Magnesium lithospermate B ameliorates renal cortical microperfusion in rats¹

Introduction

Magnesium lithospermate B (MLB) is a tetramer of caffeic acid. It was recently isolated from a plant used in Chinese herbal medicine, Salviae miltiorrhizae, and has been found to improve renal function and ameliorate experimental renal failure. The effect of MLB on renal function was first reported by Yokozawa et al^[1,2], who studied these effects in several animal models of renal failure and suggested that MLB increased renal function by improving the renal circulatory state through activation of kallikrein, promotion of prostaglandin E2 production, and scavenging radicals in rats with renal failure^[3–13]. Other researchers have confirmed the renoprotective property and free radical scavenging effect of MLB by using different animal models. Lee et al found that MLB suppressed the progression of renal injury in streptozotocin-induced diabetic rats, and inhibited reactive oxygen species generation that leads to protein kinase C activation and transforming growth factor (TGF)-1/fibronectin upregulation in mesangial cells cultured in high glucose conditions^[14]. Kang et al demonstrated the strong inhibitive effect of MLB against the production of superoxide, hydrogen peroxide, and hydroxyl radicals, the three most common oxygen radicals, and suggested that MLB ameliorated renal defects in rats with ischemia-reperfusion-induced acute renal failure via scavenging of reactive oxygen species^[15]. Wu et al reported that MLB was an inhibitor of lipid peroxidation and scavenged superoxide anions and hydroxyl radicals both in vitro and ex vivo^[16]. Soung et al demonstrated that MLB with a hydroxyl group and double bonds exerted an anti-nitration effect by scavenging peroxynitrite^[17].

Although the renoprotective effects of MLB have been primarily attributed to scavenging oxygen free radicals, other protective mechanisms may play a role as well. Taking the importance of renal microcirculation in maintaining normal renal function into account, we hypothesized that MLB might ameliorate renal microcirculation, which is a more direct way to improve renal function. The purpose of the present study, therefore, was to describe the hemodynamic action of MLB with respect to the specificity of renal hemodynamics and microcirculation in rats.

Materials and methods

Drugs MLB was provided by the Department of Phytochemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The purity of this compound was above 99.8%, which was verified by the supplier using high performance liquid chromatography methods.

Hemodynamics and renal microcirculation Studies of hemodynamics and renal circulation were carried out in 8 healthy 9−10-week-old male Sprague-Dawley rats weighing 292±6 g, which were purchased from the Shanghai SLAC Laboratory Animal Co (Certificate No SCXK 2003-0003, Shanghai, China). The rats were anesthetized with urethane (1.1 g/kg, ip) and placed on a heated operation table to maintain body temperature. The right femoral artery was exposed and an arterial catheter (PE30) was inserted into it to measure arterial blood pressure with a pressure transducer (Transpac, North Chicago, IL, USA). Left ventricle catheterization was performed to monitor systemic hemodynamic function parameters including the heart rate (HR), the left ventricular systolic pressure (LVSP), the left ventricular end-diastolic pressure (LVEDP), and the maximal velocity of pressure increase (±dp/dt_max) with another pressure transducer (P23XL, Statham; Nihon Kohden, Tokyo, Japan) connected to the Polygraph System RM-6000 (Nihon Kohden) and the MacLab data acquisition program (MacLab/8S; Analog Digital Instruments, Castle Hill, NSW, Australia). The left kidney was then exposed through a midline incision. A perivascular transonic ultrasonic transmit-time flow probe (1RB; Transonic Systems, Ithaca, NY, USA) was mounted on the left renal artery for measurement of renal blood flow, with the signals transmitted to a transmit-time flow meter (T206; Transonic Systems). A blunt superficial laser-Doppler probe (MLD-1; Nankai University, Tianjin, China) was placed on the kidney surface, and mounted on micromanipulators (Narishige Scientific Instrument Laboratory, Tokyo, Japan) so that movement artifacts were avoided. The probe was connected to a laser-Doppler flowmeter (LDM; Nankai University) to measure renal cortical microperfusion. Details of the validation of the transit time laser-Doppler method are given elsewhere^[18,19].

After surgery, the animals were allowed to recover for 30 min. Then, animals were injected intravenously with vehicle control (saline), and MLB at doses of 10, 30, and 60 mg/kg consecutively, with 45 min between injections.

Mean values for each determination were analyzed over a 0.5-min to 1-min period. Renal vascular resistance was calculated from the mean arterial pressure and the corresponding renal blood flow.

