Magnesium lithospermate B improves renal hemodynamics and reduces renal oxygen consumption in 5/6th renal ablation/infarction rats
Magnesium lithospermate B (MLB) can promote renal microcirculation. The aim of the current project was to study whether MLB improves renal hemodynamics, oxygen consumption and subsequently attenuates hypoxia in rats induced by 5/6th renal Ablation/Infarction(A/I).
Chronic renal failure (CRF) was induced in male SD rats by the 5/6 (A/I) surgery. 30 rats were randomly divided into three groups: sham group, 5/6 (A/I) + vehicle group (CRF group) and 5/6 (A/I) + MLB (CRF + MLB) group. 28 days after the surgery, rats were given with saline or 100 mg/kg MLB by i.p. injection for 8 weeks. The 24-h urinary protein (24hUp), serum creatinine (Scr), blood urine nitrogen (BUN), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured. The protein expression of Fibronectin (FN), Collagen-I (Col-I), Connective Tissue Growth Factor(CTGF) and Interleukin-6 (IL-6) were measured by Western blot. Renal blood flow (RBF) and renal O2 consumption (QO2) indicated as sodium reabsorption (QO2/TNa) were detected before sacrifice. Renal hypoxia was assessed by measuring the protein expression of nNOS, HIF-1α and VEGF.
MLB significantly reduced 24hUp, Scr, BUN, SBP and DBP levels in rats with CRF. The expression of FN, Col-I, CTGF and IL-6 were down-regulated by MLB treatment in rats with CRF. In comparison to sham operated rats, 5/6 (A/I) rats had significantly lower RBF, and MLB significantly increased RBF in rats with CRF. Moreover, QO2/TNa was higher in the CRF group as compared to that in the sham group, and it was significantly attenuated in the CRF + MLB group. MLB reversed the expression of nNOS (neuronal nitric oxide synthase), HIF-1α (hypoxia inducible factor-1) and VEGF in rats with CRF.
MLB improves renal function, fibrosis and inflammation in CRF rats induced by 5/6 (A/I), which is probably related to the increase in RBF, reduction of oxygen consumption and attenuation of renal hypoxia in the remnant kidney with CRF.
KeywordsMagnesium lithospermate B Renal blood flow Renal oxygen consumption Hypoxia CRF
- 5/6 (A/I)
5/6th renal Ablation/Infarction
Blood urine nitrogen
Chronic kidney disease
Chronic renal failure
Connective Tissue Growth Factor
Diastolic Blood Pressure
Glomerular filtration rate
Hypoxia inducible factor-1
- Male Sprague-Dawley rats
Magnesium Lithospermate B
Neuronal nitric oxide synthase
Renal blood flow
Systolic Blood Pressure
total arterial blood hemoglobin
Sodium excreted in the urine
Vascular Endothelial Growth Factor
Chronic kidney disease (CKD) is defined as the glomerular filtration rate (GFR) < 60 mL/min per 1·73 m2 or signs of renal damage, or both, for at least 3 months duration . CKD causes a substantial economic burden, and according to the estimation of WHO the death rate associated with CKD will increase to 14 per 100,000 people in 2030 [1, 2]. Renal fibrosis and inflammation, representing the unsuccessful wound-healing of kidney tissues, are the final common pathological features of different types of chronic kidney disease .
Kidney is a hypoxia-sensitive organ, and hypoxia in the kidney can be caused by inadequacy of oxygen supply and increased consumption of oxygen [3, 4]. Based on the Brenner’s hyperfiltration theory, the tubulointerstitial damages in CKD impairs blood flow in the peritubular capillary and induces ischemic injuries which leads to renal function decline and loss of nephrons [3, 5].
