"Salty Flavor Entering The Kidney" Theory And Related Experimental Study On The Treatment Of Salt Sensitive Hypertension From Kidney

Objective1. To establish rat model of high salt and high fat diet induced salt sensitive hypertension (SSHT); To observe the Renin-Angiotensin system (RAS) activity as well as insulin resistance (IR) in SSHT model rats.2. To investigate the effects of Chinese herbal medicine on reducing blood pressure, inhibiting RAS activity, improve kidney metabolism and kidney function in SSHT rats.3. To investigate the effects of Chinese herbal medicine on ameliorating IR and other aspects of SSHT rats, such as inflammatory reaction, procoagulant and etc.4. To investigate possible mechanism of Chinese herbal medicine from the point of view of RAS and IR; To pave new way to the treatment of SSHT; To scientifically elucidate the theory of "salty flavor entering the kidney".Method1. Establishing rat model of high salt and high fat diet induced salt sensitive hypertension.30male SD rat,6weeks old and acclimate for about10days before study. Randomly divided into4groups:Control group (CON, n=5); High salt group (HS, n=5); High fat group (HF, n=5); High salt and fat group (HSF, n=15). All rats are allowed access to drinking water and subjected to different dietary regimens as described below. CON-normal pellet diet; HS-high salt diet; HF-high fat diet; HSF-high salt and high fat diet. Measure blood pressure weekly by tail-cuff method. Two weeks later, rats are overnight fasting (12h) and blood was collected from retro-orbital vein, followed by glucose, insulin, blood fat, Plasma renin activity (PRA), Angiotensin Ⅱ (Ang Ⅱ), ACE (Angiotensin-converting enzyme) measurement. HOMA-IR and ISI are calculated accordingly.24h urine is collected by metabolic cages. Urine Na+and urine K+, Aldosterone (ALD), Microalbuminuria (MAU) are measured and excretion rate are calculated accordingly.2. To investigate the effects of Chinese herbal medicine on inhibiting RAS activity, improve kidney metabolism and kidney function in SSHT rats.Grouping:two weeks after diet intervention, HSF rats are randomly divided into three groups, five rats in each group and together4groups as the following:HSF group (non-diet intervention); Valsartan (VAL) group; Liu-Wei-Di-Huang-Fang (LW) group and the control groups. Doing regime is as follows:VAL,13.33mg/kg/d, i.g. QD; LW,8.13g/kg/d, i.g. QD. Both drugs are treated for6weeks. HSF diet are provided to all HSF rats during the treatment except control group with the normal diet. Body weight and blood pressure by tail-cuff method are measured weekly. Six weeks later, rats are overnight fasting (12h) and blood was collected, followed by PRA, AngⅡ, ACE measurement.24h urine are collected by metabolic cages. Urine volume, Na+, K+, ALD, MAU are measured and excretion rate of MAU, Urine Na+and K+are calculated accordingly. All rats are sacrificed at the end of study and kidney are kept at-80℃for further analysis. RT-PCR is carried for determining renin, Ang II receptor type I (AT1R), AngⅡ mRNA expression level in kidney. And HE staining was used for histopathology analysis.3. To investigate the effects of Chinese herbal medicine on ameliorating IR, inflammatory reaction and procoagulant activity.SSHT model establishment and grouping is as described above. After dietary change for8weeks (drug treated for6weeks), blood are collected after overnight fasting (12h), followed by biochemical analysis of glucose and blood fat (TC, TG, LDL-c, HDL-c). ELISA was performed to determine insulin, Leptin (LEP), Adiponectin (ADP), Tissue plasminogen activator (t-PA), Plasminogen activator inhibitor1(PAI-1), C-reactive protein (CRP), Tumor necrosis factor-a (TNF-a), Interleukin-6(IL-6) and Free fatty acid (FFA) in blood. HOMA-IR and ISI are calculated accordingly.Results1. Establish rat model of high salt and high fat induced SSHT.Compared with CON group, the blood pressure of all other groups increased significantly after2weeks of dietary change, among which HSF group shows highest blood pressure (119.4±4.7vs.94.0±2.2mmHg, p<0.05); In HSF group, AngⅡ level in the blood was significantly reduced however (66.39±3.62vs.82.16±14.68ng/L, p<0.05), there is no significant change of RAS indicator in other groups. Regarding to urine electrolyte,24h urine Na+excretion in HSF group was significantly increased.24h urine K+excretion (1.49±0.23vs.0.98±0.28mmol/L, p<0.05) in HSF group is very similar to urine Na+excretion (1.92±0.48vs.0.75±0.18mmol/L, p<0.05). The ratio of urine K+/urine Na+in HSF group is lower than CON group (0.80±0.16vs.1.35±0.49,p<0.05). UMAU excretion as another sensitive marker of early kidney injury in HSF group is also higher than CON group (22.16±4.01vs.6.66±1.17ug/L, p<0.05). In insulin sensitivity, compared with the CON group, all the other group shows obvious increase in fasting blood glucose after2weeks’ diet intervention on high salt or/and high fat and HSF group shows the highest increase (3.66±0.39vs.2.60±0.52mmol/L, p<0.05). Also HSF