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Traditional Chinese medicine believes that "kidney main water", kidney qi is the main division and regulates the water metabolism of the whole body. In patients with renal yang deficiency, the functions of transpiration, gasification, solidification, and pushing of the kidney are hindered, which affects the transport and transfer of jin liquid, mainly manifested as waist and knee soreness, long urine, frequent nocturia and other symptoms. Giving warm kidney drugs can improve the above symptoms well.
Kidney Qi Pill is a clinically effective representative prescription for warming kidney yang, mainly treating waist and knee soreness, renal deficiency edema, kidney qi deficiency, etc. Clinically, it is mostly used in patients with chronic kidney disease (CKD) such as chronic kidney disease.
In the development of CKD, peritubular capillary damage generally occurs. Angiogenesis of endothelial cells is of great significance in improving the process of kidney injury.
Aquaporin (AQP) is a group of cellular transmembrane proteins that are of great significance in maintaining the balance of water metabolism.
In the kidney, AQP1 mediates the migration of renal vascular endothelial cells after kidney injury, promotes angiogenesis and vascular damage repair, and is conducive to the recovery of renal function. In the previous study, our project team found that the level of renal AQP1 in rats with renal yang deficiency was significantly reduced, while kidney qi pills could improve the general symptoms and signs of kidney yang deficiency rats and increase the expression of kidney AQP1.
However, whether kidney qi pills improve kidney damage by promoting endothelial cell angiogenesis remains to be studied. Objective To observe the effect of renal qi pills on peritubular capillaries and the effect on human microblood tube endothelial cells in rats with adenine-induced renal yang deficiency, and to explore whether renal qi pills promote human microvascularity by regulating AQP1
Improves the condition of peritubular capillary damage. To guide the clinical treatment of renal deficiency and related diseases, it provides new ideas. Methods In animal experiments, 30 SD rats were randomly divided into normal control group, adenine group, and kidney qi pill (1.5g/kg, 3.0g/kg, 6.0g/kg) group, 6 rats each.
Rats in the adenine group were given 150mg/kgY adenine for 35 days continuously. In the renal qi pill group, kidney qi pills (1.5g/kgŸd, 3.0g/kgŸd, 6.0g/kgŸd) were given by gastric gavage from the 15th day for 21 days. The normal control group was given the same amount of normal saline.
1h after the last dose, anesthesia, blood is taken, and the heart, liver, spleen, lung, kidney, and adrenal glands are rapidly separated. Renal histopathological changes were observed by HE staining; The distribution and expression of CD34, VEGF and AQP1 proteins in rat kidney tissue were observed by immunohistochemistry.
ELISA method to detect hypothalamic CRH, blood clear content of ACTH, CORT, VCAM-1, VEGF and urine 24U-TP; Automatic biochemical detector method to detect serum BUN, Scr and urine 17-OHCS content; qPCR to detect AQP1mRNA levels in kidney tissue;
Western blot method to detect MCP-1 and AQP1 protein expression in kidney tissue. Cell experiments HMEC-1 cells were treated with different concentrations (2.5%, 5%, 10%) of renal qi pill-containing serum, and the effects of renal qi pill-containing serum on cell morphology, cell proliferation and AQP1 protein expression were observed by II microscopic observation, MTS method and qPCR method.
The effects of HMEC-1 cell proliferation, cell migration, lumen formation ability, AQP1 mRNA level and protein expression by MTS method, cell scratch method, in vitro lumen formation experiment, qPCR and Western blot method were used as control by normal rat serum.
AQP1 RNAi lentivirus infection with HMEC-1 cells detected AQP1 mRNA levels, protein expression, cell migration, lumen formation ability and intervention effect of renal qi pills containing medicated blood clearance.
Results Animal experiments 1. Compared with the normal control group, the serum BUN, urine 24h U-TP and Scr contents of rats in the adenine group were significantly increased (p<0.01). Creatinine clearance was significantly reduced (p<0.01); The contents of ACTH, CORT and 17-OHCS decreased to varying degrees (p<0.01);
The internal structure of the glomerulus collapses, the tubules expand, deposits are visible in the lumen, and the tubules are blocked. Compared with the adenine group, different doses of renal qi pills could significantly reduce BUN, urine 24h U-TP, and Scr content (p<0.01). Increase the content of CRH, ACTH, CORT (P<0.01) AND 17-OHCS (p<0.01, p<0.01, p<0.05).
And 3.0g/kg renal qi pill can significantly improve creatinine clearance (p<0.01). Compared with the normal control group, the peritubular capillary density of rats in the adenine group was significantly reduced (p<0.01).
