Renal medullary circulation has now been found to play a fundamental role in regulating long-term blood pressure control and fluid balance. Elevation of superoxide or reduction of nitric oxide (NO) in renal medulla decreases medullary blood flow and Na excretion, resulting in sustained hypertension. Angiotensin II (Ang II)-induced interaction of superoxide and NO was determined in thin tissue strips isolated from the renal outer medullary region of Sprague-Dawley rats using fluorescent microscopy techniques. Ang II can induce diffusion of NO, but not superoxide, from the medullary thick ascending limb (mTAL) to the surrounded vasa recta. However, when NO is reduced by the NO scavenger carboxy-PTIO, Ang II can induce superoxide diffusion from mTAL to vasa recta pericytes. Therefore, the physiological action of oxidative stress in renal medullary region is demonstrated as balance of superoxide and NO diffusion (“tubulo-vascular cross-talk”). These results explain how chronically hypoxic medulla can maintain blood flow. In other studies using chronically instrumented rats, we found that nearly 70% of Ang II-induced medullary renal injury was dependent on pressure determined by servo-control of renal perfusion pressure, whereas 30% of the injury was non-hemodynamic. We conclude that oxidative stress within the renal medulla can induce hypertension and also make the kidney functionally more vulnerable to the effects of Ang II.

View full abstract

Recent clinical and pre-clinical studies have indicated the utility of mineralocorticoid receptor (MR) antagonists in renal injury. We have demonstrated in rats that chronic treatment with aldosterone results in severe proteinuria and renal injury, characterized by glomerular changes, tubulointerstitial fibrosis, and collagen accumulation. We also observed increased reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPKs) activity in renal cortical tissues. Treatment with a selective MR antagonist, eplerenone, prevented elevation of ROS levels and MAPK activity, as well as ameliorating renal injury. In vitro studies revealed that MRs are highly expressed in rat glomerular mesangial cells (RMC) and rat renal fibroblasts. In RMC, aldosterone induces cellular injuries through NADPH oxidase-dependent ROS production and/or MAPK activation. Aldosterone-induced renal cellular injuries were markedly attenuated by treatment with eplerenone. These data suggest that aldosterone induces renal injury through activation of MRs and support the notion that MR blockade has beneficial effects on aldosterone-dependent renal injury through mechanisms that cannot be simply explained by hemodynamic changes. In this review, we summarized our recent findings pertaining to the roles of aldosterone and MRs in the pathogenesis of renal injury. Potential molecular mechanisms responsible for aldosterone/MR-induced renal injury were also discussed.

View full abstract

Tubulointerstitial fibrosis is a common feature of many progressive renal diseases and is a main determinant that leads to an irreversible loss of renal function. In chronic cyclosporin A nephrotoxicity, we previously reported that inflammatory responses such as macrophage infiltration preceded interstitial fibrosis. This inflammation was accompanied by an elevation in renal nuclear factor κB (NF-κB) activity. Similar findings were obtained in chronic tacrolimus nephrotoxicity and obstructive nephropathy. Inhibition of NF-κB markedly attenuated renal inflammation and interstitial fibrosis in these models. Furthermore, administration of oral adsorbent (Kremezin^®) significantly attenuated the increase in renal NF-κB activity and concomitantly reduced interstitial inflammation and renal fibrosis in chronic renal failure rats. Elimination of indoxyl sulfate by this adsorbent is likely involved in this mechanism since it is known that indoxyl sulfate activates NF-κB in renal tubular cells. It is suggested that strategy aiming at NF-κB inhibition is important to prevent the progression of renal fibrosis.

View full abstract

Among the GTP-binding proteins, Rho is known to function as a molecular switch in various cellular functions. Among the Rho effectors, the cellular function and signal transduction of Rho-kinase have been extensively studied. However, information about its in vivo functions is still limited. With the recent development of a specific Rho-kinase inhibitor such as Y-27632 and fasudil, the understanding of the role of the Rho/Rho-kinase pathway in vitro and in vivo has advanced. However, to date, there have been few studies investigating the role of Rho-kinase in renal disease. Recent studies have shown that Rho-kinase inhibitor significantly attenuated the tubulointerstitial fibrosis in kidney induced by unilateral ureteral obstruction. However, there have been few studies investigating the role of the Rho/Rho-kinase pathway in hypertensive glomerular sclerosis. In this review, we described the role of the Rho/Rho-kinase pathway in the progression of renal glomerulosclerosis in several forms of hypertensive rats. Our results suggest that chronic inhibition of the Rho-kinase pathway may be a new therapeutic approach for hypertensive glomerulosclerosis. Our results also suggest that the mechanism of the renoprotective effect of Rho-kinase inhibitor is partly mediated via inhibition of extracellular matrix gene expression, monocytes/macrophages infiltration, oxidative stress, and upregulation of eNOS gene expression.

