Hajime Nagasu

Klotho protects against mouse renal fibrosis by inhibiting Wnt signaling

Augmented Wnt signaling has been implicated in many fibrotic diseases including obstructive nephropathy. Soluble form Klotho has been reported to function as a secreted Wnt antagonist. In this study, we tested whether Klotho protein could reduce renal fibrosis by inhibition of Wnt signaling. Transgenic mice that overexpressed Klotho, wild-type mice, and Klotho hetero mutant mice underwent unilateral ureteral obstruction (UUO). In some Klotho hetero mutant mice, Klotho-encoding plasmid was transferred into the skeletal muscle by electroporation. UUO induced activation of Wnt signaling in wild-type but less in Klotho transgenic mice. Enhanced tubulointerstitial fibrosis in wild-type mice was also attenuated in Klotho transgenic mice. In contrast, Wnt signaling and concomitant tubulointerstitial fibrosis were further augmented in Klotho hetero mutant mice after UUO compared with wild-type mice. In Klotho-encoding plasmid-transfected Klotho hetero mutant mice, however, Wnt signaling was markedly reduced accompanied by a decrease in extracellular matrix deposition after UUO. In vitro studies showed that stimulation of Wnt3a induced prolonged cell cycle arrest at G(2)/M phase, with a resultant increase in production of fibrogenic cytokines. Cotreatment with Klotho bypassed the G(2)/M arrest and reduced fibrogenic cytokine production. In conclusion, Klotho is a critical negative regulator of Wnt signaling and a suppressor of renal fibrosis in the obstructed kidney model.

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.

Renal denervation reduces glomerular injury by suppressing NAD(P)H oxidase activity in Dahl salt-sensitive rats

BACKGROUND Renal sympathetic nerve activity has important effects on renal function in chronic kidney disease. Recent studies indicated that beta agonists directly stimulate NAD(P)H oxidase in endothelial cells. Therefore, we investigated whether renal denervation protects renal function through an anti-oxidative effect. METHODS The right kidney was removed from Dahl salt-sensitive hypertensive rats. Two weeks later, the rats underwent either left renal denervation (Nx-RDNx; n = 10) or a sham operation (Nx-Sham; n = 10). After a further 6 weeks, kidney function and renal tissue were assessed. In this ex vivo study, using isolated glomeruli from Sprague-Dawley rats, the direct effects of catecholamine on NAD(P)H oxidase activity were assessed. RESULTS After the Nx-RDNx or Nx-Sham surgery, urinary albumin excretion and the histologic glomerular sclerosis index were lower in the Nx-RDNx group than in the Nx-Sham group. Fluorescence 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. The levels of mRNA for NAD(P)H oxidase components and the levels of rac1 were higher in glomeruli from the Nx-Sham group than from the Nx-RDNx group. In this ex vivo study, although the NAD(P)H oxidase activity did not change with administration of either the alpha- or beta2-agonist, it increased with the beta1-agonist. CONCLUSIONS Renal sympathetic denervation helps to protect against glomerular sclerosis, possibly by suppressing NAD(P)H oxidase activity, thereby decreasing glomerular reactive oxygen species.

Tacrolimus induces glomerular injury via endothelial dysfunction caused by reactive oxygen species and inflammatory change.

Background/Aims: The immunosuppressive drug tacrolimus (FK506) is used clinically to reduce the rejection rate in patients with kidney transplantation; however, the resultant nephrotoxicity remains a serious problem. In the present study we attempted to elucidate the mechanisms of glomerular injury induced by FK506 and the renoprotective effects of the angiotensin II receptor blocker telmisartan. Methods: Seven-week-old male Wistar rats were divided into three groups: vehicle group, FK506 group, and FK506 + telmisartan group. After 8 weeks, we assessed kidney function and renal morphological changes including oxidative stress. We also assessed the effect of FK506 in human glomerular endothelial cells (hGECs) with regard to reactive oxygen species (ROS). Results: FK506 induced ROS production via activation of NAD(P)H oxidase in the glomeruli. Expression of ICAM mRNA was increased in glomeruli from the FK506 group. These effects resulted in macrophage infiltration into the glomeruli. FK506 directly promoted NAD(P)H oxidase activity and accelerated production of ROS in hGECs. Conversely, cotreatment with telmisartan inhibited both NAD(P)H oxidase activity and production of ROS. Conclusion: These findings suggest that glomerular injury resulting from FK506 is caused by oxidative stress mediated by activation of NAD(P)H oxidase and that telmisartan exerts a renoprotective effect via antioxidative activity.