Magnesium lithospermate B (MLB) is a nature product from the aqueous extracts of traditional Chinese herbal medicine, Salviae miltiorrhizae Bge. . MLB displayed renal protective effects in several rodent models of kidney disease. In CKD rats induced by 5/6thnephrectomy, MLB improved renal function as shown by decreased serum creatinine (Scr) and blood urea nitrogen (BUN) levels, which was correlated with reduced mesangial proliferation, tubulointerstitial lesions and glomerular sclerotic lesions [7, 8]. In streptozotocin-induced diabetic rats, MLB also displayed a reno-protective property as shown by reduced microalbuminuria, glomerular hypertrophy, and mesangial expansion. These beneficial effects of MLB in diabetic kidneys was associated with decreased expression of renal malondialdehyde (MDA), TGF-β1, fibronectin, and collagen . It was shown in another report that after 45 min treatment with MLB, renal cortical microperfusion was significantly increased in healthy rat kidneys . Moreover, MLB increased renal blood flow in adenine induced CKD rats . However, it is not known whether MLB improves renal hypoxia through ameliorating renal microcirculatory system in diseased kidneys.
In the current study, we aimed to study whether MLB improves renal hypoxia and protects renal function in 5/6 ablation/infarction (A/I) rats through ameliorating renal hemodynamics and attenuating renal oxygen consumption.
Male Sprague-Dawley (SD) rats (SPF grade) weighted between 190 and 210 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Animals were housed in the animal center of Shanghai University of Traditional Chinese Medicine according to local regulations and guidelines.
5/6thRenal ablation and infarction (a/I) surgery
Rats were anesthetized by sodium pentobarbital (20 mg/kg, i.p.), and placed on the temperature controlled surgical table. The renal artery of left kidney was exposed after a small flank incision. Two branches of the left renal artery were ligated with 4–0 silk sutures. The left kidney was then gently returned to the body and the incision was closed. After one week, a right flank incision was made, and the adrenal gland was separated from the right kidney. The right kidney was removed after the right renal pedicle was ligated. Control rats underwent the same anesthetic procedures and sham operation were performed on both side of kidneys. The rats were kept warm in an incubator until fully ambulatory.
The animal study protocol
30 rats were randomly divided into three groups: (I) sham group (n = 10), (II) 5/6 (A/I) + vehicle group (CRF group), and (III) 5/6 (A/I) + MLB (CRF + MLB) group. 28 days after the surgery, rats were treated with saline or 100 mg/kg MLB daily by intraperitoneally (i.p.) injection for 8 weeks. 24-h urine samples were collected one day before sacrifice. 0.8%Sodium pentobarbital was used as anesthesia before sacrificed by intraperitoneally (i.p.) injection. Blood samples and kidney tissues were collected after opening the abdominal cavity.
Animal experiments described herein were endorsed by the animal experimentation ethics committee of Shanghai University of Traditional Chinese Medicine.
Renal function and O2 consumption measurement
Serum creatinine (Scr), blood urea nitrogen (BUN) and 24-h urinary protein (24hUp) were detected by Automatic biochemical analyzer (AU680, Beckman Coulter).
Rats were anesthetized with sodium pentobarbital (20 mg/kg, i.p.) before the O2 consumption measurement. The left renal blood flow (RBF, ml/min) was monitored with a perivascular ultrasonic transit-time flow probe (Transonics T420, Ithaca, USA) which was connected to a computer for continuous recording. Proximal left renal vein was used for sampling of venous blood. Blood samples were taken from the femoral artery and renal vein for measurements of total arterial blood hemoglobin (tHb), (O2Hb), (pO2), (pCO2), pH, [Na+], [K+], [HCO3−] with the blood gas analyzer and biochemical multiple test cards (i-STAT EG7, U.S.A, Abbott). O2 content (O2ct) was calculated by the formula:O2ct (ml/ml blood) = (1.39 xtHbxO2Hb% + pO2x0.03)/100. The total left kidney O2 consumption (QO2, ml/min) was calculated from A-V difference in O2 content multiplied by RBF. TNa is equal to the total amount of sodium filtered (FNa) minus the amount of sodium excreted in the urine (UNaV). Systolic blood pressure was measured by the tail-cuff method. Cuff inflation/deflation rates and maximum cuff pressure were controlled by a programmed electro-sphygmomanometer Softron BP-2010A (Softron Biotechnology, Beijing, China). The calculated value is the mean of 3 to 5 recordings performed at the same time during seven days before sacrificed.
