Neuroimmune mechanisms and therapies mediating post-ischaemic brain injury and repair.

The nervous and immune systems control whole-body homeostasis and respond to various types of tissue injury, including stroke, in a coordinated manner. Cerebral ischaemia and subsequent neuronal cell death activate resident or infiltrating immune cells, which trigger neuroinflammation that affects functional prognosis after stroke. Inflammatory immune cells exacerbate ischaemic neuronal injury after the onset of brain ischaemia; however, some of the immune cells thereafter change their function to neural repair. The recovery processes after ischaemic brain injury require additional and close interactions between the nervous and immune systems through various mechanisms. Thus, the brain controls its own inflammation and repair processes after injury via the immune system, which provides a promising therapeutic opportunity for stroke recovery.

Arginase 2 Promotes Cisplatin-Induced Acute Kidney Injury by the Inflammatory Response of Macrophages.

Acute kidney injury (AKI) is a complex clinical syndrome with a rapid decrease in renal function caused by several different etiologies, including sepsis, ischemia, and the administration of nephrotoxic drugs. Tubular arginase 2 (ARG2), an arginine-metabolic enzyme, is a potential therapeutic target for AKI, but it has not been confirmed under various AKI conditions. The aim of this study was to investigate ARG2 as a therapeutic target for cisplatin-induced AKI. Cisplatin-treated mice with a genetic deficiency in Arg2 had significant amelioration of renal dysfunction, characterized by decreased acute tubular damage and apoptosis. In contrast, cisplatin-induced tubular toxicity was not ameliorated in proximal tubule cells derived from Arg2-deficient mice. Immunohistochemical analysis demonstrated the increased infiltration of ARG2-positive macrophages in kidneys damaged by cisplatin. Importantly, cisplatin-treated Arg2 knockout mice exhibited a significant reduction in kidney inflammation, characterized by the decreased infiltration of inflammatory macrophages and reduced gene expression of interleukin (IL)-6 and IL-1ß. The secretion of IL-6 and IL-1ß induced by lipopolysaccharides was decreased in bone marrow-derived macrophages isolated from Arg2-deficient mice. Furthermore, the lipopolysaccharide-induced elevation of mitochondrial membrane potential and production of reactive oxygen species were reduced in bone marrow-derived macrophages lacking Arg2. These findings indicate that ARG2 promotes the inflammatory responses of macrophages through mitochondrial reactive oxygen species, resulting in the exacerbation of AKI. Therefore, targeting ARG2 in macrophages may constitute a promising therapeutic approach for AKI.

Association between Serum C-Reactive Protein Concentrations and Risk of Cancer-Related Mortality in Patients Undergoing Hemodialysis: 10-Year Outcomes of the Q-Cohort Study.

INTRODUCTION: Cancer constitutes a major source of morbidity and mortality among people undergoing hemodialysis (HD). A systemic inflammatory response is associated with the incidence and prognosis of cancer in the general population. However, the effect of systemic inflammation on cancer-related mortality in patients undergoing HD remains unclear. METHODS: We analyzed 3,139 patients registered in the Q-Cohort Study, which is a multicenter, observational cohort study of patients on hemodialysis in Japan. The primary outcome was cancer-related mortality during a 10-year follow-up. The covariate of interest was serum C-reactive protein (CRP) concentrations at baseline. The patients were divided into tertiles based on their serum CRP concentrations at baseline (tertile [T] 1: ≤0.07; T2: 0.08-0.24; and T3: ≥0.25). The association between serum CRP concentrations and cancer-related mortality was calculated using the Cox proportional hazards model and the Fine-Gray subdistribution hazards model with non-cancer-related death as a competing risk. RESULTS: During the 10-year follow-up, 216 patients died of cancer. In the multivariable analysis, the risk of cancer-related mortality in the highest tertile (T3) of serum CRP concentrations was significantly higher than that in the lowest tertile (T1) (multivariable-adjusted hazard ratio [95% confidence interval]: 1.68 [1.15-2.44]). This association remained consistent in the competing risk model, in which the subdistribution hazard ratio was 1.47 and the 95% confidence interval was 1.00-2.14 for T3 compared with T1. CONCLUSION: Higher serum CRP concentrations are associated with an increased risk of cancer-related mortality in patients undergoing maintenance HD.

