137 patients on maintenance HD. ACAI was measured as an index of vascular calcification as measured by abdominal plain computed tomography. The patients were divided into a high ACAI (H) group and a low ACAI (L) group according to whether the ACAI was below or above the mean value (21.4%) of ACAI, and long-term all-cause death and cardiovascular death rates were compared between groups. Risk factors for all-cause death and cardiovascular death were examined by Cox hazard analysis. RESULTS: During follow-up (mean follow-up period 95.3 ± 50.3 months), 76 patients died, including 46 cardiovascular deaths. Deaths included 51 of 70 patients (67.1%) in Group H and 25 of 67 patients (37.3%) in Group L. Cardiovascular death rates were 51.4 and 14.9%, respectively. On Kaplan-Meier analysis, the number of all-cause deaths was significantly higher in Group H (P < 0.001, log-rank test). Similarly, the number of cardiovascular deaths was significantly higher in Group H. Multivariate Cox proportional hazards analysis showed that ACAI (%) was a significant prognostic indicator for cardiovascular death (hazard ratio 1.03; 95% confidence interval 1.00-1.06, P = 0.03). CONCLUSION: High ACAI was clearly correlated with mortality rate in HD patients, particularly cardiovascular mortality rate. ACAI was a useful long-term prognostic indicator in HD patients.

Association between serum lipids, polyunsaturated fatty acids, and prognosis in maintenance hemodialysis patients.

INTRODUCTION: Risks of mortality and cardiovascular disease (CVD) are significantly higher in hemodialysis (HD) patients than in the general population, where dyslipidemia is an established risk factor for CVD and mortality. There is no clear conclusion, however, whether dyslipidemia is a significant risk factor for CVD and mortality in HD patients. Similarly, the association between the polyunsaturated fatty acids (PUFAs) and the mortality is not clear in HD patients. METHODS: We retrospectively investigated mortality and CVD events in 420 HD patients. We classified patients into high- and low-lipid groups depending on their lipid levels. Survival rates were calculated using the Kaplan-Meier analysis and evaluated by the log-rank test. The risk estimates were computed using a multivariate Cox proportional hazard analysis. FINDINGS: During their follow-up (June 2011 to June 2016), 151 patients died (37 of CVD), and 112 patients experienced new CVD events. On Kaplan-Meier analysis, the number of all-cause deaths and CVD events were significantly higher in the low HDL-cholesterol group (P < 0.01, log-rank test). Similarly, the number of all-cause deaths was significantly higher in the high eicosapentaenoic acid/arachidonic acid ratio group (P < 0.01, log-rank test). Multivariate Cox proportional analysis showed that HDL-cholesterol was a significant prognostic indicator for new onset of CVD events (low: 0, high: 1, hazard ratio 0.66, 95% confidence interval 0.44-0.97; P = 0.04). DISCUSSION: In HD patients, LDL-cholesterol and non-HDL-cholesterol levels are not associated with mortality or CVD events. The HDL-cholesterol level, however, is an independent predictor of new CVD events even in HD patients.

Tolvaptan, a selective oral vasopressin V2 receptor antagonist, ameliorates podocyte injury in puromycin aminonucleoside nephrotic rats.

BACKGROUND: Proteinuria caused by glomerular disease is characterized by podocyte injury. Vasopressin V2 receptor antagonists are effective in reducing albuminuria, although their actions on glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of tolvaptan, a selective oral V2 receptor antagonist, on podocytes in a puromycin aminonucleoside (PAN)-induced nephrosis rat model. METHODS: Rats were allocated to a control, PAN nephrosis, or tolvaptan-treated PAN nephrosis group (n = 9 per group). Urinary protein excretion and serum levels of total protein, albumin, creatinine, and total cholesterol were measured on day 10. The influence of tolvaptan on podocytes was examined in renal tissues by immunofluorescence and electron microscopy. RESULTS: PAN induced massive proteinuria and serum creatinine elevation on day 10, both of which were significantly ameliorated by tolvaptan. Immunofluorescence studies of the podocyte-associated proteins nephrin and podocin revealed granular staining patterns in PAN nephrosis rats. In tolvaptan-treated rats, nephrin and podocin expressions retained their normal linear pattern. Electron microscopy showed foot process effacement was ameliorated in tolvaptan-treated rats. CONCLUSIONS: Tolvaptan is protective against podocyte damage and proteinuria in PAN nephrosis. This study indicates that tolvaptan exerts a renoprotective effect by affecting podocyte morphology and probably function in PAN nephrosis. Tolvaptan is a promising pharmacological tool in the treatment of renal edema.