Masson’s trichrome and Immunohistochemical staining
Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin. Four-μm-thick sections of paraffin-embedded kidney tissue were subjected to immunohistochemical staining with anti-VEGF (1:1000, A0280, Abclonal) antibodies. Masson’s trichrome staining was performed using a standard protocol as described by Livingston et al. . Briefly, the tissue was stained with hematoxylin, and then with ponceau red liquid dye acid complex, which was followed by incubation with phosphomolybdic acid solution. Finally, the tissue was stained with aniline blue liquid and acetic acid. Images were obtained with the use of a microscope (Nikon 80i, Tokyo, Japan).
Western blotting analysis
Renal protein was extracted from the medulla and cortex of rat kidneys. The protein concentration was measured by the Bradford method, and the supernatant was denatured at 95 °C for 5 min in Laemmli sample buffer. Samples were subjected to SDS-PAGE gels. After electrophoresis, proteins were electro-transferred to a polyvinylidene difluoride membrane (Merck), which was incubated in the blocking buffer (5% non-fat milk, 20 mM Tris-HCl, 150mMNaCl, PH = 8.0, 0.01%Tween 20) for 1 h at room temperature and was followed by incubation with anti-fibronectin (1:1000, ab23750, Abcam) or anti-Collagen-I (1:1000, sc-293,182, Santa Cruz) or anti-CTGF (1:1000, sc-373,936, Santa Cruz) anti-nNOS (1:1000, 4236 s, CST) or anti-HIF-1α (1:1000, ab2185, Abcam) or anti-VEGF (1:1000,A0280, Abclonal) overnight at 4 °C. Binding of the primary antibody was detected by an enhanced chemiluminescence method (BeyoECL Star, P0018A, Byotime) using horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG,1:1000, Proteintech). The quantification of protein expression was performed using Quantity One Analyzer (Bio-Rad).
Results were presented as mean ± SE. Differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) and comparison between two groups was performed by paired Student t-test or unpaired student t-test. Using statistic software SPSS 18.0 (SPSS Inc., Chicago, IL). A P value of lower than 0.05 was considered statistically significant.
The renal ablation/infarction (A/I) model of chronic kidney disease (CKD) was established in male SD rats weighted 190–210 g, which were randomly divided into three groups: (I) sham operation, (II) 5/6 renal ablation/infarction (A/I) operation, (III) 5/6 A/I operation +Magnesium Lithospermate B (MLB). 28 days after the operation, rats were treated with vehicle or 100 mg/kg MLB by i.p. once daily for 8 weeks.
Renal function decline was retarded in the CRF rats with MLB treatment
Rats with 5/6 (A/I) operation had significantly higher levels of Blood urea nitrogen (BUN) than rats in the sham group at 4 weeks after the surgery (Fig. 1). The BUN levels were 14.11% (p < 0.05) lower in the 5/6 (A/I) + MLB group than in the 5/6 (A/I) model group after 8-weeks treatment with MLB (Fig. 1b).
As showed in Fig. 1c, the 5/6(A/I) operation significantly increased the 24-h urine protein excretion as compared with the sham operation in rats at 4 weeks and 12 weeks after operation. The 24-h urine protein excretion in the 5/6 (A/I) + MLB group was 30% lower than that in the 5/6 (A/I) model group after 8-weeks intervention with MLB.
SBP and DBP were significantly increased in rats at 4 weeks and 12 weeks after the 5/6 (A/I) operation(Fig. 1d and e).With MLB treatment, SBP and DBP were reduced by 11.16% (p < 0.05) and 9.8% (p < 0.05) respectively in CRF rats at 12 weeks after the operation.
MLB treatment attenuated renal fibrosis and inflammation in the CRF rats
To further determine the effect of MLB in the CRF rats, we measured the collagen deposition by Masson’s trichrome staining and the expression of several markers for renal fibrosis and inflammation.