Non-invasive fibrosis assessments of non-alcoholic fatty liver disease associated with low estimated glomerular filtration rate among CKD patients: the Fukuoka Kidney disease Registry Study.

BACKGROUND: A growing body of evidence has shown that non-alcoholic fatty liver disease (NAFLD) is associated with chronic kidney disease (CKD). Non-invasive fibrosis assessments of NAFLD such as Fibrosis-4 (FIB-4) index and NAFLD fibrosis score (NFS) have been developed to substitute liver biopsy. Little is known about the association between FIB-4 index or NFS and the components of CKD. METHODS: In the present cross-sectional study, we assessed of 3640 Japanese CKD patients. We examined the association between FIB-4index or NFS and the odds of having low estimated glomerular filtration rate (eGFR) defined as eGFR < 60 mL/min/1.73 m2 or albuminuria defined as urinary albumin-to-creatinine ratio (UACR) ≥ 30 mg/g. Patients were divided into quartiles according to their baseline FIB-4 index and NFS levels. Linear and logistic regression analysis were conducted, with adjustment for potential confounding factors. RESULTS: FIB-4 index and NFS were negatively associated with eGFR, but not UACR, after adjustment for potential confounding factors. Both FIB-4 index and NFS were significantly associated with low eGFR after adjustment for potential confounding factors. Meanwhile, in the multivariable-adjusted model, no associations were found between FIB-4 index or NFS and albuminuria. The addition of FIB-4 index or NFS to the established clinical CKD risk factors improved diagnostic accuracy of prevalence of low eGFR. We also found that there was a significant trend of higher FIB-4 index and NFS with more advanced renal fibrosis using the kidney biopsy data. CONCLUSIONS: Higher non-invasive fibrosis assessments of NAFLD were associated with higher odds of decreased eGFR.

Estimated plasma osmolarity and risk of end-stage kidney disease in patients with IgA nephropathy.

BACKGROUND: Several experimental studies have indicated that increased plasma osmolarity caused by recurrent dehydration is involved in kidney injury via a mechanism, mediated by vasopressin secretion and activation of the aldose reductase pathway. Epidemiologic evidence linking increased plasma osmolarity and the onset of end-stage kidney disease (ESKD), in patients with primary glomerulonephritis, is lacking. METHODS: We retrospectively examined 663 patients with IgA nephropathy (IgAN) diagnosed by kidney biopsy and evaluated the association between estimated plasma osmolarity and ESKD prevalence, using a Cox proportional hazards model. RESULTS: During follow-up (median 80.4 months; interquartile range 22.2-120.1), 73 patients developed ESKD. In a baseline survey, plasma osmolarity was correlated negatively with the mean value of the estimated glomerular filtration rate, but correlated positively with the mean value of urinary protein excretion, systolic blood pressure, and pathologic severity of extracapillary proliferation, in addition to tissue fibrosis and sclerosis. The incidence rate of ESKD increased linearly with increase in plasma osmolarity (P < 0.05 for trend). In multivariate analyses, plasma osmolarity was an independent risk factor for ESKD (hazard ratio for each increment of 5 mOsm/kg in plasma osmolarity 1.56; 95% confidence interval 1.18-2.07) even after adjustment for potential confounders. CONCLUSIONS: Increased plasma osmolarity was associated significantly with an increased risk of ESKD in patients with IgAN. Maintenance of plasma osmolarity by appropriate control of the balance between salt and water may contribute to kidney protection.

Arterial Spin Labeling Magnetic Resonance Imaging for Differentiating Acute Ischemic Stroke from Epileptic Disorders.