Various dietary protein intakes and progression of renal failure in spontaneously hypercholesterolemic Imai rats.

BACKGROUND/AIM: Dietary protein restriction is known to be beneficial in the preservation of the renal function in patients with chronic renal failure. Recently, the effect of varying quantity and quality of dietary protein intakes was also studied. This study investigates the effects of different dietary animal proteins on renal function in spontaneously hypercholesterolemic Imai rats that exhibit renal lesions similar to human focal and segmental glomerulosclerosis. The sources of proteins were from casein, pork, and fish. Primary concern was the effect of fish meat protein, because the effects of fish oil are well reported. To examine whether remnants of fish oil affect the experimental results, semi-defatted fish meat and fully defatted fish meat were prepared for these experiments. METHODS: Forty-two Imai rats were placed on diets containing casein, defatted pork meat, semi-defatted fish meat, or defatted fish meat as a protein sources from 10 to 22 weeks of age. Twenty-four hour urine collections were obtained along with measurements of systolic blood pressure and drawing blood from the tail artery every 4 weeks. Finally, the kidneys were removed and prepared for histological study. RESULTS: The semi-defatted fish meat diet retarded the rise of plasma cholesterol, virtually completely prevented the development of hypertriglyceridemia, and slowed the progression of proteinuria, renal function failure, and glomerular injury as compared with the control casein diet. However, the fully defatted fish meat diet led to renal failure at the same rate as the casein diet. The defatted pork diet group exhibited a higher creatinine clearance at the end of the experiments as compared with the casein and the fully defatted fish meat diet groups. CONCLUSIONS: These data suggest that an important determinant of the protective effects of the semi-defatted fish meat diet was related to the prevention of hypercholesterolemia and hypertriglyceridemia by the remaining fish oil. Fish meat protein itself did not indicate superior beneficial effects in the regression of the renal function in Imai rats as compared with casein protein, and defatted pork showed better results than casein and fully defatted fish meat.

The efficacy of vitamin E against oxidative damage and autoantibody production in systemic lupus erythematosus: a preliminary study.

The hypothesis that reactive oxygen species (ROS) modification of DNA is involved in the development of autoantibodies in systemic lupus erythematosus (SLE) is supported by the enhanced reactivity of anti-DNA antibodies to ROS-denatured DNA. We studied the efficacy of vitamin E against both oxidative DNA damage and autoantibody production in SLE. Urinary 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, and the anti-double-stranded DNA (anti-ds DNA) antibody, a predictor of disease activity, were assayed twice, first during the season with the most intense sunlight and then later in the year. Twelve women among 36 outpatients received vitamin E (150 to 300 mg/day) together with prednisolone (PSL). No significant age or daily dose of PSL differences were evident between patient groups. Urinary 8-OHdG in the PSL with vitamin E group (15.0 +/- 10.2 ng/mg during the period of intense sunlight and 11.7 +/- 8.7 ng/mg during the remainder of the year) did not differ significantly from that in the PSL without vitamin E group (20.0 +/- 23.2 and 11.0 +/- 5.9 ng/mg, at these respective times), but the anti-ds DNA antibody titer in the PSL with vitamin E group (17.9 +/- 20.3 IU/l during the period of intense sunlight and 16.3 +/- 19.4 IU/l during the remainder of the year) was significantly lower than that in the PSL without vitamin E group for both sunlight-defined periods (66.3 +/- 76.8 and 55.8 +/- 59.0 IU/l, at these respective times; P < 0.05). The present study suggests that vitamin E can suppress autoantibody production via a mechanism independent of antioxidant activity.

