Hiroyuki Kadoya

Selective estrogen receptor modulation attenuates proteinuria-induced renal tubular damage by modulating mitochondrial oxidative status.

Proteinuria is an independent risk factor for progressive renal diseases because it initiates or aggravates tubulointerstitial injury. Clinically, females are less susceptible to progression of chronic kidney disease; however, the mechanisms underlying the renoprotective effect of estrogen receptor stimulation have yet to be clarified. Recently, inflammasome-dependent inflammatory responses were shown to be triggered by free fatty acids, and mitochondria-derived reactive oxygen species were shown to be required for this response. Albumin-bound free fatty acids trigger inflammasome activation through mitochondrial reactive oxygen species production in human proximal tubule epithelial cells in vitro, an effect inhibited by raloxifene. Female ICR-derived glomerulonephritic mice (mice with hereditary nephritic syndrome) were ovariectomized and treated with raloxifene, a selective estrogen receptor modulator. Ovariectomized mice showed activation of tubular inflammasomes and elevated levels of inflammasome-dependent cytokines. Raloxifene attenuated these changes ameliorating tubulointerstitial damage, reduced production of reactive oxygen species, averted morphological changes, and improved respiratory function in mitochondria. The expression of genes that encode rate-limiting enzymes in the mitochondrial β-oxidation pathway was reduced by ovariectomy but enhanced by raloxifene. Thus, inflammasomes may be a novel and promising therapeutic target for proteinuria-induced renal injury.

Excess aldosterone is a critical danger signal for inflammasome activation in the development of renal fibrosis in mice

High levels of aldosterone impair renal function by activating proinflammatory and profibrotic pathways. However, the molecular mechanism underlying aldosterone‐induced inflammation and fibrosis is unknown. Inflammasome activation contributes to chronic kidney disease. We hypothesized that aldosterone induces renal tubulointerstitial inflammation and fibrosis by activating the inflammasome. Infusing wild‐type mice with aldosterone (0.25 mg/kg/d) caused tubulointerstitial damage, increased expression of inflammasome components, caspase 1 activation, and overproduction of IL‐1β and IL‐18. These changes were suppressed by eplerenone treatment (100 mg/kg/d) in wild‐type mice or in mice deficient in apoptosis‐associated speck‐like protein with a caspase‐recruitment domain (ASC). Caspase 1‐positive and F4/80‐positive cells colocalized in the interstitium. Bone marrow transplantation using ASC‐deficient mice indicated that inflammasome activation in macrophages mediated aldosterone‐induced renal fibrosis. IL‐18 was detected in culture supernatants of macrophages treated with aldosterone, and mitochondria‐derived reactive oxygen species activated the inflammasome in these macrophages. Our results indicate that exposure of macrophages to high levels of aldosterone resulted in the activation of inflammasomes via the mitochondria‐derived reactive oxygen species. Thus, inflammasome activation in macrophages may serve as a new therapeutic target for chronic kidney disease.—Kadoya, H., Satoh, M., Sasaki, T., Taniguchi, S., Takahashi, M., Kashihara, N. Excess aldosterone is a critical danger signal for inflammasome activation in the development of renal fibrosis in mice. FASEB J. 29, 3899‐3910 (2015). www.fasebj.org

Klotho attenuates renal hypertrophy and glomerular injury in Ins2Akita diabetic mice.

BackgroundExpression of klotho, the renoprotective anti-aging gene, is decreased in diabetic model kidneys. We hypothesized that klotho protein attenuates renal hypertrophy and glomerular injury in a mouse model of diabetic nephropathy.MethodsKlotho transgenic (KLTG) mice were crossed with spontaneously diabetic Ins2Akita (AKITA) mice. Glomerular morphology, macrophage infiltration, urinary albumin excretion and urinary 8-hydroxy-2-deoxy guanosine excretion were examined. In vitro, human glomerular endothelial cells were stimulated with high glucose with or without recombinant klotho, and calpain activity and proinflammatory cytokine expressions were measured.ResultsWe found that klotho protein overexpression attenuates renal hypertrophy and glomerular injury in this mouse model of diabetic nephropathy. Klotho overexpression attenuated renal hypertrophy, albuminuria, glomerular mesangial expansion, and endothelial glycocalyx loss in the AKITA mice. AKITA mice exhibit high levels of urinary 8-hydroxy-2-deoxy guanosine excretion. In the presence of klotho overexpression, this effect was reversed. In addition, the glomerular macrophage infiltration characteristic of AKITA mice was attenuated in KLTG-AKITA mice. In human glomerular endothelial cells, high glucose induced calpain activity. This effect was suppressed by expression of recombinant klotho, which also suppressed the induction of proinflammatory cytokines.ConclusionOur data suggest klotho protein protects against diabetic nephropathy through multiple pathways.

