KXL improved renal function and attenuated renal injuries

The composition of KXL and the treatment duration is shown in Fig. 1A, B. The levels of BUN, Scr, and 24-UP levels were increased in the UUO group compared with the Sham group, but were reduced in both KXL medium and high dose groups (Fig. 1C–E). HE (Fig. 2A, B) staining showed that there were no abnormalities in the glomerulus, tubule, and interstitial of the sham operation group. In the UUO group, the renal tubules were dilated or contracted, and ECM were accumulated in the glomerulus, accompanied by partial glomerular atrophy or compensatory hypertrophy; tubular epithelial cells swelled and shed, and some lumens showed obvious dilation, accompanied by infiltration of inflammatory cells. The above pathological injuries were alleviated by being treated with KXL formula, indicating that KXL formula possessed the effect of protecting renal function and alleviating renal injury.

Fig. 1

A The treatment duration for UUO rats. B The composition of KXL. C–E The renal function of all groups. *P < 0.05 vs sham group; # < 0.05 vs UUO group

Fig. 2

A HE staining of glomeruli of rats in all groups, scale bar 100 µm. B HE staining of renal tubule of rats in all groups, scale bar 100 µm

KXL attenuated the formation of renal fibrosis

Both Masson and Sirus red staining (Fig. 3A–C) revealed that compared with the Sham group, abnormal collagen fiber proliferation was observed in the renal tubule interstitial area from the UUO group, and large areas of blue collagen fiber deposition were observed, indicating the area of renal tissue fibrosis in the UUO rats were significantly expanded (P < 0.05) (Fig. 3D–F). But the area of renal fibrosis in UUO rats treated with KXL formula gradually decreased (P < 0.05) (Fig. 3D–F), indicating that KXL formula produced the effect of delaying the disease progression of RF and protecting renal tissue.

Fig. 3

A Masson staining of renal tissues. B Masson staining of glomerulus. C Sirus red staining of renal tissues. D–F Positive area of fibrotic area. *P < 0.05 vs sham group; # < 0.05 vs UUO group

KXL treatment inhibited the expressions of RF-related proteins

α-SMA, TGF-β and FN are important markers of RF. The activation of α-SMA, TGF-β, and FN were contributed to promote the synthesis of collagen, accelerating the process of fibrosis. Col-III is the main component of ECM, and its expression level is closely related to the degree of organ fibrosis. IHC results (Fig. 4C, D) indicated that compared to the Sham group, the renal tissues of rats subjected to UUO exhibited a sharp increase in the positive area of TGF-β and FN, which could be reduced by KXL treatment. IF (Fig. 4A, B) results showed that compared to the Sham group, the renal tissues of rats subjected to UUO exhibited a sharp increase in the fluorescence intensity of α-SMA and Col-III, which could be reduced by KXL treatment.

Fig. 4

A, B The immunofluorescence analysis of α-SMA and COL-III expression in renal tissues. C, D The immunohistochemistry analysis of FN and TGF-β expression in renal tissues. *P < 0.05 vs sham group; # < 0.05 vs UUO group

The above results indicate that KXL exerted anti-fibrotic effect by suppressing activation of α-SMA, TGF, FN and Col-III.

KXL alleviated intestinal mucosal damage

Figure 5A and B reflects that in the Sham group, the structure of the serous layer, muscular layer, submucosal layer, and mucosal layer of the colon tissue was clear, the glandular body was arranged neatly, containing a large number of goblet cells, and the mucosal surface was covered with a large number of villi; However, in the UUO group, we found that the structure of colon was disordered, the glandular body was obviously damaged, and massive amount of inflammatory cells’ infiltration. But after KXL treatment, the intestinal mucosal epithelium was intact, the glandular body structure was arranged neatly, and less inflammatory cells’ infiltration was observed.