VCAM-1 levels were significantly increased (p<0.01) and VEGF levels were significantly reduced (p<0.05). MCP-1 protein expression was significantly elevated (p<0.01). Compared with the adenine group, different doses of renal qi pills could significantly increase the level of VEGF (p<0.05, p<0.05, p<0.01), reduce the level of VCAM-1 (p<0.01) and the expression of MCP-1 protein (p<0.01).
Clearance of 3.00g/kg renal qi pills significantly increased the density of peritubular capillaries in rats (p<0.01). 3. Compared with the normal control group, the expression of AQP1 in kidney tissue of rats in the adenine group was significantly reduced (p<0.05, p<0.01, p<0.01).
Different doses of renal qi pills can regulate AQP1mRNA levels and protein expression to varying degrees. Cell Experiment 1. Compared with the 10% fetal bovine serum group, 5% renal qi pill-containing serum could not only maintain the normal shape of cells, but also have a significant proliferative effect on HMEC-1 cells (p<0.05, p<0.01).
Significantly upregulated cellular AQP1 mRNA levels (p<0.01). Therefore, 5% renal qi pill-containing serum was selected as the optimal renal qi pill-containing serum concentration for follow-up experiments.
Compared with the 10% normal rat serum group, the cell mobility of the 5% renal qi pill-containing serum group was significantly increased, and the cell lumen formation ability was significantly improved. 3. AQP1 mRNA level and protein expression were significantly reduced after infection with cells with AQP1 RNAi lentivirus.
The ability of cell migration and lumen formation was significantly reduced, and the ability of renal qi pills containing drugs to promote cell migration and lumen formation was significantly reduced. However, the serum containing the drug in kidney qi pills can still upregulate the expression of AQP1 protein to a certain extent, promote cell migration and the formation of lumen. III Conclusion Renal qi pills can improve peritubular capillary injury in rats with renal yang deficiency.
It can improve the ability of vascular damage repair by upregulating the expression of AQP1 in human microblood tube endothelial cells. It suggests that renal qi pills may play a role in protecting peritubular capillary injury in rats with renal yang deficiency by increasing the expression of AQP1 in vascular endothelial cells.
Subject words Kidney Qi Pills; Renal yang deficiency; periductal capillaries; HMEC-1； Migrate; lumen formation; AQP kidney occupies an important position in the theory of traditional Chinese medicine, kidney is the innate foundation, internal education yuan yin yuan yang, "yin and yang secret, spirit is the cure", yin and yang balance is the basis for maintaining normal physiological activities. "Suwen • Inverse Regulation" has: "The kidney is a reservoir, the main jin liquid".
Traditional Chinese medicine believes that "kidney main water", kidney qi is the main division and regulates the water metabolism of the whole body. Kidney qi is divided into kidney yin and kidney yang, which is the foundation of yin and yang in all organs. Kidney yang is the root of human yang.
The basic theory of traditional Chinese medicine holds that kidney yang has physiological effects such as transpiration, gasification, pushing, and warming. In patients with renal yang deficiency, the transpiration and gasification, solidification, warmth, and promotion dysfunction of the kidneys affect the transport and transfer of jin liquid, and symptoms such as water retention or weight weakness, diarrhea, cough and asthma appear.
Kidney yang deficiency is one of the basic symptoms of traditional Chinese medicine, which is mainly manifested as soreness of the waist and knees, long urine, frequent nocturia, or swollen limbs and shortness of breath, short urine, fine pulse, and sluggishness. Renal yang deficiency is closely related to the development of chronic kidney disease (CKD).
CKD can be classified as "fatigue", "edema", "vertigo" and other categories of traditional Chinese medicine, and its disease location is mainly in the kidney, and the development of lesions is closely related to renal yang deficiency.
Vascular lesions play a very important role in the development of CKD. In the development of many kidney diseases, it is generally accompanied by varying degrees of sparse peritubular capillary (PTC). Periductal capillaries are sparse and are a hallmark of kidney disease.
This phenomenon occurs in both renal injury model studies and clinics, such as glomerulonephritis models, residual kidney models, aging-related nephropathy, chronic cyclosporine A nephropathy and kidney transplantation.
Renal PTC lesions are a predominant, independent risk factor for progressive renal failure. In the kidneys, peritubular capillary endothelial cells are the main source of tubular damage repair. Studies have confirmed that endothelial cells migrate and proliferate to repair their loss, reshape the structure and function of blood vessels, and form new blood vessels, thereby restoring oxygen supply to the renal tubules and the delivery of nutrients.
Therefore, angiogenesis of endothelial cells is of great significance in improving the process of kidney injury. Aquaporins (AQPs) are a family of cellular transmembrane proteins that are important in maintaining the balance of water metabolism. AQP1 is a subtype in this family that is involved in water secretion, absorption, and intracellular and intracellular water balance.
