3.1 Quality control for YGY aqueous extract
As reported by our previous study [5], the HPLC results showed that the aqueous extract of YGY mainly contained nine pharmacologically materials: Geniposidic acid, Morroniside, Chlorogenic acid, Geniposide, Loganin, Pinoresinol diglucoside, Liquiritin, Rutin, and Glycyrrhizic acid. The HPLC chromatography was shown in Supplement Fig. 1. The content determination of the nine components in YGY was performed according to the Chinese Pharmacopoeia published by the National Pharmacopoeia Commission (NPC, 2015), and all results showed in Supplement Table 1.
3.2 YGY extract improved morphological damages in the kidneys of CRF rats
The experimental design is shown in Fig. 2A. First, we observed the morphology of the kidneys and compared the renal organ index of the different groups of rats. CRF rats had “large white kidney” and significantly abnormally increased organ index(kidney) (P < 0.05), compared with that of NC rats. However, the organ index was significantly reduced by YGY extract (20 or 40 g·kg− 1) (P < 0.05), indicating that YGY extract ameliorated the kidney swelling of adenosine-induced CRF rats (Fig. 2B, C). Additionally, incompletely glomerular basement membrane and expansion glomerular mesangial cells were observed, and significantly higher renal glomerular sclerosis scoring (P < 0.05) in the CRF group rats. In contrast to that in the CRF group, YGY extract (20 g·kg− 1) improved the glomerular atrophy and inhibited the expansion of glomerular mesangial cells of the kidney significantly reduced glomerular lesion scores (Fig. 2D, E, P < 0.05). Taken together, the results indicated that YGY extract improved morphological and histopathological damage in the kidneys of CRF rats.
3.3 YGY extract ameliorated abnormal biochemical parameters in CRF rats
As it shown in Fig. 3, compared with the normal group, the CREA content in the urine and Ccr of rats in the model group were significantly lower (all P < 0.05), and the serum CREA, UREA, P, Ca and Mg contents were significantly higher (all P < 0.05). Compared with the model group, urine CREA was significantly increased, and serum CREA content, Ccr and serum UREA, P, Ca and Mg content were significantly decreased in the rats in the group of YGY (10, 20 or 40 g·kg− 1) (Fig. 3A-G, all P < 0.05). These results suggested that YGY extract delayed the progression of adenine-induced CRF.
3.4 YGY extract promoted angiogenesis and enhances vascular density in the renal cortex in CRF rats
Revascularization is a promising treatment strategy for slowing the progression of CRF. To observe angiogenesis within the renal cortex, we used CD31/CD105 to label newly generated vessels (Fig. 4A). The vascular density in the kidney cortex of rats in the model group was significantly lower compared with the NC group (P < 0.01). Vascular density, CD105 and CD31+/CD105+-double positive cell expression were significantly increased in the kidney cortex in the YGY (10, 20 or 40 g·kg− 1) group compared with the CRF group (Fig. 4B-D, all P < 0.05).
Next, we provided more pieces of evidence to support that YGY promoted angiogenesis in kidney of CRF rats by performing photoacoustic imaging (PAI) experiments (Fig. 5A). After indocyanine green was injected into rats, we performed PA signal acquisition in the kidney by using a photoacoustic imaging device. We found that the PA signal in the kidney cortex was significantly reduced in the CRF group compared to that in the NC group. However, after the treatment with YGY extract (10, 20 or 40 g·kg− 1), a significant increased PA signal was observed in the kidney cortex (Fig. 5B, C, P < 0.05), indicating enhanced neovascularisation in the kidney cortex. This result was in agreement with the CD31/CD105 immunofluorescence data. Overall, these results suggested that YGY extracts protect the kidney by promoting angiogenesis and increasing vascular density.
3.5 YGY extract improved renal vascular endothelial cell function and reduced vascular injury in CRF rats
To further determine the vascular protection effects of YGY extract, we measured the expression of von Willebrand Factor (vWF) in kidney, a marker which indicated endothelial injury, by immunohistochemistry (Fig. 6A). And also, we tested the expression levels of Ang-1 and Ang-2 in serum and the expression levels of eNOS and NO in rat kidney tissues. We found that the expression levels of vWF and Ang-2 were significantly increased in the CRF group rats compared to that in the NC group (both P < 0.05), while the expression levels of Ang-1, eNOS and NO were significantly reduced in rats from YGY groups (all P < 0.05). The expression levels of vWF and Ang-2 were significantly decreased in the YGY extract (10, 20 or 40 g·kg− 1) groups (Fig. 6B, D), compared to those in the CRF group (P < 0.01, P < 0.05), while the expression levels of Ang-1, Ang-1/Ang-2, eNOS and NO were greatly increased (Fig. 6C, E, F, G, all P < 0.05). All the results indicated YGY extract improves the function of renal microvascular endothelial cells by regulating the functional factors of renal vascular endothelial cells, thus maintaining the stability of vascular structure.
