Study on the regulation of VEGF pathway and inhibition of contralateral renal lymphangiogenesis in rats with unilateral ureteral ligation by traditional Chinese medicine for tonifying qi, promoting blood circulation and detoxification
In recent years, the incidence rate of chronic kidney disease (CKD) has increased significantly, ranging from 7% to 12% depending on the country and ethnic group, of which the incidence rate in China is 10.8%. In terms of the etiology of CKD, the continuous progression of unilateral obstructive kidney injury can lead to the occurrence of chronic kidney failure, and its pathological change is renal interstitial fibrosis (RIF). The traditional Chinese medicine for tonifying qi, promoting blood circulation, and detoxifying is based on the experience of Professor Zhao Yuyong, a nationally renowned traditional Chinese medicine practitioner, in treating kidney disease. It combines renal pathological changes and inflammatory damage mechanisms, and is composed of 15g Huangqi, 10g Poria cocos, 12g Paeonia lactiflora, 6g Dilong, 6g Bombyx mori, 10g Huangqin, 10g Honeysuckle, and 6g Rhubarb. It has the effects of tonifying deficiency and supporting the body, detoxifying and unblocking collaterals, and has achieved satisfactory clinical results in the treatment of chronic kidney disease. Animal experimental studies have confirmed that traditional Chinese medicine for nourishing qi, promoting blood circulation, and detoxifying can inhibit oxidative stress, reduce inflammatory damage, and alleviate damage in obstructive kidney disease. It can also regulate autophagy and other pathways to reduce damage to the contralateral kidney. This study found that traditional Chinese medicine for nourishing qi, promoting blood circulation, and detoxifying can regulate the VEGF pathway, inhibit contralateral renal lymphangiogenesis in rats with unilateral ureteral obstruction (UUO), and slow down the progression of kidney disease. This study used UUO to prepare an experimental animal model of obstructive kidney disease, which was treated with traditional Chinese medicine that tonifies qi, promotes blood circulation, and detoxifies, as well as aldosterone receptor blocker eplerenone. The study observed lymphatic vessel neogenesis related indicators such as Na+- Cl cotransporter (NCC), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3), lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1), podophyllin (PDPN), etc. Alpha smooth muscle actin (α – SMA) The expression of cell phenotype transformation indicators such as vimentin was investigated to explore the pathway of contralateral renal lymphangiogenesis and involvement in renal interstitial fibrosis in obstructive nephropathy rats, as well as the protective mechanism of traditional Chinese medicine.

In recent years, the incidence rate of chronic kidney disease has increased significantly, and it has become a public health problem of global concern. According to epidemiological data, the most common cause of chronic kidney disease in rural areas of China is obstructive kidney disease, which is characterized by renal interstitial fibrosis. Our preliminary experiments have shown that cell proliferation, pyroptosis, macrophage aggregation, and phenotype transformation are involved in contralateral kidney damage. Recent studies have also found that lymphangiogenesis plays an important role in contralateral kidney fibrosis.

In normal kidneys, lymphatic vessels are only distributed between the lobules and around the arcuate arteries, and there are few lymphatic vessels in the cortical renal tubulointerstitial region if there is no damage. However, in the areas of renal tubulointerstitial fibrosis and inflammatory injury, lymphangiogenesis is evident and the lumen is filled with monocytes, and the quantity generated is related to the degree of renal tubulointerstitial injury. The currently recognized lymphatic markers include lymphatic hyaluronic acid receptor 1 (LY-VE-1), podocyte protein (PDPN), and vascular endothelial growth factor receptor 3 (VEGFR-3). LYVE-1 is a membrane protein mainly expressed on lymphatic endothelial cells, widely distributed on the inner and outer surfaces of lymphatic vessels, and involved in the process of lymphatic endothelial cells taking up and transporting hyaluronic acid from tissues into lymphatic fluid; PDPN is a mucinous transmembrane glycoprotein present on renal tubular epithelial cells, mainly expressed in human renal podocytes, lung type I epithelial cells, osteoblasts, and lymphatic endothelial cells, but only in small lymphatic vessels; VEGFR-3 belongs to the receptor tyrosine protein kinase family of lymphatic endothelial markers and is a specific receptor for vascular endothelial growth factor VEGF-C. It is mainly expressed in lymphatic endothelial cells. Animal experiments have shown that 10 days after UUO surgery, the expression of endothelial markers LYVE-1, PDPN, and VEGFR-3 in the contralateral renal lymphatic vessels of rats in the UUO group significantly increased, indicating early occurrence of lymphangiogenesis; Considering that cell phenotype transformation such as renal tubular epithelial cells, podocytes, vascular endothelial cells, etc. involves myofibroblast like transformation in the formation of renal interstitial fibrosis, in order to confirm whether there are similar changes in lymphatic endothelial cells, we co stained LYVE-1 with cell phenotype transformation markers α – SMA and Vim entin. The expression level of α – SMA in renal tissue can indirectly reflect the number of myofibroblasts, therefore α – SMA is widely used as an indicator of phenotypic transformation towards myofibroblasts; Vimentin is a product of epithelial cell to myofibroblast transformation. In renal disease, the expression level of Vimentin is correlated with tubular injury. Therefore, Vimentin is a hallmark protein of myofibroblasts expressed in interstitial cells of interstitial fibrosis. The results showed that LYVE-1, α – SMA, and Vimentin were co expressed in UUO group rats, indicating that lymphatic endothelial cells underwent myofibroblast like transformation and were involved in the formation of contralateral renal fibrosis in obstructive nephropathy.