group shows obvious increase in HOMA-IR (p=0.000) and decreased in ISI (p=0.001). All the above data like blood pressure, RAS activity, kidney function and insulin sensitivity shows that we successfully establish rat model of SSHT induced by HSF, which can be used in the following pharmacology evaluation of Chinese herbal medicine.2. To investigate the effects of Chinese herbal medicine on reducing blood pressure, inhibiting RAS activity, improve kidney metabolism and kidney function in SSHT rats.After one week treatment, compared with HSF group, VAL shows greater decrease in blood pressure (89.2±4.8vs.117.6±9.3mmHg, p<0.05) and after two weeks treatment, LW shows effect in decreasing blood pressure (105.2±8.8vs.118.6±5.3mmHg,p<0.05); After6weeks treatment, blood pressure returned to the normal level after LW treatment (103.0±8.1vs.118.4±6.3mmHg,p<0.05).Regarding to circulating RAS, after2weeks’diet, compared with CON group, HSF group shows obvious decrease in Ang Ⅱ (66.39±3.62vs.82.16±14.68ng/L, p<0.05) and on the eighth week on diet intervention, Ang Ⅱ has come back to base line. However, there’s no significance difference in ACE, PRA and others RAS indicators. After2weeks’diet, compared with CON group, HSF group shows obvious rise in UALD (135.02±9.93vs.101.27±13.30ng/L, p<0.05) and after6weeks treatment, both of LW and VAL can bring down UALD to the base line.Regarding to local kidney RAS, after8weeks diet intervention on high salt and high fat, compared to CON group, HSF group shows obvious mRNA expression on Ang II, rennin and AT1R. After6week drug treatment, compared with HSF group, LW and VAL show the equal effect on inhibiting kidney RAS activation.Concerning kidney function, after8week diet intervention on high salt and high fat, compared with CON group,24h urine Na+(p=0.007) and urine K+(p=0.017) in HSF group is significantly increased, while the ratio urine Na+/urine K+is relatively low, which indicates the retention of Na+when having diet intervention on high Na+treatment. After6week drug treatment, compared with HSF group, LW and VAL can have equal effect on promoting excretion of urine Na+and decreasing the loss of urine K+. The MAU data shows that, after6weeks drug treatment, compared with HSF group, LW and VAL can obvious decrease MAU level. HE results show that, HSF group shows slight hyaline degeneration in glomerular afferent arteriole and there is no significant change in glomerular afferent arteriole, arterioles and each kidney unit. Another two kidney injury indexes, Serum Creatinine and Urea Nitrogen were not seen significant change among the groups and indicate our rat model is created to cause blood pressure rise and slight kidney function injuries, lesions of which is similar to the actual clinical patients.3. To investigate the effects of Chinese herbal medicine on ameliorating IR and other aspects of SSHT rats.After8weeks of diet intervention, blood glucose and insulin level are continuing rising in HSF group, which shows obvious difference compared with CON group (5.55±0.62mmol/L vs.2.87±0.77mmol/L, p<0.05and4.92±1.34mIU/L vs.2.79±0.26mIU/L, p<0.05), but no significance difference for TC、TG、LDL-c; After6weeks drug treatment, compared with HSF group, blood glucose (p=0.000) and insulin level (p=0.005) are back to base line in VAL and blood glucose (p=0.000) is significantly deceased in LW. Regarding ISI and HOMA-IR results, both of LW and VAL can improve insulin sensitivity and IR (p<0.05).Regarding adipose cell cytokine, after6weeks treatment, obvious increase in LEP (p=0.047) and PAI-1(p=0.031) and decrease in t-PA (p=0.037) in HSF group compared to the CON group; there’s no significant difference between CRP、TNF-α、 IL-6、ADP、FFA in each group. After6week drug treatment, compared with HSF group, LW and VAL have equal effect on significantly decreasing LEP and PAI-1and rise t-PA back to base line.Conclusion1. HSF diet can be used to induce reliable rat model of SSHT. It is easy to carry out and mimic modern dietary habit, which can be used to improve anti-SSHT drug as well as mechanism study.2. LW can significantly reduce blood pressure of SSHT model rats which is as good as VAL; inhibit local RAS activation vigorously; reduce urine ALD level; LW can also improve the high urine Na+excretion and urine K+loss. LW has effect on UMAU too.3. LW shows significant effect on improving IR, deducing blood glucose, insulin and HOMA-IR; It has good effect on the cytokine release and fibrinolytic function, too.In summary, HSF diet can induce reliable rat model of SSHT. LW can be used for the treatment of SSHT. It has obvious effect on the reducing blood pressure, inhibit renal RAS activation and improving IR.The underlying mechanism is probably related to RAS antagonism, improve kidney function, improve the glucose metabolism and fibrinolytic function etc, which reflects the multiple factors and target of Chinese herbal medicine. It provides reliable evidence to the treatment of SSHT from the point of view of "Salty flavor entering the kidney".