View full abstract

Rho/Rho-kinase plays an important role not only in the vasoconstrictor mechanism but also in cellular morphology, motility, adhesion, and proliferation. This pathway also serves to modulate the structure and function of various kidney cells including tubular epithelial cells, mesangial cells, and podocytes. The inhibition of the Rho/Rho-kinase pathway elicits marked increases in renal blood flow in vivo and dilates both afferent and efferent arterioles preconstricted by angiotensin II in vitro. In renal injury, intrarenal angiotensin II is reported to be activated, which subsequently would upregulate the Rho-kinase pathway. A selective Rho-kinase inhibitor, fasudil, has recently been shown to improve renal damage resulting from hypertensive glomerulosclerosis, unilateral ureteral obstruction (for interstitial renal fibrosis) and subtotal nephrectomy. Of interest, fasudil upregulated the expression of p27^kip1, a cyclin-dependent kinase inhibitor, and increased the p27^kip1 immuno-positive cells in both glomeruli and tubulointerstitium with the use of immunohistochemistry. Collectively, the Rho-kinase pathway is involved in the pathogenesis of renal injury. Clinical application of this type of therapy however awaits further investigations.

View full abstract

Radial artery (RA) is increasingly used as graft for coronary artery bypass grafting due to its good long-term patency. However, the mechanism of peri- and post-operative spasm is still unclear. Because of that, the aim of our study is to analyze the contractility of RA and to determine whether the presence of functional endothelium alters its contractile properties. Contractions of isolated RA rings were provoked by exogenously applied vasoconstrictors or by electrical field stimulation (EFS, 20 Hz). The order of vasoconstrictors potency based on their EC₅₀ values was as follows: angiotensin II > phenylephrine > 5-hydroxytriptamine. Presence of endothelium increased both EC₅₀ and maximal contraction to phenylephrine and angiotensin II, but inhibited reactivity of RA to 5-hydroxytriptamine. Spontaneous rhythmic contractions (SRC, <4 mHz) and EFS-induced contractions of RA are endothelium-independent and weaker than contractions induced by exogenously applied vasoconstrictors. Our study concludes that RA shows marked sensitivity and reactivity to angiotensin II, phenylephrine, and 5-hydroxytriptamine. Further investigations are necessary to answer why angiotensin II and phenylepehrine induce stronger contractions in the presence of endothelium. In addition, SRC as well as contractions of neurogenic origin may take part in developing vascular spasm of RA.

View full abstract

Anti-inflammatory and anti-arthritic effects of water distillates of Ephedra sinica STAPF (ES), in herb-acupuncture, on the inflammatory responses of arthritis was investigated using phorbol 12-myristate 13-acetate (PMA)/lipopolysaccharide (LPS)-induced human macrophage and adjuvant-induced arthritic rat. The luciferase reporter vectors driven by the tumor necrosis factor (TNF)-α and cyclooxygenase-2 promoters were transiently transfected into U937 cells, which were then differentiated and stimulated by PMA and LPS, respectively, to develop an in vitro anti-inflammation assay system. The luciferase activities, observed in the activated U937 cells, were significantly inhibited by ES herb-acupuncture, compared to those of PD98509 and berberine. To evaluate ES herb-acupuncture as a novel anti-arthritic therapy, a polyarthritic rat model was developed using heat-killed Mycobacterium tuberculosis, and 50 μl of ES distillate was subcutaneously injected into the ST36 acupoint on each knee joint. While the articular indexes of arthritic rats were evidently decreased by ES herb-acupuncture, their body weights did not regain their initial levels. This may be due to the accelerating effects of ES on weight-loss and fat consumption. The mRNA expressions of TNF-α and interleukin (IL)-6 genes, which were closely stimulated in the arthritic rat joints, were found to be restored to the normal levels through the ES treatment. In the case of IL-1β, the recovery was not significant but substantial. The anti-arthritic effect of ES herb-acupuncture was not found in the ES-treated/non-acupoint group. In conclusion, the ES herb-acupuncture into the ST36 acupoint was found to be effective in alleviating the inflammatory response and thus arthritic symptoms in adjuvant-induced arthritic rats.

View full abstract

To investigate the possibility that natural medicines affect lipid metabolism by regulating lipoprotein lipase (LPL) expression, a green fluorescent protein (GFP) gene was constructed downstream of the peroxisome proliferator response element (PPRE) and the constructed plasmid was microinjected into Xenopus oocytes to establish a PPRE regulatory reporter system. Using this system, hawthorn flavonoids were quickly selected from a panel of natural medicines and found to up-regulate GFP expression by an effect on PPRE. To confirm the effect of hawthorn flavonoids, we treated mice orally with water (control), hawthorn flavonoids, and pioglitazone and measured the LPL levels in serum, adipose tissue, and muscle by an enzyme-linked immunosorbent assay. The serum LPL levels were no different from the controls after treatment with either hawthorn flavonoids or pioglitazone, but LPL increased significantly in muscular tissues and decreased in adipose tissues. These results demonstrate that hawthorn flavonoids meditate LPL expression in mice with tissue-specific differences. A novel PPRE regulatory report system was established for rapid and effective selection and evaluation of LPL-mediating drugs.