Among numerous signaling pathways, the VEGF-C/VEGFR-3 pathway is the most critical pathway for lymphangiogenesis. VEGF has six subtypes, A, B, C, D, and E. Among them, C and D are mainly involved in lymphangiogenesis. Renal tubular epithelial cells secrete VEGF-C and VEGF-D, which act on the homologous receptor VEGFR-3 of lymphatic endothelium, inducing the generation of lymphatic vessels and blood vessels. VEGFR-3 is a tyrosine kinase expressed in the endothelium of lymphatic vessels and is a biomarker of lymphatic vessels. Both VEGF-C and VEGF-D can bind to it to stimulate its expression, with the role of VEGF-C being more pronounced. The experimental results confirmed that ligating the unilateral ureter of rats significantly increased the expression of VEGF-C in the contralateral kidney, which showed the same trend as the expression of lymphatic endothelial markers LYVE-1, VEGFR-3, and PDPN; Inhibiting the secretion of VEGF-C can reduce the generation of renal lymphatic vessels, suppress inflammatory damage, and alleviate renal interstitial fibrosis in UUO rats.

The overexpression of VEGF-C is related to tissue edema, ischemia and hypoxia. Water sodium retention is a common factor that causes ischemia and hypoxia. The overexpression of NCC is involved in this pathophysiological change. NCC is a sodium chloride ion co transporter protein, mainly expressed in the distal tubules. Its hydrophilic core is located on the luminal side of the renal tubules, and based on the difference in sodium ion gradient between the lumen and cells, sodium and chloride ions are transported together into the tubular cells. Aldosterone regulates the expression of NCC, which can cause excessive absorption of sodium and chloride ions, leading to tissue edema, ischemia and hypoxia, stimulating the secretion of VEGF-C by renal tubular epithelial cells, and promoting lymphangiogenesis.

Through clinical verification, traditional Chinese medicine plays an important role in the treatment of chronic kidney disease. Chronic kidney disease belongs to the category of edema, hematuria, and cloudy urine in traditional Chinese medicine. In the later stages of the disease, it progresses to symptoms such as chronic obstructive pulmonary disease and obstructive pulmonary disease, and is generally caused by spleen and kidney deficiency, dampness, evil, turbidity, and toxin accumulation. Based on the traditional Chinese medicine theories of “chronic disease leads to deficiency”, “chronic disease leads to stasis”, and “chronic disease enters the meridians”, combined with clinical practice and modern research, we believe that “deficiency, toxicity, and stasis” are the basic pathogenesis of chronic kidney disease. “Deficiency is the foundation, stasis is the fruit, and toxicity is the culprit”. Therefore, we have formulated the treatment of tonifying qi, promoting blood circulation, and detoxifying traditional Chinese medicine. The “holistic concept” is a fundamental characteristic of traditional Chinese medicine, where the various components of the human body are physiologically inseparable and pathologically interdependent. Unilateral kidney damage caused by kidney disease, especially ureteral obstruction, not only affects itself but also the contralateral side. contralateral damage is an important factor leading to chronic kidney failure, and protecting the “healthy” kidney has clinical reference value for the treatment of chronic kidney disease.

Our previous research has confirmed that in long-term experiments, macrophages in the contralateral kidney of UUO rats transform into myofibroblasts, exacerbating fibrosis in the contralateral kidney; In short-term experiments, UUO rats exhibited phenomena such as cell proliferation and apoptosis in the contralateral kidney, which were involved in contralateral kidney injury. This experiment further proves that the changes in the contralateral kidney after unilateral kidney injury are related to lymphangiogenesis. Changes occur in the early stage after obstruction, although the degree is mild, with significant fibrosis changes occurring as the obstruction time prolongs. After MR activation, the expression of NCC, a sodium chloride ion cotransporter, is up-regulated, leading to water and sodium retention, causing tissue ischemia and hypoxia, stimulating renal tubular epithelial cells to secrete VEGF-C to induce lymphangiogenesis, and proliferative lymphatic endothelial cells undergo phenotypic transformation (α – SMA, Vimentin), participating in interstitial fibrosis. Traditional Chinese medicine for nourishing qi, promoting blood circulation, and detoxifying can regulate the VEGF pathway, downregulate the expression of LYVE-1, VEGFR-3, and PDPN, inhibit the phenotypic transformation of lymphatic endothelial cells, and thus achieve the effect of reducing renal interstitial fibrosis.