Moreover, the expression of IL-6,an inflammation marker, was up-regulated in the 5/6 (A/I) model group as compared with that in the sham group (p < 0.05). The treatment with MLB for 8-weeks significantly down-regulated the expression of IL-6 in the rat kidney with 5/6 (A/I) operation (Fig. 2b).
Deterioration of the renal blood flow and remnant renal oxygen consumption (QO2 /TNa) was improved in the CRF rats with MLB treatment
Effects of MLB on the protein expression of nNOS, HIF-1α and VEGF in the remnant kidneys
In the current study, we showed that MLB improved renal function and attenuated renal fibrosis and inflammation in the 5/6 (A/I) rat model of CRF, which was correlated with the increase in renal blood flow and reduction in remnant renal oxygen consumption (QO2/TNa). Moreover, MLB reversed the expression of nNOS, HIF-1α and VEGF protein in the kidney of 5/6 (A/I) rats.
The kidney is one of the major organs with well-supplied oxygenated blood, and it is vulnerable to ischemic insults leading to impaired renal function .The ischemic conditions can be induced by acute kidney injuries and by damages associated with chronic kidney diseases . Hypoxia, resulting from decreased blood flow or increased oxygen consumption, affects the expression of a wide array of genes, including many fibrogenic factors, leading to the progressive loss of renal function [4, 14, 15].MLB exerts beneficial effects in several rodent models of chronic kidney disease, and the protective mechanism of MLB in the kidney is related to its anti-oxidative and anti-fibrotic potentials [7, 8, 9]. It has been shown that MLB promoted renal circulatory state in healthy rat kidneys and adenine induced CKD kidneys [11, 15]. We therefore hypothesized that MLB protects renal function in chronic kidney disease through improving renal hemodynamics and subsequently attenuating hypoxia in 5/6 kidneys. In our study, we firstly showed that MLB attenuated renal function decline, renal fibrosis and inflammation in the 5/6 (A/I) rat model of renal failure. Secondly, we showed that MLB significantly increased renal blood flow. To further confirm that the renal hypoxia was reduced by MLB, we measured the expression of HIF-1α, the hypoxia marker, in the kidney tissues . We found that MLB reduced the expression of HIF-1αin 5/6 rat kidneys suggesting that renal hypoxia was attenuated by MLB. Moreover, we measured the expression of VEGF which is a downstream target of HIF-1α in 5/6 and the renal expression of VEGF is tightly regulated by hypoxia  Our study showed that the renal expression of VEGF in CRF rats was reduced by the MLB treatment. Interestingly, in the present study, we found that MLB reduced renal oxygen consumption in 5/6 (A/I) rats. We further measure the expression of nNOS, a negative regulator of oxygen utilization in mitochondria, and we found that MLB restored the expression of nNOS in 5/6 (A/I) rat kidneys .
In conclusion, MLB is renal protective and attenuates renal hypoxia in 5/6 (A/I) rats, and the mechanism is probably caused by improving renal hemodynamics and attenuating renal oxygen consumption.
This work was supported by National Natural Science Foundation of China General Projects(81573946), Scientific Research Foundation of Science and Technology Commission of Shanghai Municipal Government (16401931700) and Scientific Research Foundation of Shanghai Municipal Commission of Health and Family Planning (201540199)to CW, Scientific Research Foundation of Shanghai Municipal Commission of Health and Family Planning(201740193) to MW, Key Disciplines Group Construction Project of Pudong Health Bureau of Shanghai (PWZxq2017–07) to CY, and Science Foundation of Shuguang Hospital Affiliated to Shanghai University of TCM (SGKJ-201712) to PL.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
CW conceived and coordinated the study. PL, MW, CY and CW wrote the paper. PL, MW, JQ, JY designed, performed and analyzed the animal experiments. PL, JQ, JY performed and analyzed the Western blotting. All authors reviewed the results and approved the final version of the manuscript.
Ethics approval and consent to participate
Not applicable of human participate. Animals experiment were kept under local guidelines and endorsed by the animal experimentation ethics compliance of Shanghai University of Traditional Chinese Medicine.
Consent for publication
All authors reviewed the results and approved the final version of the manuscript, they have no conflicts of interest with the contents of this article.
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