View full abstract

Cabergoline is used in the treatment of Parkinson’s disease (PD). Clarithromycin is a potent inhibitor of CYP3A4 and P-glycoprotein and is often co-administered with cabergoline in usual clinical practice. We studied the effect of clarithromycin co-administration on the blood concentration of cabergoline in healthy male volunteers and in PD patients. Study 1: Ten healthy male volunteers were enrolled and were randomized to take a single oral dose of cabergoline (1 mg/day) for 6 days or a single oral dose of cabergoline plus clarithromycin (400 mg/day) for 6 days. Study 2: Seven PD patients receiving stable cabergoline doses were enrolled. They were evaluated for the plasma cabergoline concentration before and after the addition of clarithromycin 400 mg/day for 6 days, and again 1 month after discontinuation of clarithromycin. The dose and duration of clarithromycin were decided according to usual clinical practice. In healthy male volunteers, mean C_max and AUC_{0–10 h} of cabergoline increased to a similar degree during co-administration of clarithromycin. Mean plasma cabergoline concentration over 10 h post-dosing increased 2.6-fold with clarithromycin co-administration. In PD patients, plasma cabergoline concentration increased 1.7-fold during clarithromycin co-administration. Co-administration with clarithromycin may increase the blood concentration of cabergoline in healthy volunteers and in PD patients.

View full abstract

We investigated the involvement of reactive oxygen species (ROS) and intracellular calcium in nephrotoxicity related to an antitumor agent, cisplatin. In this study, we employed cultured renal epithelial cells (LLC-PK₁). Cisplatin at 500 μM significantly increased the production of ROS 5 h and caused cell injury. This agent significantly increased the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner 1 h or more after exposure. DPPD (N,N'-diphenyl-p-phenylenediamine), an antioxidant, inhibited a cisplatin-related increase in active oxygen production and cell injury but did not inhibit an early increase in the [Ca²⁺]_i level. An intracellular calcium-chelating compound BAPTA-AM (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester) inhibited an increase in ROS production and cell injury induced by cisplatin. Furthermore, BAPTA-AM suppressed the rise of [Ca²⁺]_i level in 1 h after exposure; however, an extracellular calcium chelator EGTA and a calcium antagonist nicardipine did not inhibit the rise in [Ca²⁺]_i level in the early phase. An NADPH oxidase inhibitor inhibited a cisplatin-related increase in ROS production and cell disorder. These results suggest that cisplatin-related calcium release from the site of intracellular calcium storage in the early phase causes oxidative stress in renal tubular epithelial cells. Cisplatin may increase the intracellular production of ROS via NADPH oxidase.

View full abstract

The expressions of the immediate early genes, c-fos and c-jun, and their product proteins C-FOS, C-JUN, and P-JUN were examined in the hippocampal CA1 subfield after global ischemia and reperfusion in rats treated with cyclosporin A. More than 90% neuronal cell death was seen in hippocampal CA1 7 days after global ischemia in control animals, but only 5% cell death after ischemia was seen in the CsA-treated animals. The expressions of c-fos and c-jun mRNA in the control animals were detected with an increase from 1 to 48 h after ischemia. On the other hand, they showed significant suppression in the CsA-treated animals. Increased expressions of C-FOS were found 1, 24, and 48 h after reperfusion in the control animals. In the CsA-treated animals C-FOS expression was found to increase, but the expression level reduced to a statistically insignificant level within 48 h after the ischemia. C-JUN and P-JUN expressions increased in control animals, but were almost completely suppressed in the CsA-treated animals. The present study demonstrated that the suppressant effects of CsA on IEGs and their products might have causal relationship to the dramatic protecting effect of the drug against delayed neuronal cell death.

View full abstract

Lysophosphatidic acid (LPA), the simplest of the water-soluble phospholipids, can evoke various biological responses. The present study examined the activity of LPA to induce plasma exudation and histamine release in mice. Plasma exudation was assessed by extravasation of Evans blue. Subcutaneous administration of LPA (1 – 100 μg/site) led to increased plasma exudation in the skin. The LPA-induced plasma exudation was inhibited by ketotifen, a histamine H₁-receptor antagonist, and diacylglycerol pyrophosphate (DGPP), a LPA₁/LPA₃-receptor antagonist. Moreover, pretreatment with pertussis toxin and DGPP inhibited the histamine release from peritoneal mast cells induced by LPA. These findings indicate that plasma exudation induced by LPA is mediated by histamine release from mast cells via LPA receptor(s), presumably LPA₁ and/or LPA₃, coupled to G_i/o proteins. Moreover, these findings point to a role of LPA in the pathomechanisms of various allergic disorders.

View full abstract

Extents of the sparfloxacin (3 – 10 mg/kg, i.v.)-induced QT interval prolongation under normokalemic and hypokalemic conditions were assessed in halothane-anesthetized beagle dogs (n = 5). The hypokalemic condition was induced by an oral administration of furosemide (200 mg/kg per day) for 3 days, which decreased the serum potassium concentration from 3.65 ± 0.13 to 2.35 ± 0.13 mM (P<0.05). However, the decrease of potassium concentration by itself did not affect the extent of the sparfloxacin-induced QT interval prolongation. These results indicate that acute hypokalemia may not severely sensitize the in situ heart for drug-induced long QT syndrome as previously thought.

View full abstract