BACKGROUND: Differential diagnosis between acute ischemic stroke (AIS) and epilepsy-related stroke mimics is sometimes difficult in the emergency department. We investigated whether a combination of diffusion-weighted imaging (DWI) and arterial spin labeling imaging (ASL) is useful in distinguishing AIS from epileptic disorders. METHODS: The study included suspected AIS patients who underwent emergency MRI including both DWI and ASL, and who exhibited DWI high-intensity lesions corresponding to neurological symptoms. We investigated the relationship between the ASL results from within and/or around DWI lesions and the final clinical diagnosis. RESULTS: Eighty-five cases were included (mean age, 71 ± 13 years; 47 men). The time from onset to the MRI examination was 493 ± 536 minutes. ASL showed hyperintensity in 13 patients, isointensity in 43, and hypointensity in 29. All ASL hyperintensities were observed in the cortex, with 4 patients (31%) presenting with AIS and 9 (69%) with an epileptic disorder. All of the AIS patients with ASL hyperintensity were diagnosed with cardioembolic stroke (4/4, 100%), with magnetic resonance angiography demonstrating recanalization of the occluded artery in all cases (4/4, 100%). In the 9 patients with an epileptic disorder, the area of ASL hyperintensity typically extended beyond the vascular territory (7/9, 78%) and involved the ipsilateral thalamus (7/9, 78%). All patients with ASL isointensity and hypointensity were diagnosed with AIS; none had epileptic disorders. CONCLUSIONS: Although cortical ASL hyperintensity can indicate cardioembolic stroke with recanalization, hyperintensity beyond the vascular territory may alternatively suggest an epileptic disorder in suspected AIS patients with DWI lesions.

Secular trends in the incidence of end-stage renal disease and its risk factors in Japanese patients with immunoglobulin A nephropathy.

Background: There are limited data on secular trends in the incidence of end-stage renal disease (ESRD) and frequencies of its risk factors or treatment modalities in patients with immunoglobulin A nephropathy (IgAN). Methods: This study divided 1255 patients with IgAN into three groups according to the timing of renal biopsy: 1979-89 (n = 232), 1990-99 (n = 574) and 2000-10 (n = 449). The age-adjusted incidence rates, incidence rate ratios and 95% confidence intervals (CIs) for ESRD were calculated by the person-year method and compared using Poisson regression analysis. Results: A total of 63 patients (5.0%) developed ESRD. The age-adjusted incidence of ESRD decreased significantly over time, i.e. 11.5 per 1000 person-years (95% CI 5.4-24.6) in 1979-89, 6.5 per 1000 person-years (95% CI 1.0-25.2) in 1990-99 and 4.2 per 1000 person-years (95% CI 1.0-17.7) in 2000-10. The proportions of patients with preserved renal function and acute-stage inflammatory histologic changes (i.e. endocapillary hypercellularity and extracapillary proliferation) at the timing of biopsy increased over time, as did the rates of prescriptions of renin-angiotensin system blockers and corticosteroids (all P for trend <0.05). The effect of acute inflammatory histologic lesions on renal prognosis was drastically reduced over time. Conclusions: These findings suggest that early diagnosis in the acute inflammatory phase and subsequent aggressive treatment may have contributed to the significant downward trend in the incidence of ESRD in patients with IgAN over three decades.

Factors Associated with the Serum Myostatin Level in Patients Undergoing Peritoneal Dialysis: Potential Effects of Skeletal Muscle Mass and Vitamin D Receptor Activator Use.

