3.1 Structural characterization of Ds
The functional groups of Ds were analyzed using FT-IR (Fig. S1). The vibrational region detected between 3200 cm− 1 and 3600 cm− 1 corresponds to the hydroxyl group characteristic band, which represents the polysaccharide's characteristic peak. The fluctuations observed at 2925 cm− 1 represent the absorption peaks of C-H bonds. These include stretching vibrations of C-H, C-H2 and C-H3. The absorption peak at 1622 cm− 1 is indicative of the bending vibration of C = O and suggests the presence of uronic acid in Ds [15].In addition, the vibrations in the range of 1000 cm− 1-1200 cm− 1 are caused by CO-C and C-O-H of the pyranose ring, which proves that Ds contains pyranose. The α-configuration of glucans was responsible for the vibrations within the 860 − 830 cm− 1 range [16].
The monosaccharide composition of Ds was quantified using high-performance anion-exchange chromatography (HPAEC) (Figs. S2A and S2B). The results revealed that Ds consisted mainly of Glc and Gal with minor amounts of Ara, Man, Gal-UA, Glc-UA, Xyl, and Rha monosaccharides, as displayed in Table S1. The composition percentages were 89.75%, 3.36%, 1.49%, 1.37%, 1.30%, 1.25%, 0.75% and 0.73%, respectively.
3.2 Presence of renal fibrosis in stone kidney tissue
We collected renal cortex tissue form three kidney cancer patients with renal stones (no other kidney disease such as hydronephrosis, severe urinary tract infection, renal atrophy, etc.) and normal renal cortex tissue sample were obtained from three kidney cancer patients who underwent nephrectomy at the First Affiliate hospital of Guangzhou Medical University. Renal cortex tissue was excised from normal adjacent tissues > 5 cm distance from the tumor tissue, and subsequent pathological examination confirmed the absence of tumor invasion.
The fibrosis marker protein α-SMA exhibited notably heightened expression levels in the kidney tissues of patients with renal stones (Fig. 1A). Subsequently, we corroborated this phenomenon using a rat kidney stone model, wherein we observed a significantly elevated MASSON-stained collagen volume fraction in the stone group in comparison to the control group of rat kidney tissues (Fig. 1B). These findings suggest the occurrence of differing degrees of fibrosis in the stone kidneys.
3.3 Screening of pivotal genes associated with renal fibrosis induced by kidney stones
To further elucidate the molecular mechanisms underpinning renal fibrosis resulting from kidney stones, we conducted a transcriptome sequencing analysis of RNA expression differences in the kidney tissues of kidney stone model rats compared to normal rats. This data was subsequently analyzed using bioinformatics techniques. In the stone model group, we identified 1396 significantly altered differential genes compared to the control group (Fig. 1C). Subsequently, we retrieved a gene set containing the keyword "fibrosis" from five databases, namely GeneCards, OMIM, DrugBank, TTD, and PharmGkb, and intersected it with above 1396 differential genes, resulting in 11 differential genes (Fig. 1D). Following this, we conducted protein interaction analysis (Fig. 1E) and excluded two proteins with no interactions, leaving us with nine genes for further analysis. Using the Cytoscape algorithm in Cytohub software (Fig. 1F), we determined that TGF-β1 emerged as the core gene in this context.
3.4 Ds accumulates within the kidney and affords protection to renal function
The distribution and metabolism of Ds within organisms were initially investigated in normal mice. The in vivo distribution of the drug was monitored using an animal in vivo imaging system at intervals of 1h, 2h, 4h, 8h, and 12h (Fig. 2A). These observations revealed that the highest fluorescence intensity was detected in the kidneys at the 4-hour mark (Fig. 2B), after which the fluorescence intensity gradually diminished. Furthermore, we conducted a comparative analysis of the metabolic disparities of Ds in the kidneys of normal and stone-model mice. Notably, the fluorescence intensity in the kidneys of the stone-model group was markedly higher than that in the normal mice (Fig. 2D). This finding was subsequently validated through immunofluorescence staining of frozen sections, confirming the enrichment of Ds within the kidneys of stone-model mice (Figs. 2C and 3E).
Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining results revealed that the kidneys of the control group were morphologically intact, with no swelling or damage to the glomeruli and tubules. In contrast, the kidneys in the model group displayed compromised integrity, marked by various degrees of tubular swelling and crystal deposition. Conversely, the kidneys in the Ds-treated group demonstrated preserved structural integrity, and there was a notable reduction in tubular swelling and damage compared to the model group (Figs. 3A and 3D).
Polarization microscopy was employed to observe crystal deposition within the renal tissues. The renal tubules in the model group contained crystals of varying sizes. In contrast, the Ds treatment group exhibited a substantial reduction in both the size and quantity of crystals (Figs. 3B, 3E, and 3F). Additionally, MASSON staining unveiled a notable increase in the collagen volume fraction within the kidneys of the model group in comparison to the control group. The areas expressing collagen were primarily concentrated around the region surrounding the damaged renal tubules. Conversely, the collagen volume fraction in the kidneys of the rat was markedly reduced in the Ds treatment group, accompanied by a significant decrease in collagen fiber staining around the renal tubules (Figs. 3C and 3G).
Immunohistochemistry was employed to assess changes in oxidative stress damage, inflammation, and adhesion factors following DS treatment. The results indicated that in the kidney stone model rats, kidney injury factor (KIM-1), monocyte chemoattractant protein-1 (MCP-1), adhesion-related molecules (CD44 and OPN), and oxidative stress-related molecules (NOX-1 and NOX-4) were up-regulated to varying degrees. Simultaneously, antioxidant-related molecules (NRF-2 and SOD-1) were down-regulated in these rats. However, Ds treatment was effective in reversing these alterations in renal stone risk factors induced by the kidney stone, ultimately preserving kidney function (Figs. 4A, 4B and 4F).
Furthermore, fibrosis-related proteins (FN, N-cadherin, Vimentin, α-SMA, and TGF-β) were obviously up-regulated, and the epithelial cell marker E-cadherin in the model rats were obviously down-regulated (Figs. 4C, 4D, 4E, and 4G). Which suggests that there may be a phenomenon of functional transformation of epithelial cells (EMT) in the renal stone model rats. Conversely, Ds treatment effectively reduced the alterations in fibrosis-associated factors.
3.5 Ds efficiently mitigates cellular damage induced by nano-COM
In order to better simulate the model of crystallization-induced renal injury and fibrosis, we prepared nano-scale calcium oxalate monohydrate (nano-COM) crystals in vitro by direct precipitation. Scanning electron microscope images of CaOx crystals are shown in Fig. 5A, and the crystals exhibit a rhombic shape. By using Nano Measurer software (v1.2.5), the diameter of the prepared crystals was counted to be about 244 ± 77 nm (Fig. 5B). Figure 5C shows the XRD spectrum of the synthesized nano COM crystals, diffraction peaks with crystal face spacing d = 0.593, 0.365, 0.296, 0.249, and 0.235 nm were detected, which were attributed to the (-101), (020), (-202), (112), and (130) crystal faces of the COM, respectively (PDF Card No. 20–231), which indicated that the synthesized crystals were pure phase COM crystal.
Toxicity testing revealed that concentrations of Ds below 150 µg/mL had no significant impact on HK-2 cells and NRK-52E cells (Fig. 5D). The cytotoxicity of nano-COM displayed a positive correlation with the crystal concentration (Fig. 5E). The optimal therapeutic effect was observed when the concentration of Ds was set at 80 µg/mL (Fig. 5F). Microscopic examination unveiled that a substantial quantity of nano-COM crystals adhered to the cell surfaces in the modeling group. Consequently, the cells adopted an elongated, spindle-shaped morphology, and there was a noticeable reduction in cell numbers. In contrast, the Ds-treated group showed an obvious increase in the number of cells and a markedly decrease in the proportion of spindle-shaped cells (Fig. 5G).