Fig. 5

A HE staining of colon tissues of rats in all groups. B Masson staining of colon tissues of rats in all groups

KXL maintained the integrity of intestinal barrier

E-cadherin, Occuludin and ZO-1 are three vital intestinal tight junction proteins essential for protecting integrity of intestinal barrier. The reduced expressions of Occuludin and ZO-1 are contributed to the loss of integrity of the intestinal barrier. IHC results (Fig. 6C, F) showed that colon tissues of UUO rats suffered a decreased positive area of Occuludin compared with the rats in Sham group, which could be restored by KXL treatment. IF results showed the colon tissues of UUO rats displayed less fluorescence intensity of ZO-1 (Fig. 6A, D) and E-cadherin (Fig. 6B, E) compared with the sham group, which could be upregulated by KXL intervention. These outcomes indicate that KXL can repair the damaged intestinal barrier permeability by restoring the expressions of intestinal tight junction proteins.

Fig. 6

A IF detected the expression of ZO-1. B IF detected the expression of E-cadherin. C IHC detected the expression of Occuludin. D–F The positive area of ZO-1, E-cadherin and Occuludin calculation. *P < 0.05 vs sham group; #< 0.05 vs UUO group

The regulatory effect of KXL on gut microbiota diversity

The Venn diagram (Fig. 7A) displayed that there were 2453 OUTs across all three groups, including 1563 OUTs in the Sham group, 956 OUTs in the UUO group, and 749 OUTs in the KXLH group. Notably, 173 of these OUTs were shared among all three groups. Subsequently, we used α diversity analysis to analyze the diversity of gut microorganisms. As shown in Fig. 7B, C, compared to Sham group, the Chao, Ace, Simpson and Shannon indices in the UUO rats were significantly reduced. However, after KXL intervention, the aforementioned indices in the KXL group displayed a further decrease.

Fig. 7

A Venn diagram of Sham group, UUO group and KXLH group. B The PCA of Sham group, UUO group and KXLH group. C The Chao, Ace, Simpson and Shannon of Sham group, UUO group and KXLH group

The in-depth analysis of gut microbiota composition revealed that at the phylum level (Fig. 8A), Bacteroidota, Firmicutes, Proteobacteria and Desulfobacterota were identified the most occupied gut microbiota in Sham, UUO and KXLH group. In comparison with the Sham group, a higher abundance of Proteobacteria and a lower abundance of Actinobacteriota were found in rats subjected to UUO. After KXL intervention, the abundance of Proteobacteria was reduced, while the abundance of Actinobacteriota was increased. At order level (Fig. 8B), Lachnospirales, Oscillospirales and Lactobacillales were identified the most occupied gut microbiota in Sham, UUO and KXLH group. In comparison with the Sham group, a lower abundance of Lachnospirales and Lactobacillales was observed in UUO rats, which could be increased by KXL intervention. At family level (Fig. 8C), in comparison with the Sham group, a lower abundance of Prevotellaceae and a higher abundance of Ruminococcaceae was observed in UUO rats. But KXL intervention could reduce abundance of Ruminococcaceae and increase abundance of Prevotellaceae.

Fig. 8

The analysis of gut microbiota composition revealed that at the phylum A, order B and family level C, respectively

LEfSe analysis

LEfSe analysis can select species or genes that exhibit significant differences and elucidate their distinctions, making it a crucial application in the analysis of gut flora. By utilizing LEfSe analysis, it becomes possible to pinpoint biomarker species that are linked to disease states, providing deeper insights into the function of gut flora in the different stages of diseases development. As shown in Fig. 9A, B, the main bacteria communities of UUO group were Enterobacteriaceae, Ruminococcaceae, Desulfobacterota, Acetatifactor, Rikenellaceae and Alistipes, which are pathogenic bacteria that promotes the release of pro-inflammatory factors contributing to the disruption of gut barrier. While Actinobacteria, Lactobacillales, Burkholderiales, Bifidobacteriales and Prevotella, the important beneficial bacteria were identified in the KXL group that promote the production of SCFAs conferring health benefits on host.

Fig. 9

A The LEfSe analysis of Sham group and UUO group. B The LEfSe analysis of UUO group and KXLH group