Myostatin is a member of the transforming growth factor-ß family, which regulates synthesis and degradation of skeletal muscle proteins and is associated with the development of sarcopenia. It is up-regulated in the skeletal muscle of chronic kidney disease patients and is considered to be involved in the development of uremic sarcopenia. However, serum myostatin levels have rarely been determined, and the relationship between serum myostatin levels with clinical and metabolic factors remains unknown. This cross-sectional study investigated the association between serum myostatin level and clinical factors in 69 outpatients undergoing peritoneal dialysis. Serum myostatin level was determined by commercially available enzyme-linked immunosorbent assay (ELISA). Univariable and multivariable analysis were conducted to determine factors associated with serum myostatin levels. The factors included age, sex, diabetes mellitus, dialysis history, body mass index, residual kidney function, peritoneal dialysate volume, serum biochemistries, and the use of vitamin D receptor activators (VDRAs). Mean serum myostatin level was 7.59 ± 3.37 ng/mL. There was no association between serum myostatin level and residual kidney function. Serum myostatin levels were significantly and positively associated with lean body mass measured by the creatinine kinetic method and negatively associated with the use of VDRAs after adjustment for potential confounding factors. Our study indicated that serum myostatin levels are associated with skeletal muscle mass and are lower in patients treated with VDRAs. Further studies are necessary to determine the significance of measuring serum myostatin level in patients undergoing peritoneal dialysis.

Comparison of oral versus intravenous vitamin D receptor activator in reducing infection-related mortality in hemodialysis patients: the Q-Cohort Study.

BACKGROUND: Hemodialysis patients who receive vitamin D receptor activator (VDRA) reportedly have better survival after infection than those who do not. However, the optimal route of its administration for minimizing death from infection remains unclear. METHODS: This prospective cohort study aimed to compare the effectiveness of oral versus intravenous VDRA regarding infection-related mortality in 3372 hemodialysis patients. Eligible subjects were divided into the following three groups by route of administration of VDRA: oral (n = 1868), intravenous (n = 492) and not administered (n = 1012). The effect of VDRA on infection-related mortality was examined using a Cox regression model with propensity score-based adjustments. RESULTS: During follow-up (median, 4.0 years), 118 study patients died of infection. There was a significantly lower incidence of death from infection in subjects who received intravenous VDRA than in those who did not receive VDRA; however, oral VDRA did not significantly reduce the risk of mortality from infection compared with those who did not receive VDRA [hazard ratio (HR) for intravenous VDRA, 0.16; 95% confidence interval (CI), 0.10-0.25, and HR for oral VDRA, 0.78; 95% CI, 0.60-1.01]. Direct comparison between the oral and intravenous VDRA groups showed that the intravenous group had significantly better survival than the oral group (HR, 0.39; 95% CI, 0.27-0.62). CONCLUSIONS: Treatment with intravenous VDRA more effectively reduces the incidence of mortality from infection than oral VDRA in hemodialysis patients.

[DAMPs (damage-associated molecular patterns) and inflammation].

Post-ischemic inflammation is re-appraised as an important player in the progression of ischemic stroke. Activation of inflammatory cells via Toll-like receptor 2 (TLR2) and TLR4 is caused by several damage-associated molecular patterns (DAMPs), including high mobility group box-1 (HMGB-1) and heat shock proteins. We have recently found that peroxiredoxin (Prx) is one of the strong DAMPs and activates infiltrating macrophages in brain ischemia. We have also found that interleukin-23 (IL-23) from the activated macrophages stimulates γδT cells which release IL-17, thereby causing the delayed expansion of infarct lesions. Further investigation of the innate immune response would lead to development of novel stroke treatment with a broad therapeutic time window.

Fetuin-A decrease induced by a low-protein diet enhances vascular calcification in uremic rats with hyperphosphatemia.

Although dietary phosphate restriction is important for treating hyperphosphatemia in patients with chronic kidney disease, it remains unclear whether a low-protein diet (LPD), which contains low phosphate, has beneficial effects on malnutrition, inflammation, and vascular calcification. The effects of LPD on inflammation, malnutrition, and vascular calcification were therefore assessed in rats. Rats were fed a normal diet or diets containing 0.3% adenine and low/normal protein and low/high phosphate. After 6 wk, serum and urinary biochemical parameters, systemic inflammation, and vascular calcification were examined. The protective effect of fetuin-A and albumin were assessed in cultured vascular smooth muscle cells. Rats fed the diet containing 0.3% adenine developed severe azotemia. LPD in rats fed high phosphate induced malnutrition (decreases in body weight, food intake, serum albumin and fetuin-A levels, and urinary creatinine excretion) and systemic inflammation (increases in serum tumor necrosis factor-α and urinary oxidative stress marker). LPD decreased the serum fetuin-A level and fetuin-A synthesis in the liver and increased serum calcium-phosphate precipitates. A high-phosphate diet increased aortic calcium content, which was enhanced by LPD. Reduced fetal calf serum in the medium of cultured vascular smooth muscle cells enhanced phosphate-induced formation of calcium-phosphate precipitates in the media and calcification of vascular smooth muscle cells, both of which were prevented by fetuin-A administration. Our results suggest that phosphate restriction by restricting dietary protein promotes vascular calcification by lowering the systemic fetuin-A level and increasing serum calcium-phosphate precipitates and induces inflammation and malnutrition in uremic rats fed a high-phosphate diet.