A case of MPO-ANCA-positive polyarteritis nodosa complicated by exudative otitis media, mononeuritis multiplex, and acute renal failure

In December 2008, a 69-year-old Japanese woman was admitted to the Department of Otorhinolaryngology because of hearing impairment due to bilateral exudative otitis media, and was discharged without complete recovery despite conventional treatment. Two weeks later, she was readmitted for worsened deafness, numbness, gait disturbance, and general fatigue. She was referred to our department for general investigation. On admission, laboratory examination revealed severe inflammatory signs and active nephritic urinary sediments. Cranial computed tomography (CT) revealed progressive exudative otitis media and sinusitis. Initially, Wegener’s granulomatosis was suspected. Nasal cavity biopsy, however, showed no granuloma formation or vasculitis. Serology revealed high titer of myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA), suggestive of microscopic polyangitis (MPA). However, contrast CT identified stenosis of a celiac artery, and renal biopsy showed tubulointerstitial changes with minor glomerular abnormalities. Therefore, polyarteritis nodosa (PAN) was suspected and treatment with intravenous methylprednisolone was initiated. However, a lacunar infarct developed followed by cerebral hemorrhage, and the patient died 19 days after readmission. Autopsy revealed fibrinoid necrosis, neutrophilic infiltration, and giant cell reaction in small to medium-sized arteries in multiple organs. These findings led to diagnosis of systemic vasculitis anatomically compatible with PAN. This was a rare case of a patient with MPO-ANCA-positive PAN who may have developed bilateral exudative otitis media and hearing loss as the initial manifestation of PAN.

Hypertension promotes islet morphological changes with vascular injury on pre-diabetic status in SHRsp rats.

Abstract Hypertensive patients have a higher incidence of new-onset diabetic mellitus than normotensive subjects, and we hypothesized that hypertension induces morphological changes in islets via vascular injury. To test our hypothesis, we administrated hydralazine or irbesartan to spontaneously hypertensive stroke-prone (SHRsp) rats. A greater islet fibrosis was observed in SHRsp rats compared with controls, and irbesartan significantly ameliorated the fibrosis. High fat diet induced glucose intorelance in SHRsp rats and irbesartan but not hydralazine improved glucose torelance. We demonstrate islet morphological changes in hypertensive rats, and our data suggest that angiotensin receptor blockers have the potential to prevent islet injury.

The eNOS-NO pathway attenuates kidney dysfunction via suppression of inflammasome activation in aldosterone-induced renal injury model mice

Hypertension causes vascular complications, such as stroke, cardiovascular disease, and chronic kidney disease (CKD). The relationship between endothelial dysfunction and progression of kidney disease is well known. However, the relationship between the eNOS–NO pathway and chronic inflammation, which is a common pathway for the progression of kidney disease, remains unexplored. We performed in vivo experiments to determine the role of the eNOS–NO pathway by using eNOS-deficient mice in a hypertensive kidney disease model. All mice were unilateral nephrectomized (Nx). One week after Nx, the mice were randomly divided into two groups: the aldosterone infusion groups and the vehicle groups. All mice also received a 1% NaCl solution instead of drinking water. The aldosterone infusion groups were treated with hydralazine to correct blood pressure differences. After four weeks of drug administration, all mice were euthanized, and blood and kidney tissue samples were collected. In the results, NLRP3 inflammasome activation was elevated in the kidneys of the eNOS-deficient mice, and tubulointerstitial fibrosis was accelerated. Suppression of inflammasome activation by knocking out ASC prevented tubulointerstitial injury in the eNOS knockout mice, indicating that the eNOS–NO pathway is involved in the development of kidney dysfunction through acceleration of NLRP3 inflammasome in macrophages. We revealed that endothelial function, particularly the eNOS–NO pathway, attenuates the progression of renal tubulointerstitial injury via suppression of inflammasome activation. Clinically, patients who develop vascular endothelial dysfunction have lifestyle diseases, such as hypertension or diabetes, and are known to be at risk for CKD. Our study suggests that the eNOS–NO pathway could be a therapeutic target for the treatment of chronic kidney disease associated with endothelial dysfunction.