Transmission electron microscopy unveiled that a substantial number of crystals adhered to the cell membrane, and numerous crystals were endocytosed into the cells in the modeling group. This led to swollen and ruptured mitochondria, as well as distorted nuclei. Conversely, in the Ds treatment group, there was a reduction in the crystals adhering to the cell membrane, along with a significant decrease in the number of cellular endocytosed crystals. Additionally, the morphology of mitochondria appeared more intact, with a noticeable decrease in the proportion of swollen mitochondria compared to the modeling group (Fig. 5H). The evaluation of oxidative stress-related parameters indicated that nano-COM crystals induced oxidative stress in HK-2 cells. This was evidenced by an elevation in intracellular reactive oxygen species (ROS) generation, a decline in mitochondrial membrane potential (Δψm), an increase in the number of dead cells, and heightened levels of LDH and MDA. Conversely, these aforementioned indicators demonstrated significant improvement following Ds treatment (Figs. 6A-6F). The results from the Western blot assay on NRF-2 and Keap-1 further corroborated these findings (Figs. 6G and 6H).
3.6 Ds delays the EMT process in HK-2 cells triggered by nano-COM
In the preliminary stage of this study, TGF-β was found to be a key factor in kidney stones leading to fibrosis, and the cell morphology suggested that HK-2 cells might have undergone EMT (Fig. 5G). Subsequently, we delved into investigating whether nano-COM induced EMT in HK-2 cells. The results show that nano-COM stimulation of cells at graded concentrations and durations triggers expression of TGF-β and N-cadherin from epithelial cells (Figs. 7D, 7E and 7G-7I). Cellular immunofluorescence analysis revealed the co-expression of the epithelial marker protein CK-18 and the mesenchymal cell marker Vimentin in HK-2 cells following nano-COM stimulation. This co-expression of both epithelial and mesenchymal cell markers indicated the occurrence of EMT in HK-2 cells (Figs. 7A and 7B). Additionally, the expression of the mesenchymal cell marker protein Vimentin was significantly elevated in these cells, and their morphology changed to a long spindle shape. Conversely, in the Ds treatment group, the cell morphology remained largely unchanged, and the expression of Vimentin was significantly lower than that in the modeling group. This observation indicates that Ds effectively suppressed the EMT process in HK-2 cells induced by nano-COM stimulation.
The secretion of the TGF-β protein was assessed in the culture medium of nano-COM-stimulated HK-2 cells utilizing an Elisa kit. The outcomes indicated that nano-COM stimulation markedly increased the secretion of TGF-β protein. Conversely, the secretion of TGF-β protein from the Ds treatment group was significantly lower than that from the modeling group, signifying the effectiveness of Ds in inhibiting the secretion of TGF-β protein (Fig. 7C). Subsequently, we examined the key proteins involved in the major signaling pathways associated with TGF-β-induced fibrosis. The Western blot results revealed that nano-COM stimulation led to a decrease in the expression of the epithelial phenotypic protein E-cadherin in HK-2 cells. Simultaneously, there was a corresponding increase in the expression of mesenchymal phenotypic proteins, including N-cadherin and Vimentin. The expression of TGF-β protein, a key activator of fibrosis, was also elevated. Moreover, the expression of phosphorylated Smad2 and Smad3 (P-Smad2 and P-Smad 3) was found to be up-regulated. These findings suggest that following stimulation of HK-2 cells with nano-COM, the cells expressed TGF-β and concurrently activated the TGF-β/Smad pathway, leading to EMT in renal tubular epithelial cells (Fig. 7F). Remarkably, the epithelial phenotypic proteins in Ds-protected HK-2 cells remained at high levels compared to the modeling group. Conversely, mesenchymal phenotypic proteins were at low levels, and the expressions of TGF-β, P-Smad2, and P-Smad 3 were noticeably reduced. This observation suggests that Ds effectively inhibited the overexpression of TGF-β in HK-2 cells induced by nano-COM stimulation. Additionally, it inhibited the activation of the TGF-β/Smad pathway, consequently suppressing the EMT process in HK-2 cells (Figs. 7F-7J). These findings were further supported by subsequent RT-qPCR results (Figs. 7K-7M). Our experimental results indicate that Ds effectively reduces the synthesis and secretion of TGF-β in HK-2 cells due to nano-COM stimulation, thereby inhibiting the activation of the TGF-β/Smad pathway and, consequently, the EMT process in HK-2 cells.