Factors associated with serum soluble inhibitors of Wnt-ß-catenin signaling (sclerostin and dickkopf-1) in patients undergoing peritoneal dialysis.

AIM: Sclerostin and dickkopf-1 (Dkk-1) are soluble inhibitors of Wnt-ß-catenin signaling and are involved in decreased bone formation and bone volume in patients with various bone diseases. The clinical characteristics of sclerostin and Dkk-1 and their impacts on mineral and bone metabolism remain undetermined in patients undergoing peritoneal dialysis (PD). METHODS: This cross-sectional study investigated the association between serum sclerostin and Dkk-1 levels and mineral disorders in 74 outpatients under PD treatment. Levels of sclerostin and Dkk-1 in serum, urine, and peritoneal dialysate were determined using enzyme-linked immunosorbent assay kits. The associations between serum sclerostin and Dkk-1 levels and biochemical parameters were evaluated by linear regression analyses. RESULTS: Median serum sclerostin and Dkk-1 levels were 138 pmol/L (range, 98.3-195.9 pmol/L) and 38.8 pmol/L (range, 28.5-47.1 pmol/L), respectively. Both sclerostin and Dkk-1 were excreted into urine and peritoneal dialysate. Multivariable linear regression analyses showed that serum sclerostin level was significantly associated with age, sex, parathyroid hormone level, and renal Kt/V. In contrast, serum Dkk-1 level was associated with platelet count and serum fibroblast growth factor 23 level but not with any of the bone metabolic markers. CONCLUSION: Serum sclerostin was associated with serum intact parathyroid hormone, while Dkk-1 was associated with serum fibroblast growth factor 23 in patients undergoing PD. The utility of determining soluble Wnt-ß-catenin inhibitors levels in patients undergoing PD requires further investigation.

Phosphate overload directly induces systemic inflammation and malnutrition as well as vascular calcification in uremia.

Hyperphosphatemia contributes to increased cardiovascular mortality through vascular calcification (VC) in patients with chronic kidney disease (CKD). Malnutrition and inflammation are also closely linked to an increased risk of cardiovascular death in CKD. However, the effects of Pi overload on inflammation and malnutrition remain to be elucidated. The aim of the present study was to investigate the effects of dietary Pi loading on the interactions among inflammation, malnutrition, and VC in CKD. We used control rats fed normal diets and adenine-induced CKD rats fed diets with different Pi concentrations ranging from 0.3% to 1.2% for 8 wk. CKD rats showed dietary Pi concentration-dependent increases in serum and tissue levels of TNF-α and urinary and tissue levels of oxidative stress markers and developed malnutrition (decrease in body weight, serum albumin, and urinary creatinine excretion), VC, and premature death without affecting kidney function. Treatment with 6% lanthanum carbonate blunted almost all changes induced by Pi overload. Regression analysis showed that serum Pi levels closely correlated with the extent of inflammation, malnutrition, and VC. Also, in cultured human vascular smooth muscle cells, high-Pi medium directly increased the expression of TNF-α in advance of the increase in osteochondrogenic markers. Our data suggest that dietary Pi overload induces systemic inflammation and malnutrition, accompanied by VC and premature death in CKD, and that inhibition of Pi loading through dietary or pharmacological interventions or anti-inflammatory therapy may be a promising treatment for the prevention of malnutrition-inflammation-atherosclerosis syndrome.

