Yuji Sogawa

Infiltration of M1, but not M2, macrophages is impaired after unilateral ureter obstruction in Nrf2-deficient mice

Chronic inflammation can be a major driver of the failure of a variety of organs, including chronic kidney disease (CKD). The NLR family pyrin domain-containing 3 (NLRP3) inflammasome has been shown to play a pivotal role in inflammation in a mouse kidney disease model. Nuclear factor erythroid 2-related factor 2 (Nrf2), the master transcription factor for anti-oxidant responses, has also been implicated in inflammasome activation under physiological conditions. However, the mechanism underlying inflammasome activation in CKD remains elusive. Here, we show that the loss of Nrf2 suppresses fibrosis and inflammation in a unilateral ureter obstruction (UUO) model of CKD in mice. We consistently observed decreased expression of inflammation-related genes NLRP3 and IL-1β in Nrf2-deficient kidneys after UUO. Increased infiltration of M1, but not M2, macrophages appears to mediate the suppression of UUO-induced CKD symptoms. Furthermore, we found that activation of the NLRP3 inflammasome is attenuated in Nrf2-deficient bone marrow–derived macrophages. These results demonstrate that Nrf2-related inflammasome activation can promote CKD symptoms via infiltration of M1 macrophages. Thus, we have identified the Nrf2 pathway as a promising therapeutic target for CKD.

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.

5-aminolevulinic acid exerts renoprotective effect via Nrf2 activation in murine rhabdomyolysis-induced acute kidney injury

Acute kidney injury (AKI) is associated with chronic kidney disease, as well as high mortality, but effective treatments for AKI are still lacking. A recent study reported the prevention of renal injury, such as ischemia‐reperfusion injury, by 5‐aminolevulinic acid (ALA), which induces an antioxidant effect. The current study aimed to investigate the effect of ALA in a rhabdomyolysis‐induced mouse model of AKI created by intramuscular injection of 50% glycerol.

MPS 01-07 ENDOTHELIAL CELLS REGULATE INFLAMMASOME ACTIVATION THROUGH NITRIC OXIDE IN HYPERTENSIVE KIDNEY DISEASE

Objective: It is well known that chronic inflammation is an important common pathway of various progressive kidney disease. It has also been reported that inflammasome activation, which is part of the innate immune system, plays an important role in such chronic inflammation. However, the mechanism involved in the prolongation of inflammasome activation is unknown. We focused on nitric oxide (NO) as a regulatory mechanism of inflammasome. To test the hypothesis that “Endothelial cells-induced nitric oxide inhibits inflammasome activation and regulates chronic inflammation,” we used wild-type (WT) and eNOS knockout (eNOSKO) mice. Design and Method: [1] After unilateral nephrectomy (Nx), aldosterone (Ald) was continuously administered (0.25 mg/kg/day) using an infusion pump with 1% NaCl drinking water. The mice were sacrificed after 4 weeks of Ald administration, and the kidney tissue was examined. [2] We used bone-marrow-derived macrophages (BMDMs) to examine the molecular mechanism. After LPS priming (200 ng/mL 3 h), ATP (5 mM 60 min) was added to activate NLRP3 inflammasome. At the same time, we also performed priming with the NO donor, S-nitrosoglutathione (GSNO) to confirm the role of NO. Results: [1] In the WT mice, administration of Ald-Nx-Nacl caused an increase in blood pressure, kidney tissue fibrosis and inflammasome activation. The eNOSKO mice showed a similar tendency, but the inflammation in kidney was even worse. Since blood pressure was higher in the eNOSKO mice, we administered hydralazine to both groups to lower the blood pressure to the same extent before the examination. Even when the blood pressure was lowered to the same extent, renal inflammasome activation was significantly enhanced in the eNOSKO mice. [2] BMDM stimulation with LPS-ATP caused NLRP3 inflammasome-dependent cell death and IL1beta secretion. GSNO inhibited this activation. Conclusions: Endothelial cells-induced NO inhibits inflammasome activation to regulate chronic intrarenal inflammation. Endothelial dysfunction might promote chronic inflammation in CKD.