[Fish meat retarding the progression of chronic renal failure in spontaneously hypercholesterolemic rats].

OBJECTIVE: To investigate the effects of different dietary proteins of fish meat and casein upon the progression of chronic renal failure (CRF) in spontaneously hypercholesterolemic (SHC) rats. METHODS: 48 SHC rats of 10 weeks of age were randomly divided into four groups, and placed on diets containing 20% and 40% casein or fish protein respectively till they were 22 weeks of age. 24-hour urine collections were obtained along with measurements of systolic blood pressure and blood were drew from their tail vein evry 4 weeks. Finally, the kidneys were removed and prepared for histological and immunohistochemistry study . RESULTS: Fish meat diet slowed the progression of proteinuria, renal function failure and glomerular injury compared with the control casein diet in both 20% and 40% dietary groups. CONCLUSION: Fish meat retarded the progression of CRF in SHC rats compared with casein.

Effects of antioxidants on kidney disease.

Kidney mesangial cells (MCs) and vascular smooth muscle cells (VSMCs) are closely related in terms of origin, microscopic anatomy, histochemistry, and contractility. This relationship suggests a similarity between kidney glomerular sclerosis and atherosclerosis. Vitamin E appears beneficial in the prevention and treatment of coronary disease and also inhibits the proliferation of VSMCs in vitro. We used vitamin E and probucol to treat glomerular sclerosis and MC-proliferative glomerulonephritis (GN) in two animal models of glomerular disease. Using rats, a remnant kidney model accelerated with hyperlipidemia was employed to reflect progressive glomerular sclerosis leading to chronic renal failure, and an anti-thymocyte serum treatment was used to model acute MC-proliferative GN. Supplemental dietary antioxidants suppress MC proliferation and glomerular sclerosis in models of glomerular disease in rats. These results suggest that treatment with antioxidants may be a promising intervention to prevent progression of kidney disease.

Effect of environmental changes on oxidative deoxyribonucleic acid (DNA) damage in systemic lupus erythematosus.

In a study conducted in Japan, the authors used urinary 8-hydroxydeoxyguanosine (8-OHdG) to study the effects of high-intensity and low-intensity sunlight on oxidative damage to deoxyribonucleic acid (DNA) in patients who had systemic lupus erythematosus (SLE). During late May through early September (i.e., a period of high-intensity sunlight), the mean urinary 8-OHdG level in SLE patients was significantly higher than in controls (31.0 +/- 20.6 [standard deviation] ng/mg vs. 15.4 +/- 7.2 ng/mg, respectively [p < .05]). During late November through early March (i.e., low-intensity sunlight season), however, no significant differences were noted (15.4 +/- 5.5 ng/mg vs. 16.3 +/- 4.6 ng/mg, respectively). The mean urinary 8-OHdG level in SLE patients during the period of high-intensity sunlight was significantly higher than during the period of low-intensity sunlight (21.3 +/- 20.6 ng/mg vs. 12.6 +/- 6.7 ng/mg, respectively; p < .01), although no such seasonal changes were observed among controls (16.2 +/- 8.0 ng/mg vs. 15.7 +/- 5.1 ng/mg, respectively). The effect of sunlight intensity (i.e., season) may require consideration when oxidative DNA damage occurs in individuals who have SLE.

[Levels of serum ascorbate and its metabolites in hemodialysis patients].