A Case of Copper Deficiency-Induced Pancytopenia With Maintenance Hemodialysis Outpatient Treated With Polaprezinc

Zinc deficiency has been reported in hemodialysis (HD) patients and causes erythropoietin-resistant anemia (3). These HD patients sometimes receive oral adjuvant zinc therapy using polaprezinc (Promac, Zeria Pharmaceuticals, Tokyo, Japan). We report a rare case of pancytopenia due to polaprezinc-induced copper deficiency in a HD patient without a history of gastrointestinal surgery. Erythropoietin-resistant anemia was improved by drinking hot chocolate. We found that hot chocolate was useful to correct copper deficiency safely in a HD patient. A 46-year-old Japanese non-diabetic woman who had been receiving dialysis therapy including peritoneal dialysis for more than 10 years complained of low appetite and general fatigue. She had no history of an abnormal blood cell count prior to the event. On physical examination, her conjunctiva was pale but other examinations were normal. Laboratory data showed pancytopenia. Further serological study showed that zinc was remarkably elevated (182 μg/dL), while copper was significantly decreased (4 μg/dL). She had been taking oral polaprezinc (zinc content, 34 mg/day) for more than 10 years. We diagnosed polaprezinc-induced pancytopenia associated with copper deficiency and polaprezinc was discontinued. Although the serum zinc level gradually decreased, pancytopenia was not improved because of the lowered serum copper level. Hence, we urged her to drink hot chocolate (containing about 0.23 mg copper/cup) as a copper substitution therapy. After that, pancytopenia gradually resolved with improvement of serum zinc and copper levels (Fig. 1). There are few case reports of anemia due to copper deficiency in HD patients (2). Those cases involved enteral nutrition, which was different from our case. We thought that expansion of the zinc/copper ratio was an important factor in the progression of copper deficiency-induced pancytopenia. This patient had a low appetite before pancytopenia appeared. However, we had continued oral administration of polaprezinc. It may have caused by rapid expansion of the zinc/copper ratio. We need to consider the possibility of copper deficiency in outpatient dialysis treatment in the setting of erythropoietin-resistant anemia. Knowledge of the detailed mechanism by which copper is absorbed from the diet is incomplete. It is generally known that copper and zinc are absorbed in the stomach and small intestine (5). Excessive serum zinc levels cause an upregulation of synthesis of metallothionein (MTO) in enterocytes. MTO has a high affinity for transition metals, forming mercaptide bonds through its multiple cysteine residues. Copper displaces zinc because of its higher affinity for MTO (4). When there is an excess of zinc, copper is not absorbed into the intestinal tract and leads to hypocupremia. Another important point to note is that serum copper could be corrected without elevation of potassium and phosphate levels by drinking a cup of hot chocolate. We suggest that hot chocolate can safely correct serum copper levels in dialysis patients. In conclusion, this case suggests that serum trace elements in long-term HD patients should be closely monitored to prevent over-supplementation. We FIG. 1. Clinical course of patient. Cu, copper; DA, darbepoetinalfa; ESA, erythropoiesis stimulating agent; Hb, hemoglobin; Plt, platelets; WBC, white blood cells; Zn, zinc. 422 Letters to the Editor

[PS 01-17] HYPERTENSION PROMOTES ISLET MORPHOLOGICAL CHANGES WITH VASCULAR INJURY ON PRE-DIABETIC STATUS IN SHR-SP RATS

Objective: Type 2 diabetes causes not only insulin resistance but also failure of insulin producing &bgr; cells resulting in insufficient insulin secretion. Hypertensive patients have a higher incidence of new-onset diabetic mellitus than normotensive subjects, and we hypothesized that hypertension induces morphological changes in islets via vascular injury. To test our hypothesis, we administrated hydralazine or irbesartan to spontaneously hypertensive stroke-prone (SHRsp)/Izm male rats. Design and Method: SHRsp rats were randomly divided into three groups (SHRsp, SHRsp+Hyd, and SHRsp+Irb). The SHRsp+Hyd group was administrated hydralazine (5.0 mg body–1 d–1) by the gavage method, and the SHRsp+Irb group was similarly administrated irbesartan (50 m g body–1 d–1) from the age of 6 weeks until 20 weeks. Systolic blood pressure (SBP) and heart rate were measured weekly by the tail-cuff method. Blood samples were obtained via cardiac puncture to measure serum creatinine levels, and the pancreas was excised and fixed in 4% paraformaldehyde after sacrificing the rats with Azan staining. Results: A marked increase in systolic blood pressure was observed in SHRsp rats compared with controls (224 ± 12 mmHg vs. 120 ± 8 mmHg, respectively), which was significantly reduced by hydralazine and irbesartan administration (SHRsp+Hyd, 166 ± 12 mmHg; SHRsp+Irb, 180 ± 9 mmHg). There was no significant change in oral glucose tolerance test results in any groups. However, a greater Azan staining area was seen in SHRsp rats compared with controls, and irbesartan significantly decreased the Azan staining of islets in SHRsp rats. Irbesartan also decreased the number of ED1-positive islet cells compared with SHRsp and SHRsp+Hyd rats. Conclusions: We demonstrate islet morphological changes in hypertensive rats, and our data suggest that angiotensin receptor blockers have the potential to prevent islet injury through independent blood pressure-lowering effects.