Statistical analysis Data were given as mean±SD, from 8 animals in each group. The statistical significance of differences in the hemodynamic parameters was assessed using one-way analysis of variance (ANOVA). Student’s t-test was used for comparison of the parameters with their baseline values. Statistical significance was set at P<0.05.

Results

Effects on renal microcirculation and hemodynamics There was no difference between groups with respect to the baseline values of renal hemodynamic and microcirculation parameters (Figure 1). Neither vehicle nor MLB administration had any significant effect on renal blood flow or renal vascular resistance. In contrast, MLB administration increased renal cortical microperfusion significantly, whereas the vehicle had no effect (Figure 1, Tables 1, 2). This microcirculation improving effect reached its peak 15 min after injection and returned to baseline after 45 min. Fifteen minutes after injection with 10, 30, or 60 mg/kg MLB, renal cortical microperfusion increased by 38.7%±27.3%, 51.4%±22.2%, and 62.4%±20.2%, respectively (changes relative to baseline; P<0.01), whereas renal blood flow (2.3%±19.9%, 1.6%±20.4%, 3.7%±9.7%, respectively) and renal vascular resistance (-8.4%±13.8%, -1.6%±12.4%, -1.4%±9.1%, respectively) did not change significantly.

Figure 1 Renal hemodynamic effects of magnesium lithospermate B (MLB) in rats. Rats were injected consecutively with one vehicle control and three doses of MLB with 45 min between injections. (A) Renal cortical microperfusion (RCM). (B) Renal blood flow (RBF) and renal vascular resistance (RVR). n=8. Mean±SD. ^bP<0.05, ^cP<0.01 vs baseline, paired Student’s t-test. ^eP<0.05, ^fP<0.01 vs vehicle control, one-way ANOVA.

Table 1 Effects of MLB on renal cortical microperfusion (unit: BPU) in rats. n=8. Mean±SD. ^bP<0.05, ^cP<0.01 vs baseline. ^eP<0.05, ^fP<0.01 vs vehicle control.
Full table

Table 2 Effects of MLB on renal hemodynamics 15 min after MLB iv administration in rats. n=8. Mean±SD. ^cP<0.01 vs baseline. ^eP<0.05, ^fP<0.01 vs vehicle control.
Full table

Effects on systemic hemodynamics There was no difference between groups with respect to the baseline values of systemic hemodynamic parameters (Figure 2). Vehicle administration had no effect on these parameters. Although MLB administration had some effect on these parameters at some time points, this effect was neither time-dependent nor dose-dependent. Fifteen minutes after injection with MLB 60 mg/kg, when the effect of MLB on renal cortical micro-perfusion had reached its peak, mean arterial pressure (1.9%±10.2% vs baseline), heart rate (0.2%±5.4% vs baseline), LVSP (0.4%±7.8% vs baseline), LVEDP (7.8%±33.4% vs baseline) and +dp/dt_max (3.0%±6.8% vs baseline) had not changed in a statistically significant way, whereas -dp/dt_max increased slightly (5.7%±6.2%; P<0.05 vs baseline).

Figure 2 Systemic hemodynamic effects of MLB in rats. Rats were injected consecutively with one vehicle control and three doses of MLB with 45 min between injections. (A) Heart rate (HR); (B) Mean arterial pressure (MAP); (C) Left ventricular systolic pressure (LVSP); (D) Left ventricular end-diastolic pressure (LVEDP); (E) +dp/dt_max; (F) -dp/dt_max. n=8. Mean±SD. ^bP<0.05, ^cP<0.01 vs baseline, paired Student’s t-test. ^eP<0.05, ^fP<0.01 vs vehicle control, one-way ANOVA.

Discussion

The kidneys play a central role in the regulation of the body’s salt and water balance. A highly regulated microcirculatory and interstitial environment is essential for optimum function of the kidneys. Although the renal function improving property of MLB has been studied extensively, there has been no report concerning its effect on renal microcirculation. We demonstrated here, to our knowledge for the first time, that MLB could ameliorate renal microcirculation while causing no other significant changes to hemo-dynamics.

In our study, the effect of intravenously administered MLB on renal cortical microperfusion was dose-dependent, and reverted back to the baseline level 45 min after MLB administration. Li et al reported the pharmacokinetic parameters of MLB after iv administration in 6 beagle dogs, and showed that MLB was distributed and eliminated quickly^[21]. The mean T_1/2β values for MLB at doses of 3 mg/kg, 6 mg/kg, and 12 mg/kg were 43±9 min, 42±7 min, and 42±10 min, respectively. Therefore, the time-response curve of MLB was correlated with its serum concentration-time profiles.