Disturbance in the potential cardiovascular-bone-skeletal muscle axis and morbidity and mortality in patients undergoing haemodialysis: the Q-Cohort Study.

Background: Disturbances in the cardiovascular system, bone and skeletal muscle are independent risk factors for death among patients receiving haemodialysis (HD). However, the combined impact of disorders of these three organs on morbidity and mortality is unclear in the HD population. Methods: A total of 3031 Japanese patients on maintenance HD were prospectively followed. The outcomes were all-cause mortality, major adverse cardiovascular events (MACE) and bone fracture. Patients were divided into four groups (G1-G4) according to the baseline number of diseased organs represented as histories of cardiovascular disease and bone fractures and the presence of low skeletal muscle mass as follows: G1, no organ; G2, one organ; G3, two organs; G4, three organs. Multivariable-adjusted survival models were used to analyse associations between the number of diseased organs and outcomes. Results: During a 4-year follow-up, 499 deaths, 540 MACE and 140 bone fractures occurred. In the Cox proportional hazards model, the risk for all-cause mortality was significantly higher in G2, G3 and G4 than in G1 as the reference {hazard ratio: G2, 2.16 [95% confidence interval (CI) 1.65-2.84], G3, 3.10 [95% CI 2.27-4.23] and G4, 3.11 [95% CI 1.89-5.14]}. Similarly, the risks for developing MACE and bone fractures were significantly elevated as the number of organ disorders increased. Conclusions: Multiple disorders of the cardiovascular-bone-skeletal muscle axis are strong predictors of morbidity and mortality in patients undergoing HD.

Novel therapeutic strategies targeting innate immune responses and early inflammation after stroke.

Post-ischemic inflammation is an essential step in the progression of ischemic stroke. This review focuses on the function of infiltrating immune cells, macrophages, and T cells, in ischemic brain injury. The brain is a sterile organ; however, the activation of Toll-like receptor (TLR) 2 and TLR4 is pivotal in the beginning of post-ischemic inflammation. Some endogenous TLR ligands are released from injured brain cells, including high mobility group box 1 and peroxiredoxin family proteins, and activate the infiltrating macrophages and induce the expression of inflammatory cytokines. Following this step, T cells also infiltrate into the ischemic brain and mediate post-ischemic inflammation in the delayed phase. Various cytokines from helper T cells and γδT cells function as neurotoxic (IL-23/IL-17, IFN-γ) or neuroprotective (IL-10, IL-4) mediators. Novel neuroprotective strategies should therefore be developed through more detailed understanding of this process and the regulation of post-ischemic inflammation.

Impact of Multivascular Disease on Cardiovascular Mortality and Morbidity in Patients Receiving Hemodialysis: Ten-Year Outcomes of the Q-Cohort Study.

AIM: Multivascular disease, indicating concurrent arteriosclerotic lesions in a number of different vascular beds, is an independent risk factor for recurrent ischemic events in the general population. However, the impact of multivascular disease on the risk of developing cardiovascular disease has not been fully evaluated in patients receiving hemodialysis. METHODS: A total of 3,504 hemodialysis patients were prospectively followed for 10 years. In this study, multivascular disease was defined as the coexistence of coronary artery disease and stroke. We examined the relationship between multivascular disease and the occurrence of composite cardiovascular endpoint, consisting of cardiovascular death, nonfatal coronary artery disease, nonfatal stroke, and peripheral artery disease. RESULTS: The proportion of participants with multivascular disease was 5.7% (n=200) at baseline. During follow-up (median, 106.6 months; interquartile range, 50.1-121.8 months), 1,311 patients experienced the composite endpoint, which was defined as at least one of the following: cardiovascular death (n=620), nonfatal coronary artery disease (n=318), nonfatal stroke (n=340), and peripheral artery disease (n=257). Compared with the group with no history of cardiovascular disease, the risk of experiencing the composite endpoint increased significantly with higher numbers of injured vascular beds in patients with single vascular disease (hazard ratio, 1.68; 95% confidence interval, 1.49-1.89) and in those with multivascular disease (hazard ratio, 2.11; 95% confidence interval, 1.71-2.60). In a multivariable analysis, multivascular disease was an independent predictor of cardiovascular events, in addition to diabetes, aging, and hypertension. CONCLUSIONS: This study clearly demonstrated that multivascular disease was a powerful predictor for cardiovascular mortality and morbidity in patients receiving hemodialysis.