The status of ascorbic acid (AA) in dialysis patients is the subject of debate. Some reports have found AA to be deficient in dialysis patients, while others have found that AA is not deficient. In an attempt to confirm AA serum concentrations in dialysis patients, we analyzed the concentrations of AA as well as its metabolites using the specific determination of AA with chemical derivatization and the HPLC method. We studied 131 patients under maintenance hemodialysis therapy (HD), 23 patients with chronic renal failure (CRF) and 48 healthy controls (C). Serum concentrations of AA and the AA metabolites dehydroascorbic acid (DHA) and 2, 3-diketogulonate (DKG) were measured by HPLC. Nine HD patients were taking AA supplements. Seventy-six (62.3%) of the 122 HD patients not taking AA supplements exhibited deficient levels of AA (< 20 microM), while 13 (56.5%) of the 23 CRF patients and 9 (18.8%) of the 48 C showed deficient levels of AA. Analysis of AA metabolites in the normal-range AA (20-80 microM) group revealed that the DHA/AA ratio in HD patients was significantly higher than in C (3.3 +/- 2.6% and 1.2 +/- 2.2%, respectively). The DKG/AA ratio in HD patients was higher than in CRF patients (3.6 +/- 5.2% vs. 0.9 +/- 1.9%), whereas DKG was not detected in C. When compared to serum levels before the start of dialysis, serum AA, DHA and DKG concentrations at the end of the dialysis session decreased by an average of 74.2, 84.0 and 78.8% respectively. In HD patients, serum levels of thiobarbituric reactive substances (TBARS) were significantly lower in the higher AA (> 80 microM) group than in the deficient and normal-range AA groups. In 12 AA-deficient patients, after 1 month of taking AA supplements (200 mg/day), serum AA levels rose to 79.9 microM, while serum TBARS level declined when compared with levels before supplementation. In conclusion, the frequency of AA deficiency in dialysis patients is extremely high. AA deficiency in HD patients may result in high TBARS levels, which reflect increased oxidative stress. Adequate AA supplementation should therefore be considered in such patients.

[Interstitial nephritis induced by mesalazine].

This report concerns the first case in Japan of interstitial nephritis induced by mesalazine, a new therapeutic agent for inflammatory bowel disease, such as ulcerative colitis. Twenty-two cases have already been reported in other countries. The patient, a 27-year-old woman, was treated with mesalazine for her ulcerative colitis at another hospital. At the beginning of her treatment, her serum creatinine level was within the normal range. After 12 months, this level increased up to 5.7 mg/dl. She was then referred to our hospital for renal investigation and therapy. A renal biopsy revealed that severe tubulo-interstitial nephritis had occurred. Her mesalazine treatment was withdrawn and prednisolone was administered. Her serum creatinine level decreased gradually. However, this level remained at about 2.8 mg/dl and stabilized at that level. She was then discharged from the hospital. Glomeruli appeared to have minor glomerular abnormalities except for one globally sclerosed glomerulus as observed by light microscopy. However, IgM and C3 deposition on glomeruli were also observed. Glomerular lesions were suspected from these histological findings. A similar case that showed IgM. C3 depositions in glomeruli has previously been reported. The possibility of glomerular lesions being induced by mesalazine should be further researched. From the summary of reported cases, a delay of diagnosis of interstitial nephritis induced by mesalazine has resulted in permanent irreversible renal failure. Intensive monitoring of renal function is required when a patient is treated with mesalazine.

Improved assessment of aortic calcification in Japanese patients undergoing maintenance hemodialysis.

OBJECTIVE: Vascular calcification is a feature of arteriosclerosis and in hemodialysis (HD) patients it may be severe, even at a relatively young age, and is closely related to the overall prognosis. We used the aortic calcification area index (ACAI), derived from the aortic calcification index (ACI), to evaluate and analyze the risk factors for abdominal aortic calcification in HD patients. PATIENTS AND METHODS: Subjects comprised 137 patients on maintenance HD. ACAI was measured on abdominal plain computed tomography: 10 slices of the abdominal aorta were obtained at 1-cm intervals from the bifurcation of the common iliac artery and the area of the aortic cross-section and calcification was measured using image software. The calcification area was divided by the cross-sectional area and expressed as a percentage (%). The mean value for the 10 slices was also calculated. Patients were divided into 2 groups according to ACAI being lower or higher than the mean value and the risk factors in each group were compared by multivariate analysis. Results Group comparison showed significant differences in age, systolic blood pressure, serum calcium, and lipoprotein(a). On multiple regression analysis, age, systolic blood pressure, and serum calcium were independent risk factors. On logistic regression analysis, age, duration of dialysis, systolic blood pressure, and serum calcium were independent risk factors. CONCLUSION: Risk factors for abdominal aortic calcification in HD patients include age, systolic blood pressure, and serum calcium, according to ACAI evaluation. The ACAI was accurate and useful for evaluating abdominal calcification.