Mitochondrial damage-induced impairment of angiogenesis in the aging rat kidney

Decreased expression of vascular endothelial growth factor (VEGF) in the renal tubules is thought to cause progressive loss of the renal microvasculature with age. Mitochondrial dysfunction may be a principal phenomenon underlying the process of aging. The relation between VEGF expression and mitochondrial dysfunction in aging is not fully understood. We hypothesized that mitochondrial dysfunction blocks VEGF expression and contributes to impaired angiogenesis in the aging kidney. The aim of this study was to assess the role of mitochondria in VEGF expression in the aging rat kidney. We evaluated the accumulation of 8-hydroxy-2′-deoxyguanosine in mitochondrial DNA, as well as mitochondrial dysfunction, as assessed by electron microscopy of mitochondrial structure and histochemical staining for respiratory chain complex IV, in aging rat kidney. An increase in hypoxic area and a decrease in peritubular capillaries were detected in the cortex of aging rat kidneys; however, upregulation of VEGF expression was not observed. The expression of VEGF in proximal tubular epithelial cells in response to hypoxia was suppressed by the mitochondrial electron transfer inhibitor myxothiazol. Mitochondrial DNA-deficient cells also failed to upregulate VEGF expression under hypoxic conditions. These results indicate that impairment of VEGF upregulation, possibly as a result of mitochondrial dysfunction, contributes to impaired angiogenesis, which in turn leads to renal injury in the aging rat kidney.

Endothelial dysfunction promotes the transition from compensatory renal hypertrophy to kidney injury after unilateral nephrectomy in mice

Loss of functional nephrons associated with chronic kidney disease induces glomerular hyperfiltration and compensatory renal hypertrophy. We hypothesized that the endothelial nitric oxide synthase (eNOS) [soluble guanylate cyclase (sGC)] protein kinase G (PKG) pathway plays an important role in compensatory renal hypertrophy after unilateral nephrectomy. Analysis of mice subjected to unilateral nephrectomy showed increases in kidney weight-to-body weight and total protein-to-DNA ratios in wild-type but not eNOS knockout (eNOSKO) mice. Serum creatinine and blood urea nitrogen increased after nephrectomy in eNOSKO but not in wild-type mice. Furthermore, Bay 41-2272, an sGC stimulator, induced compensatory renal hypertrophy in eNOSKO mice and rescued renal function. The NO donor S-nitrosoglutathione (GSNO) and Bay 41-2272 stimulated PKG activity and induced phosphorylation of Akt protein in human proximal tubular cells. GSNO also induced phosphorylation of eukaryotic initiation factor 4E-binding protein and ribosomal protein S6. Our results highlight the importance of the eNOS-NO-PKG pathway in compensatory renal hypertrophy and suggest that reduced eNOS-NO bioavailability due to endothelial dysfunction is the underlying mechanism of failure of compensatory hypertrophy and acceleration of progressive renal dysfunction.

Overexpression of klotho protein modulates uninephrectomy-induced compensatory renal hypertrophy by suppressing IGF-I signals

The klotho gene is highly expressed in the distal convoluted tubule of the kidney, while its encoded protein has many physiological and pathophysiological renal roles. We investigated the effect of klotho protein on physiological compensatory renal hypertrophy after nephrectomy in klotho transgenic (KLTG) mice. Renal hypertrophy was suppressed in KLTG mice compared with wild-type mice, and this was associated with suppression of insulin growth factor-1 (IGF-1) signaling by klotho protein. In vitro, IGF-1 signaling was suppressed in human proximal tubular cells transfected with the klotho plasmid. Our data suggest that klotho modulates compensatory renal hypertrophy after nephrectomy via suppression of the IGF-1 signaling pathway, indicating a novel physiological role for klotho protein in the kidney.

Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice.

Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes.

Azelnidipine attenuates glomerular damage in Dahl salt-sensitive rats by suppressing sympathetic nerve activity.

Dihydropyridine-type calcium channel blockers (CCBs) exert potent antihypertensive effects. The CCB azelnidipine decreases heart rate by suppressing sympathetic nerve activity, which affects afferent and efferent arterioles in the glomeruli. We examined whether azelnidipine can improve progressive glomerular injury in comparison with amlodipine by suppressing renal sympathetic nerve activity in Dahl salt-sensitive rats. Glomerular circulation in Dahl salt-sensitive rats was monitored with a charge-coupled device camera before and after administration of amlodipine (0.5 mg kg−1, bolus injection) or azelnidipine (0.1 mg kg−1, bolus injection). Systemic sympathetic nerve activity was also compared by analysis of heart rate variability with a telemetry blood pressure monitoring system after crossover administration of amlodipine (1.0 mg kg−1 per day) and azelnidipine (3.0 mg kg−1 per day) for 1 week. To investigate renoprotective effects, rats were treated with amlodipine (1.0 mg kg−1 per day) or azelnidipine (3.0 mg kg−1 per day) for 3 weeks with or without renal denervation. The efferent arteriole contracted in response to acute amlodipine but not azelnidipine treatment. The low frequency/high frequency ratio, an index of parasympathetic nerve activity, decreased in response to azelnidipine but not amlodipine treatment. In response to chronic treatment, proteinuria and glomerular injury improved to a greater extent with azelnidipine compared with amlodipine. The renoprotective effects of azelnidipine were diminished by renal denervation. Azelnidipine decreased glomerular damage in Dahl salt-sensitive rats to a greater extent than amlodipine. Azelnidipine appeared to decrease intraglomerular pressure by suppressing sympathetic nerve activity.

Azelnidipine exerts renoprotective effects by improvement of renal microcirculation in angiotensin II infusion rats

BACKGROUND Hypoxia-induced tubulointerstitial injury caused by loss of peritubular capillary (PTC) blood flow may be associated with progressive renal disease. Therefore, the maintenance of blood flow in PTCs may protect against loss of renal function. A long-acting calcium channel blocker, azelnidipine, has been shown to be useful in the treatment of progressive renal disease. However, its mechanism of action remains unclear. The aim of the present study was to elucidate whether azelnidipine maintains PTC blood flow and to compare it to nifedipine in its ability to improve tubulointerstitial injury caused by angiotensin II (AII) infusion in rats. METHODS PTC blood flow was initially monitored using a pencil-lens interval microscope before and after intravenous AII (30 ng/kg/min) infusion with or without azelnidipine (10 microg/kg/min). Next, Wistar rats were treated with chronic infusion of AII (500 ng/kg/min) via an osmotic minipump with or without azelnidipine (3 mg/kg/day, orally) or nifedipine (60 mg/kg/day, orally) for 14 days, and tubulointerstitial damage (PTC loss, interstitial fibrosis, tubular atrophy) was examined. RESULTS PTC blood flow was reduced after AII infusion but improved after a bolus injection of azelnidipine. Tubulointerstitial damage observed in chronically AII-treated kidneys was associated with hypoxic conditions, as indicated by the measurement of hypoxia biomarkers (intracellular hypoxyprobe-1 adducts). These tubulointerstitial injuries in AII-infused rats were more effectively reduced by azelnidipine than by nifedipine. The area showing hypoxic conditions in the kidney was also more reduced with azelnidipine than nifedipine treatment. CONCLUSIONS Azelnidipine may increase PTC blood flow and improve renal hypoxia and tubulointerstitial injury induced by AII infusion.