3.7 Ds inhibits cellular EMT through regulation of TGF-β synthesis and secretion
To further delve into the therapeutic mechanism of Ds, we used human recombinant protein TGF-β to stimulate HK-2 cells, and explored whether Ds could inhibit EMT in HK-2 cells induced by human recombinant protein TGF-β. HK-2 cells were cultured for 48 hours in the presence of recombinant TGF-β. The cell morphology was observed using phase-contrast microscopy, and it was observed that the recombinant TGF-β notably induced a morphological transformation of HK-2 cells, causing them to adopt a long shuttle-like shape. However, Ds effectively maintained the elliptical cell morphology of HK-2 cells (Fig. 8A). Cellular immunofluorescence analysis revealed that HK-2 cells stimulated with human recombinant TGF-β displayed high expression of the mesenchymal cell marker Vimentin, while Ds effectively maintained low levels of vimentin in HK-2 cells (Fig. 8B). Subsequently, we conducted a Western blot assay for fibrosis-related proteins and observed that recombinant TGF-β significantly reduced the epithelial marker protein E-cadherin, elevated the mesenchymal phenotypic protein N-cadherin, and increased the protein level of TGF-β in HK-2 cells (Figs. 8C and 8D). Intriguingly, Ds effectively attenuated the rise in fibrosis-associated proteins induced by human recombinant TGF-β in HK-2 cells, while reducing the cellular TGF-β protein expression level. This was further supported by RT-qPCR results (Fig. 8E), confirming that Ds inhibited fibrosis in HK-2 cells induced by human recombinant TGF-β.
In the subsequent functional rescue experiments, we delved deeper into the action targets of Ds. Prior to administering Ds treatment, human recombinant TGF-β was added to stimulate the cells for 1 hour, and changes in damage-related and fibrosis-related markers were assessed. Phase-contrast microscopy revealed that the cell morphology in the Ds treatment group following the addition of human recombinant TGF-β differed from that of the Ds treatment alone group. It displayed more mesenchymal cell characteristics, with cells adopting a long spindle shape, similar to the COM group (Fig. 9A). Subsequently, we assessed pertinent injury markers, including intracellular ROS generation, mitochondrial membrane potential, and the count of viable and non-viable cells. It was observed that the TGF-β pretreatment led to an elevation in intracellular ROS production, a disruption in mitochondrial membrane potential equilibrium, and an increase in the count of non-viable cells compared to the COM + Ds group (Figs. 9B, C, D, and E). This suggests that TGF-β pretreatment counteracts some of the therapeutic effects of Ds. Additionally, fibrosis-related parameters in the cells were assessed. Cellular immunofluorescence revealed that in the group pre-treated with TGF-β, the cells expressed a higher level of the mesenchymal cell phenotypic protein Vimentin, along with corresponding alterations in cellular morphology, in contrast to the COM + Ds group (Fig. 9F). These findings were consistent with the results obtained from the Western blot analysis (Fig. 9G and 9I). The cellular oxidative stress-related protein NRF-2 was analyzed, and the results showed that NRF-2 protein was significantly down-regulated in the COM + Ds + TGF-β group compared to the COM + Ds group (Figs. 9G and 9H). This indicates that Ds's protective effect against oxidative stress was partially reversed. Similar results were also observed in the RT-qPCR data (Figs. 9J and 9K). Consequently, we concluded that human recombinant protein TGF-β partially counteracted the protective effect of Ds on HK-2 cells in vitro.