Renal protective effects of pitavastatin on spontaneously hypercholesterolaemic Imai Rats.

BACKGROUND: Independent of their lipid-lowering effects, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have renal protective effects on various models of progressive renal diseases, therefore, additional therapeutic advantages have been considered. In the present study, using spontaneously hypercholesterolaemic Imai rats, we examined the protective effects of pitavastatin on renal injuries and the oxidative modification of the low-density lipoprotein (LDL) and high-density lipoprotein (HDL), since oxidized lipoproteins are speculated to be involved in the mechanism of this rat strain's renal injuries. METHODS: Male Imai rats were treated with pitavastatin (n = 11) at a dose of 100 mg/kg diet or received no specific therapy as controls (n = 11) from 10 to 22 weeks of age. Body weight, urinary protein excretion and serum constituents were evaluated every 4 weeks. At the end of the study, the effects of pitavastatin on the susceptibility of serum LDL and HDL to oxidation, and renal histology were examined. RESULTS: Pitavastatin treatment did not affect hyperlipidaemia, but significantly reduced proteinuria and preserved creatinine clearance deterioration. At the end of the study, lag times for LDL and HDL oxidation were prolonged by the treatment of pitavastatin to 126 and 153%, respectively, compared with the controlled group. The glomerulosclerosis index (SI) for untreated controlled rats was significantly higher than that for the pitavastatin-treated group. An immunohistochemistry study showed significantly lower numbers of ED-1 positive macrophages in the glomeruli and interstitium in pitavastatin-treated rats compared with those controlled. CONCLUSION: Pitavastatin treatment prevented renal injuries in Imai rats independent of lipid-lowering effects. Prevention of oxidative modification of LDL and HDL may play an important role on the beneficial effects of pitavastatin treatment.

Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes.

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H-303]+) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of approximately 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress). These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.

Deletion of the kinase domain in death-associated protein kinase attenuates tubular cell apoptosis in renal ischemia-reperfusion injury.

Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase localized to renal tubular epithelial cells. To elucidate the contribution of DAPK activity to apoptosis in renal ischemia-reperfusion (IR) injury, wild-type (WT) mice and DAPK-mutant mice, which express a DAPK deletion mutant that lacks a portion of the kinase domain, were subjected to renal pedicle clamping and reperfusion. After IR, DAPK activity was elevated in WT kidneys but not in mutant kidneys (1785.7 +/- 54.1 pmol/min/mg versus 160.7 +/- 60.6 pmol/min/mg). Furthermore, there were more TUNEL-positive nuclei and activated caspase 3-positive cells in WT kidneys than in mutant kidneys after IR (24.0 +/- 5.9 nuclei or 9.4 +/- 0.6 cells per high-power field [HPF] versus 6.3 +/- 2.2 nuclei or 4.4 +/- 0.7 cells/HPF at 40 h after ischemia). In addition, the increase in p53-positive tubule cells after IR was greater in WT kidney than in mutant kidneys (9.9 +/- 1.4 cells/HPF versus 0.8 +/- 0.4 cells/HPF), which is consistent with the theory that DAPK activity stabilizes p53 protein. Finally, serum creatinine levels after IR were higher in WT mice than in mutant mice (2.54 +/- 0.34 mg/dl versus 0.87 +/- 0.24 mg/dl at 40 h after ischemia). Thus, these results indicate that deletion of the kinase domain from DAPK molecule can attenuate tubular cell apoptosis and renal dysfunction after IR injury.