This effect of MLB on renal circulation is consistent with previous studies. However, some differences exist. Yokozawa et al reported that MLB increased renal blood flow^[20], which we did not find in our study. The different animal models and techniques we used may account for this difference. Yokozawa et al used rats with renal failure, which had significantly lower renal blood flow than normal rats, whereas we used normal Sprague-Dawley rats with normal renal blood flow. The techniques we used to measure renal blood flow were also different. Yokozawa et al used a needle-type bipolar electrode electrolytic organ rheometer, whereas we used a transit-time ultrasonic-Doppler flow meter with a much higher precision (±5%).

The effect of MLB on renal microcirculation may be attributed to several factors. The primary contributor may be its potent antioxidant properties. Recent studies suggest that free radicals may play a key role in regulating renal microvascular tone. Although studies investigating the roles of oxygen radicals in the physiological regulation of renal microcirculation have only recently begun, it is evident that oxygen radicals have important direct and indirect actions in both cortical and medullary microcirculation^[22–25]. Because O₂^– and NO both contain unpaired electrons in their outer orbits, they undergo extremely rapid, diffusion-limited radical-radical reactions, leading to the formation of peroxynitrite anions (ONOO^–), strong oxidants that could prompt the generation of hydroxyl radicals (OH^–). In the renal microva-sculature, free radicals can cause vasoconstriction, mediate the vasoconstriction of other agonists, and modulate the action of vasodilators (inactivate nitric oxide and blunt endothelium-dependent vasodilation)^[26–28]. These findings have led to the idea that antioxidants might be used therapeutically as part of a nephroprotective strategy^[29–31]. Our current study supports this idea: MLB was proven to be a potent inhibitor of the production of superoxides, hydrogen peroxide, and hydroxyl radicals, the three most common oxygen radicals in the renal microvasculature. Here we propose that MLB, as a potent antioxidant, scavenges free radicals, blocks the O₂^–_ONOO^–_OH^– cascade, promotes NO bioavailability and thus ameliorates renal microcirculation.

Some other factors may play a role too. MLB has been reported to improve the renal circulatory state through activation of kallikrein and promotion of prostaglandin E₂ production^[7,9,13]. Tissue kallikrein cleaves the kininogen substrate to release the vasoactive peptide kinin, which binds to endothelial bradykinin B₂ receptors and stimulates the release of potent vasodilators, including prostacyclin, nitric oxide, and endothelium-derived hyperpolarizing factor^[32,33]. The paracrine agent PGE₂ is the predominant cyclooxygenase metabolite of arachidonic acid in the kidney^[34]. PGE₂ plays an important role in tubular reabsorption of salt and water as well as in the control of renal vascular resistance and the maintenance of glomerular hemodynamics. Despite several reports of PGE₂-induced vasoconstriction^[35,36], there is convincing evidence that PGE₂ acts primarily on the preglomerular vasculature to counteract the effects of the vasoconstricting hormones and protect the kidney from excessive vasoconstriction^[37–41]. Although the effect of MLB on cyclooxygenase has not been studied, it is reported to be a potent inhibitor of 5-lipoxygenase, and such inhibition of lipoxygenase causes a shift of arachidonic acid from the lipoxygenase to the cyclooxygenase pathway, which is thought to result in increased formation of cyclooxygenase metabolites^[3]. Indeed, Yokozawa et al reported that MLB increased urinary excretion of prostaglandin E₂ (PGE₂) and 6-keto-PGF_1α, while thromboxane B2 (TXB₂) remained unchanged or decreased in rats with renal failure^[3]. Activation of kallikrein, promotion of PGE₂ production, and scavenging of radicals could act simultaneously to increase the bioavailability of NO and prostacyclin, the major vasodilators in the kidney. These three effects are suggested to be the major contributors to increased renal microcirculation after MLB administration. Nonetheless, the exact mechanism by which MLB ameliorates renal cortical microperfusion is not completely clear, and should be evaluated further.

In conclusion, the major finding of this study is that intravenously administered MLB dose-dependently ameliorates renal microcirculation. This finding suggests that the renal protective properties of MLB may be mediated in part by vasodilation of the renal microvasculature.

References

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