Renal AQP1 is mainly distributed in the proximal tubule, apical and basement membrane cells of Henry's descending loops, and straight small vascular endothelial cells, which transport water to 80% of all total absorption and are an important part of the urine concentration mechanism.
Studies have shown that AQP1 plays an important role in the angiogenesis process. This suggests that AQP1-mediated angiogenesis of endothelial cells is of particular value in renal injury repair.
Kidney Qi Pill is a clinically effective representative formula for warming kidney yang, from Zhang Zhongjing's "Outline of Jin Kuang", which is composed of 2 cinnamon, aconite, rehmania, yam, dogwood, ze lax, poria and peony peel eight flavor medicines, the main effect of which is to warm kidney yang.
Mainly treats renal deficiency edema caused by renal yang deficiency, waist and knee soreness, unfavorable urination and other diseases. Clinically, it is mostly used in patients with renal qi deficiency such as chronic nephritis, adrenal insufficiency, chronic bronchial asthma, diabetes and hypothyroidism.
The project team is carrying out the National Natural Science Foundation of China project "Exploring the molecular mechanism of regulating the water metabolism of eight flavor and six flavor dihuang pills from aquaporin" (project number: 30973921; It was found that the level of kidney AQP1 in rats with renal yang deficiency was significantly reduced, while kidney qi pills could correct the symptoms of reduced water intake and increased urine (less drinking and more urine) in rats with kidney yang deficiency, and increase the expression of kidney AQP1.
Previous in vitro experiments in our group showed that the presence of serum in renal qi pills can promote the migration of renal tubular epithelial cells and improve the expression of AQP1. However, whether kidney qi pills enhance the repair effect of kidney damage by promoting endothelial cell angiogenesis remains to be studied.
This study uses modern scientific technology and methods to further explore the molecular mechanism of renal injury repair, aiming to elucidate the intrinsic target and mechanism of action of renal qi pills in improving kidney injury, and provide new ideas for guiding the clinical treatment of renal deficiency and related diseases.
The use of adenine mixed with rat feed as a model for the study of kidney injury has been widely recognized by scholars at home and abroad. When Yokozawa et al. first created this model, they believed that the model rats prepared by this method would have metabolic disorders similar to those manifested by human chronic renal failure.
Later, after the improvement of domestic scholars, the method of giving adenine suspension by gavage to the state gradually began to prepare a kidney injury model in China. Adenine is a component of nucleic acids, and uric acid is its final metabolite.
When the concentration of adenine is too high in the body, metabolites that are extremely difficult to dissolve in water will be produced, deposited in the renal tubules, causing blockage of the renal tubular lumen, and eventually leading to renal function failure.
On the other hand, the enzyme activity related to the energy metabolism of renal tissue was inhibited by adenine metabolites, and then showed a series of renal yang deficiency symptoms, such as cold intolerance and vertical hair arching in rats. Therefore, adenine-induced SD rat renal yang deficiency model has been widely recognized and meets the diagnostic criteria for animal models of kidney yang deficiency in the "Dialectical Reference Standard for Hypocrisy in Traditional Chinese Medicine".
Moreover, it is considered that the model is more in line with the morphological performance of renal yang deficiency in traditional Chinese medicine in terms of symptom main evidence and objective indicators, and is better than the renal yang deficiency model induced by hydrocortisone.
According to the preliminary research basis of the research group, a kidney injury model was prepared by using SD rats, giving 150mg/kg•d adenine suspension at 1mL/100g of gavage and continuous gavage for 14 days. Combined with the results of the previous research, it was found that after adenine induction, SD rats had a decrease in diet, water drink, and increased urine output, that is, "less drinking and more urine", accompanied by body temperature, reduced grip, vertical hair, arched back and curled up phenomena.
The levels of ACTH, CORT, and 17-OHCS were significantly reduced in the main pathological basis of renal yang deficiency. HE and Masson staining showed collapse of the internal structure of the glomerulus, dilation of the renal tubules, obstruction of the renal tubules, partial protein casts, and fibrosis of the renal interstitium.
Therefore, we believe that the adenine-induced kidney injury model is an ideal model for studying peritubular capillary injury repair in rats with renal yang deficiency
Chronic kidney disease affects millions of people around the world, and finding more effective treatments for chronic kidney disease and promoting the repair of kidney tissue has become an urgent problem in the field of nephrology research.
Peritubular capillary damage is closely related to the occurrence and development of chronic kidney disease. Therefore, the correlation between peritubular capillary injury repair and renal injury repair and the molecular mechanism of peritubular capillary injury repair are further understood.
It is urgent to find a better early treatment strategy to improve the damage of the peritubular capillaries, thereby enhancing the regenerative repair ability of the kidneys themselves. This article reviews the latest research progress on the mechanism of peritubular capillary injury repair, in order to find potential therapeutic targets for patients with chronic renal diseases