Seiji Itano

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.

Feasibility of fluorescence energy transfer system for imaging the renoprotective effects of aliskiren in diabetic mice

Introduction: We investigated the feasibility of using a fluorescence resonance energy transfer system to image enzymatic activity in order to evaluate the effects of aliskiren (a direct renin inhibitor) on diabetic nephropathy. Materials and methods: 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 four 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. Conclusion: Fluorescence resonance energy transfer could provide a new tool for evaluating tissue and plasma enzymatic activity.

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.

Distinct characteristics and outcomes in elderly-onset IgA vasculitis (Henoch-Schönlein purpura) with nephritis: Nationwide cohort study of data from the Japan Renal Biopsy Registry (J-RBR)

Background The clinical presentation and prognosis of adult and elderly patients with IgA vasculitis (Henoch-Schönlein purpura) accompanied by nephritis (IgAV-N) have not been investigated in detail. We therefore surveyed the features and outcomes of IgAV-N based on nationwide data derived from the Japan Renal Biopsy Registry (J-RBR). Methods This multi-center cohort study compared the clinicopathological parameters at diagnosis, initial therapies and outcomes between 106 adult (age 19–64 years) and 46 elderly (≥65 years) patients with IgAV-N who were registered in the J-RBR between 2007 and 2012. The primary end-points comprised a 50% increase in serum creatinine (sCr) values or end-stage kidney disease. Factors affecting a decrease in renal function were assessed using Cox proportional hazards models. Results Rates of hypertension, impaired renal function, hypoalbuminemia and crescentic glomerulonephritis were significantly higher among the elderly, than the adult patients. About 80% and 60% of the patients in both groups were respectively treated with corticosteroid and a renin-angiotensin system (RAS) blockade. Both groups had favorable renal survival rates for nine years (93.6% and 91.4% of the adult and elderly patients, respectively). Significantly more elderly than adult patients developed a 50% increase in sCr during a mean observation period of 3.9 years (21.7% vs. 4.7%, p = 0.012), and significantly fewer elderly, than adult patients achieved clinical remission (23.9% vs. 46.2%, p = 0.016). Multivariate analysis selected advanced age (≥65 years) and lower serum albumin values as independent prognostic factors for a decline in renal function, whereas steroid pulse therapy helped to preserve renal function. Conclusions The renal prognosis of adult and elderly patients with IgAV-N was favorable when treated aggressively with corticosteroid and RAS blockade. However, the course of renal function should be carefully monitored in patients aged over 65 years and those with hypoalbuminemia.

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.

[PS 01-21] RENAL DENERVATION PROTECTS RENAL FUNCTION BY SUPPRESSING NAD(P)H OXIDASE ACTIVITY IN DAHL SALT-SENSITIVE RATS

Objective: Sympathetic activity is increased in CKD patients and plays an important and distinct role in renal disease-associated hypertension. Indeed, sympathetic activation is of crucial importance for both the raised blood pressure and the high morbidity and mortality of end-stage renal disease. So we investigated whether renal denervation protects renal function through an anti-oxidative effect. Design and Method: Dahl salt sensitive rats in two groups had the right kidney removed (uninephrectomy; Nx) and those in the other two groups had sham surgery (DL group or DH group). Two weeks later, half of the Nx group underwent left renal denervation (Nx-RDNx group; n = 10) and the other half had sham surgery (Nx-Sham; n = 10). The DL group received a 0.3% salt diet and the DH, Nx-RDNx, and Nx-Sham groups received an 8% salt diet after renal denervation or sham operation. After a further 6 weeks, kidney function and the renal tissue were assessed. Results: Blood pressure, heart rate, body weight, kidney weight and urinary sodium excretion did not differ between Nx-RDNx and Nx-Sham group. Creatinine clearance and urinary albumin excretion were significantly lower in the Nx-RDNx group than in the Nx-Sham group. Desmin staining was lower in Nx-RDNx compared with Nx-Sham. Fluorescent staining for reactive oxygen species in isolated glomeruli was significantly weaker in the Nx-RDNx group. A lucigenin assay of NAD(P)H oxidase activity in isolated glomeruli indicated that renal denervation may have caused the reduction in reactive oxygen species through suppression of the activity of NAD(P)H oxidase. Plasma renin activity was significant increased in the Nx-Sham group compared with DH, and renal denervation suppressed this activity. Conclusions: Renal sympathetic denervation helps protect against glomerular sclerosis, possibly by suppressing NAD(P)H oxidase activity, thereby decreasing glomerular reactive oxygen species.

MPS 01-08 CYCLIC GMP DEPENDENT PROTEIN KINASE-AKT PATHWAY IS ESSENTIAL FOR COMPENSATORY RENAL HYPERTROPHY AND MAINTENANCE RENAL FUNCTION AFTER UNILATERAL NEPHRECTOMY

Objective: Renal hypertrophy as an early compensatory mechanism to replace the loss of functioning tissue ultimately turns into a maladaptive process leading to progressive deterioration of renal function. We previously reported that nitric oxide (NO) production in the kidney is increased in the early phase after nephrectomy. However, the precise mechanism of increased NO production and its role in compensatory renal hypertrophy following nephrectomy remain unclear. We therefore hypothesize that the eNOS-soluble guanylate cyclase (sGC)-protein kinase G (PKG) pathway plays a crucial role in compensatory renal hypertrophy following unilateral nephrectomy. Design and Method: We used the following three groups of mice (male; background: C57BL6;): wild-type (WT), eNOS knockout (eNOS-KO) and endothelial specific eNOS transgenic (ECeNOS-TG) mice. These mice underwent left unilateral nephrectomy (i.e., WT-Nx, eNOS-KO-Nx and ECeNOS-TG-Nx), and their right kidney and serum were obtained for examination 2 weeks after nephrectomy. Next, we investigated the mechanism of NO-induced hypertrophy in vitro using human proximal tubular epithelial cells (hPTECs). hPTECs were stimulated with sGC stimulator BAY 41-2272 (Bay), and the activity of PKG and the downstream pathway were examined. In addition, we examined the effects of BAY in vivo by treating eNOS-KO mice with BAY (eNOS-KO-Nx-BAY) after unilateral nephrectomy. Results: Two weeks after nephrectomy, compensatory renal hypertrophy was markedly suppressed in the eNOS-KO-Nx group compared with the WT-Nx group. And serum CRN was increased in eNOSKO mice after nephrectomy compared with WT mice. Furthermore eNOS-KO-Nx also was increased excretion of albunuria after uni-nephrectomy. On the other hand, the ECeNOS-TG-Nx group exhibited enhanced compensatory renal hypertrophy than the other two groups. Treatment with BAY significantly enhanced compensatory renal hypertrophy in the eNOS-KO-Nx mice. Conclusions: Reduction of eNOS-NO bioavailability, as a endothelial dysfunction, may induce failure of compensatory hypertrophy and accelerate progressive renal dysfuction.

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.

[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.

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.