3.6 YGY improves renal hemodynamics in CRF rats
The Doppler ultrasound probe was placed on the kidney and targeted to the interlobular arterial vessels in the cortical region of the kidney. The angle of the collected data in the probe was adjusted to coincide with the direction of blood flow, and the blood flow velocity was visualized to obtain a Doppler spectrogram, displayed as time-flow rate (Fig. 7). Peak systolic velocity (PSV) and end-diastolic velocity (EDV) of the renal interlobular arteries were labeled to analyze renal inter-arterial vascular resistance (RI) as an indicator of vascular function[16]. Generally, an elevated RI is considered to be an indicator of renal disease. Compared with the NC group, the renal interlobular artery PSV was significantly increased and the renal interlobular artery EDV was significantly decreased in the CRF group rats, resulting in a significant increase in interlobular artery RI (Fig. 7A). As expected, PSV and interlobular artery RI were significantly decreased and EDV was significantly increased in the YGY extract (10, 20, or 40 g·kg-1) group (Fig. 7A-D). Next, we measured serum levels of renin, Ang-II and ET-1 to comprehensively assess the hemodynamic effects of YGY on CRF rats. We found that serum levels of renin, angiotensin II and ET-1 were significantly increased in CRF rats compared with the NC group (all P < 0.05). However, YGY extracts (10, 20 or 40 g·kg-1) significantly downregulated serum renin, angiotensin II and ET-1 levels compared with the CRF group (Fig. 7E-G, all P < 0.05). This seems to suggest that YGY can modulate renal hemodynamics in CRF rats and ameliorate the damage caused by changes in blood velocity and other factors to the kidney.
3.7 YGY extract improved renal blood flow distribution and renal oxygen saturation in CRF rats
Renal blood irrigation can, to some extent, reflect renal vascular blood filling and effective vascular function, while oxygen saturation reflects the ability of blood to carry oxygen and is closely related to blood substance exchange.To observe the blood supply and oxygen saturation in the kidney cortex, ultrasound Doppler mode and blood oxygenation mode of the photoacoustic imaging unit were used. The percentage of blood distribution (Fig. 8A) and oxygen saturation (Fig. 8B) in the kidney cortex were both significantly lower in the CRF group than the NC group (both P < 0.05). However, after the treatment with YGY extract (10, 20 or 40 g·kg− 1), the percentage of blood distribution and oxygen saturation in the cortical areas of the kidneys were significantly increased (Fig. 8C, D, both P < 0.05). Overall, these results suggested that YGY extract improved the blood supply to the kidneys of CRF rats.
3.8 Significant renal pathological damage in EPO-/- mice
The appearance and histopathology of the kidneys were just as expected (Fig. 9). Compared to wild-type (WT) mice, the kidneys of EPO-/- mice were significantly smaller in size, lighter in color (Fig. 9B), and had thickened glomerular basement membranes, dilated thylakoid membranes (Fig. 9C), and other conditions with significantly increased glomerulosclerosis scores (Fig. 9D, P < 0.05).
3.9 The renal microcirculatory was significantly damaged in EPO-/- mice
Monitoring of renal blood irrigation by Doppler ultrasound and blood oxygen saturation by 750 nm and 850 nm revealed that renal cortical blood flow and blood oxygen saturation were significantly lower in EPO-/- mice compared with wild-type (WT) mice (Fig. 10A-D, P < 0.05). To some extent, this indicates a significant decrease in renal cortical blood flow and impaired blood circulation in the kidney.
The maximum systolic flow velocity (PSV) and end-diastolic flow velocity (EDV) of the interlobular arteries in the renal cortex of EPO-/- mice were significantly lower (Fig. 10E-H, P < 0.05) compared to wild-type (WT) mice. This result implies that knockout of EPO has a significant effect on the hemodynamics of the kidney, and this lesion manifests as a significant impairment of renal circulation.
3.10 YGY extract upregulates EPO in the kidney cortex and activates PI3K/AKT pathway to upregulate VEGF expression
Additionally, We found that there is also a relationship between EPO and VEGF by examining the renal vasculature of the EPO−/− mouse model and the adenine-induced chronic renal failure rat model. After EPO stimulation of endothelial cells, EPOR dimerization is induced and then EPOR is phosphorylated, which activates PI3K/AKT [17], activating specific downstream effects [18, 19]. VEGF promotes angiogenesis by acting on endothelial cell-specific receptors (DH et al., 2005; Urbich and Dimmeler, 2004). Daniela [21] showed that the PI3K/AKT pathway promotes VEGF expression, suggesting that VEGF is not only an activator of the PI3K/AKT signaling pathway but that the activated PI3K/AKT signaling pathway also promotes VEGF transcription, creating positive feedback. The renal cortical EPO, p-EPOR, p-PI3K, p-AKT, and VEGF protein expression levels were significantly lower in EPO−/− mice compared with WT group (Supplement Fig. 11A, B, P < 0.05). We hypothesized that YGY could play a role in improving renal microcirculatory disorders through the EPO/VEGF pathway. Compared with the NC group, VEGF fluorescence intensity was significantly reduced in the renal cortical area of the model group rats, which inhibited angiogenesis. Meanwhile, the expression of EPO, EPOR, p-EPOR, p-PI3K, p-AKT, and VEGF were significantly reduced (all P < 0.05), and the overall expression of EPO-VEGF pathway was decreased. Compared with the model group, the fluorescence intensity of VEGF was significantly increased in the renal cortical area of rats in the YGY group, implying that YGY significantly activated the expression of VEGF and promoted the probability of angiogenesis. Meanwhile, YGY significantly upregulated the expression of p-EPOR, p-PI3K, p-AKT and VEGF (Fig. 12A-D, all P < 0.05), which successfully activated the expression of EPO-VEGF pathway. These studies support the hypothesis that YGY regulates EPO production, which in turn regulates key proteins of the PI3K-AKT pathway and affects VEGF expression, and that this pathway is involved in promoting renal angiogenesis in rats with chronic renal failure, which in turn improves renal microcirculatory impairment.