MPS 13-08 EXCESS ALDOSTERONE IS A CRITICAL DANGER SIGNAL FOR INFLAMMASOME ACTIVATION IN THE DEVELOPMENT OF RENAL FIBROSIS IN MICE

Objective: High levels of aldosterone impair renal function by activating proinflammatory and profibrotic pathways. However, the molecular mechanism underlying aldosterone-induced inflammation and fibrosis is unknown. Inflammasome activation contributes to chronic kidney disease. We hypothesized that aldosterone induces renal tubulointerstitial inflammation and fibrosis by activating the inflammasome. Design and Method: We used ASC-deficient mice (ASCKO) to investigate the role of inflammasome, which ASC is a critical component of the inflammasomes. C57Bl/6 mice (WT) were used for control. All animals were received left uninephrectomy and given drinking water with 1% NaCl. The mice were divided into the following groups: WT-vehicle (WT-vehi), WT-Aldo (WT-Aldo; 0.25 mg/kg/day, osmotic pump), WT-Aldo treated with eplerenone (WT-Aldo + Eple; 100 mg/kg/day, gavage), ASCKO-vehicle (ASCKO-vehi), ASCKO-Aldo (ASCKO-Aldo). Three weeks after drug administration, mice were sacrificed. In vitro assay, we investigated the mitochondrial superoxide production by Aldo, which is a trigger of inflammasome activation. Results: Infusing wild-type mice with aldosterone caused tubulointerstitial damage, increased expression of inflammasome components, caspase-1 activation, and overproduction of interleukin-1&bgr; and interleukin-18. These changes were suppressed by eplerenone treatment in wild-type mice or in mice deficient in apoptosis-associated speck-like protein with a caspase-recruitment domain. Caspase-1-positive and F4/80-positive cells colocalized in the interstitium. Bone marrow transplantation using ASC-deficient mice indicated that inflammasome activation in macrophages mediated aldosterone-induced renal fibrosis. Interleukin-18 was detected in culture supernatants of macrophages treated with aldosterone, and mitochondria-derived reactive oxygen species activated the inflammasome in these macrophages. Conclusions: Our results indicate that exposure of macrophages to high levels of aldosterone resulted in the activation of inflammasomes via the mitochondria-derived reactive oxygen species. Thus, inflammasome activation in macrophages may serve as a new therapeutic target for chronic kidney disease.

[PS 01-29] COMBINATION IRBESARTAN/AMLODIPINE VERSUS IRBESARTAN/CILNIDIPINE FOR ATTENUATION OF ALBUMINURIA IN RATS WITH STREPTOZOTOCIN-INDUCED DIABETIC NEPHROPATHY

Objective: Excessive urinary albumin excretion is associated with hypertension and diabetic nephropathy. Calcium channel blockers (CCBs) used as antihypertensives suppress such albuminuria with variable efficacy. While hypertension benefits from the addition of angiotensin receptor blockers (ARBs), it is unknown if ARBs alter the effects of CCBs on albuminuria. This study compared the efficacy of combined ARB irbesartan with either CCB amlodipine or CCB cilnidipine on albuminuria associated with experimental diabetic nephropathy. Design and Method: Male Sprague-Dawley rats with streptozotocin-induced diabetes were treated with a CCB alone (amlodipine 2.0 mg/kg/d or 2.0 mg/kg/d cilnidipine 2.0 mg/kg/d), an ARB alone (irbesartan 20.0 mg/kg/d), or combinations. In the acute protocol, changes in glomerular afferent and efferent arteriole diameters were examined by a charge-coupled device video microscope following single doses. In the chronic protocol, urinary albumin excretion, glomerular reactive oxygen species, and endothelial surface layer (ESL) condition were evaluated after 2 weeks of daily treatment. Results: In the acute protocol, cilnidipine monotherapy caused a greater dilation in glomerular efferent arterioles than amlodipine monotherapy, while combination therapy with irbesartan induced comparable efferent arteriole dilation. In the chronic protocol, cilnidipine monotherapy suppressed albuminuria, reduced glomerular oxidative stress, and protected the glomerular ESL against degeneration to a much greater extent that amlodipine monotherapy. However, addition of irbesartan reduced albumin excretion, oxidative stress, and ESL degeneration to the same extent in both groups. Conclusions: While cilnidipine is more effective alone, the combinations of irbesartan with cilnidipine or amlodipine are equally effective for reducing albuminuria and other pathological sequela of experimental diabetic nephropathy.