Change in intracellular pH causes the toxic Ca2+ entry via NCX1 in neuron- and glia-derived cells.

Brain hypoxia or ischemia causes acidosis and the intracellular accumulation of Ca(2+) in neuron. The aims of the present study were to elucidate the interaction between intracellular pH and Ca(2+) during transient acidosis and its effects on the viability of neuronal and glial cells. Intracellular Ca(2+) and pH were measured using the fluorescence of fura-2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester in neuroblastoma (IMR-32), glioblastoma (T98G), and astrocytoma (CCF-STTG1) cell lines. The administration of 5 mM propionate caused intracellular acidification in IMR-32 and T98G cells but not in CCF-STTG1 cells. After the removal of propionate, the intracellular pH recovered to the resting level. The intracellular Ca(2+) transiently increased upon the removal of propionate in IMR-32 and T98G cells but not in CCF-STTG1 cells. The transient Ca(2+) increase caused by the withdrawal of intracellular acidification was abolished by the removal of external Ca(2+), diminished by a reduction of external Na(+), and inhibited by benzamil. Transient acidosis caused cell death, whereas the cells were more viable in the absence of external Ca(2+). Benzamil alleviated cell death caused by transient acidosis in IMR-32 and T98G cells but not in CCF-STTG1 cells. These results suggest that recovery from intracellular acidosis causes a transient increase in cytosolic Ca(2+) due to reversal of Ca(2+) transport via Na(+)/Ca(2+) exchanger coactivated with Na(+)/H(+) exchanger, which can cause cell death.

Amiloride inhibits hydrogen peroxide-induced Ca2+ responses in human CNS pericytes.

The aims of the present study were to investigate the mechanisms of Ca(2+) signaling caused by hydrogen peroxide in CNS pericytes. In cultured human brain microvascular pericytes, cytosolic Ca(2+) concentration was measured by means of fura-2 fluorescence. Reverse transcription and polymerase chain reaction was performed to examine the expression of mRNA. Knockdown of Na(+)/H(+) exchanger (NHE) was done by transfecting the cells with specific double-strand siRNAs for NHE. Externally applied hydrogen peroxide dose-dependently (100 microM-10 mM) increased cytosolic Ca(2+) in human CNS pericytes. Cytosolic Ca(2+) remained high after wash-out of hydrogen peroxide. However, the addition of dithiothreitol rapidly reversed cytosolic Ca(2+) to the resting level. The hydrogen peroxide-induced Ca(2+) increase was not inhibited by nicardipine, Gd(3+), La(3+), or omission of external Ca(2+). Neither thapsigargin nor carbonyl cyanide 4-trifluoromethoxyphenylhydrazone attenuated the hydrogen peroxide-induced Ca(2+) rise. Amiloride and its derivatives, benzamil and hexamethylene amiloride reversed the hydrogen peroxide-induced Ca(2+) increase. Human CNS pericytes expressed acid sensing ion channel (ASIC) 1a, Na(+)/Ca(2+) exchanger (NCX) 1, Na(+)/H(+) exchanger (NHE) 1, and NHE7. However, the removal of external Na(+), treatment with KB-R 7943 and mibefradil, or knockdown of NHE1 and NHE7 did not affect the hydrogen peroxide-induced Ca(2+) increase. Hydrogen peroxide releases Ca(2+) from intracellular Ca(2+) pool via an amiloride-sensitive protein, which is controlled by oxidation of thiol group in human CNS pericytes.