ISH NIA PS 01-02 Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice

Objective: Increased generation of reactive oxygen species (ROS) is a common pathogenic mechanism underlying vascular and renal complications in diabetes. Endothelial NAD(P)H oxidase is a major source of vascular ROS and plays important role in endothelial dysfunction. We hypothesize that activation of endothelial NAD(P)H oxidase would initiate and accelerate diabetic nephropathy, especially development of albuminuria, in diabetic milieu. Design and Method: We used endothelial specific NOX2 transgenic (Es-NOXTg) mice, AKITA type1 diabetic (AKITA) mice, NOX2Tg crossbred with AKITA mice, and wild type (WT) mice. Es- NOXTg was generated in which NOX2, gp91 phox of NAD(P)H oxidase, under the control of Tie2 promoter, was overexpressed in the endothelium. All mice were back-crossed into C57BL/6J strain. These mice were sacrificed at 6 and 12-week-old of ages for molecular and histological analysis. We applied the in vivo live imaging techniques with multi-photon laser microscopy and various sizes of FITC labeled dextrans to analyze alterations in permeability of glomerular capillary walls in disease conditions. Results: Urinary albumin excretion was increased only in NOXTG-AKITA mice but not in WT and AKITA mice at 6-week-old. At 12-weeks-old, serum creatinine level was significantly elevated only in NOXTg-AKITA but not AKITA and WT mice. No significant morphological changes were detected in glomeruli from all groups by light microscopic examinations. But slight degree of structural changes in podocytes and mesangial cells were observed only in NOXTg-AKITA mice under the electron microscope. The in vivo live imaging techniques revealed increased filtration of 40kDa dextran in glomeruli in AKITA and NOXTg-AKITA, but not in WT mice. Moreover, increased permeability of larger molecules, 70 KDa dextran, were detected in NOXTg-AKITA mice. Lectin (WGA lectin) staining was decreased along glomerular endothelium in NOXTg-AKITA mice. Conclusions: Activation of endothelial NAD(P)H oxidase in hyperglycemic milieu initiated and accelerated diabetic nephropathy characterized by development of albuminuria and hyperfiltration of macromolecules.

ISH NIA PS 01-07 Possible implication of xanthine oxidase activation on the pathogenesis diabetic nephropathy

Objective: Endothelial dysfunction represents a predominant early feature of diabetes and makes diabetic patients prone to renal complications. Recent evidence has indicated possible role of xanthine oxidase (XO) in the pathogenesis of vascular dysfunction associated with diabetes. However, it is not clear whether XO activity is involved in pathogenesis of diabetic nephropathy (DN). We investigated the contribution of XO activation on the progression of mouse DN by selective XO inhibitors, Topiroxostat (Top) and Febuxostat (Feb). Design and Method: Male Ins2Akita heterozygote (Akita; 10 weeks old) mice were used. Wild-type (WT) mice were used for control. Akita mice were treated with Top (3 mg/kg/day), Feb (1 mg/kg/day) or Vehicle (Vehi) for 4weeks. Serum uric acid and urinary albumin excretion (UAE) were measured. Glomerular pathological changes were also examined by light microscope and electron microscope. Glomerular permeability was assessed using 2 photon microscopy and fluorescent labeling albumin. Results: Serum uric acid levels showed no significant difference between all groups. Akita + Top or Akita + Feb groups showed significant reduction of UAE in comparison with Akita + Vehi group. Mesangial expansion, glomerular collagen IV deposition, and glomerular endothelial injury (examined by lectin stain and transmission electron microscope) were ameliorated in Akita + Top or Akita + Feb group compared with Akita + Vehi group. Furthermore, glomerular permeability was deteriorated in Akita + Vehi group compared with WT group. These changes were ameliorated with addition of Top or Feb. Conclusions: XO inhibitors preserved glomerular endothelial function and improved deteriorated glomerular permeability, indicating that XO activation is involved in pathogenesis of DN.

MPS 07-04 FEASIBILITY OF FLUORESCENCE ENERGY TRANSFER SYSTEM FOR IMAGING THE RENOPROTECTIVE EFFECTS OF ALISKIREN IN DIABETIC MICE

Objective: Although systemic renin activity is suppressed in diabetes, renin-angiotensin system (RAS) inhibitors exert renoprotective effects. Aliskiren, a renin inhibitor, is renoprotective in diabetic animal models. While it is possible to measure tissue RAS activity, no reports of in vivo images exist. We investigated the feasibility of using a fluorescence resonance energy transfer system to image enzymatic activity in order to evaluate the effects of aliskiren on diabetic nephropathy. Design and Method: First, we induced diabetes in C57BL/6J mice using streptozotocin, then treated them with either aliskiren (25 mg/kg/day) or the angiotensin type 1 receptor blocker valsartan (15 mg/kg/day) for 4 weeks. Finally, we utilized renin fluorescence resonance energy transfer substrate to assess renin activity. Results: Renin activity was much higher in the kidneys of diabetic mice compared to those of the non-diabetic control mice. While aliskiren inhibited this activity, valsartan did not. We noted that production of reactive oxygen species intensified and the bioavailability of nitric oxide diminished in the glomeruli of diabetic mice. Aliskiren and valsartan significantly ameliorated these effects. They suppressed glomerular production of reactive oxygen species and urinary albumin excretion. In fact, urinary albumin excretion in diabetic mice treated with aliskiren or valsartan was lower than that in untreated diabetic mice. Furthermore, aliskiren and valsartan significantly reduced glomerular permeability by maintaining the glomerular endothelial surface layer. Conclusions: We found that injecting renin fluorescence resonance energy transfer substrate intravenously enabled us to visualize renin activity in the glomeruli of living animals. Our imaging technique helped reveal that although both aliskiren and valsartan significantly inhibited urinary albumin excretion in diabetic mice, only aliskiren significantly inhibited renin bioactivity. Fluorescence resonance energy transfer could provide a new tool for evaluating tissue and plasma enzymatic activity.