The animal study was approved by the Ethics Committee of Hebei University of Chinese Medicine. Thirty specific pathogen-free (SPF) male C57BL/6 mice, weighing 24.7 $\pm$ 1.1 g and aged 6–8 weeks, were provided by Liaoning Changsheng Biotechnology Co., Ltd., Changchun, China. The mice were maintained under a 12-hour light/dark cycle at a controlled ambient temperature of 25 °C, with ad libitum access to standard rodent chow and tap water. All animal care and experimental procedures were conducted in accordance with the guidelines provided by the Animal Experimentation Ethics Committee of Hebei University of Chinese Medicine and were approved by the Animal Care and Use Committee of the same institution. All efforts were made to minimize animal distress and ensure ethical treatment.

Thirty mice were divided randomly into 3 groups: the control (CON) group, the ALD group (The mice were received continuous ALD infusion at 0.75 µg/h via a mini-osmotic pump and the pumps were replaced every 6 weeks for the entire duration of the study; CAS NO: 52-39-1, Cayman Chemical, Ann Arbor, MI, USA), and the esaxerenone-treated group (ESA group) (aldosterone infusion + esaxerenone administration via diet at a dosage of 1 mg/kg/day, kindly provided by Daiichi Sankyo Co., Ltd., Tokyo, Japan). The mice were acclimatized and fed for 7 days before modeling. The mice were anesthetized via chamber induction with 4% isoflurane in 100% oxygen (1 L/min) until loss of righting reflex. Surgical anesthesia was maintained via nose cone with 1.5–2% isoflurane in oxygen (0.5 L/min), confirmed by absent pedal reflex and stable respiration. The modeling method has been described previously [8]. After twelve weeks, animals were initially anesthetized with a low concentration of isoflurane, followed by a rapid transition to a high concentration (5%) to induce immediate loss of consciousness, after which kidney tissue samples and blood were collected.

Renal pathological specimens were obtained as previously described [8]. Hematoxylin and eosin (H&E) staining, Masson staining, and immunohistochemistry were performed with antibodies against F4/80 (1:100; Servicebio, Wuhan, China, Cat# GB113373), CD68 (1:100, Abcam, Cambridge, UK, Cat# ab53444), $\alpha$-SMA (1:100, Servicebio, Cat# GB13044), vimentin (1:100, Abcam, Cat# ab92547), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) (1:50, Novus, Centennial, CO, USA; Cat# NB600-1008), podoplanin (1:50, Bioss, Woburn, MA, USA, Cat# bs-1048R), vascular endothelial growth factor receptor-3 (VEGFR3) (1:50, Abcam, Cat# ab27278), type III collagen (COL III) (1:100, Abcam, Cat# ab283694), IL-1$\beta$ (1:100, Abcam, Cat# ab283822), TNF-$\alpha$ (1:100, Abcam, Cat# ab307164), TGF-$\beta$1 (1:100, Abcam, Cat# ab215715), and VEGFC (1:100, Immunoway, Plano, TX, USA, Cat# Y75297). The sections were viewed under a Leica inverted microscope (DMI6000-CS; Leica Microsystems, Wetzlar, Germany). A Leica TCS SP8 instrument equipped with LAS AF software was used for observation and imaging.

On H&E-stained slides, inflammatory cell infiltration and tubulointerstitial changes were graded semiquantitatively by two investigators in a blinded fashion. A 0–6 scoring scale was used to evaluate the two items, where scores of 0, 1, 2, and 3 represented normal, mild, moderate, and severe conditions, respectively [9]. The percentage of the collagen positive area was semiquantitatively graded by Masson staining. The images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The kidneys were perfused with saline, and dehydrated in 20% and 30% sucrose solutions, and the OCT complex was frozen for immunofluorescence staining (Sakura, Torrance, CA, USA). After being taken from the frozen samples, 6 µm kidney sections were stained with Alexa Fluor 555-coupled $\alpha$-SMA (1:300, Abcam, Cat# ab175651) or the following noncoupled antibodies for staining: anti-LYVE-1 (1:50, Novus, Cat# NB600–1008), vimentin (1:100, Affinity, Nottingham, UK, Cat# BF8006), COL III (1:100, Abcam, Cat# ab283694), anti-VEGFC (1:100, Immunoway, Cat# Y75297), and anti-F4/80 (1:100, Servicebio, Cat# GB113373). Second or third fluorescence staining of the sections was performed. After sections were stained, nuclei were stained with or without DAPI, sealed and photographed for observation under a confocal microscope (CTS SP8, Leica Microsystems, Wetzlar, Germany).

Kidney tissues, RAW264.7 cells, and human lymphatic endothelial cells (HLECs) were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors, and total protein was extracted. Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‒PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk at room temperature for 2 h, the membranes were incubated with the following primary antibodies at 4 °C overnight: vimentin (1:1000, Abcam, Cat# ab8978), IL-1$\beta$ (1:300, Abcam, Cat# ab283822), IL-1$\beta$ (1:500, Affinity, Cat# AF5103), TGF-$\beta$1 (1:1000, Abcam, Cat# ab215715), VEGFR3 (1:500, Abcam, Cat# ab27278), $\alpha$-SMA (1:1000, ABclonal, Wuhan, China, Cat# A17910), TNF-$\alpha$ (1:1000, ABclonal, Cat# A0277), podoplanin (1:1000, ABclonal, Cat# A13261), LYVE-1 (1:1000, ABclonal, A4352), VEGFC (1:1000, Immunoway, Cat# Y75297), and NR3C2 (1:300, Proteintech, Wuhan, China, Cat# 21854-1-AP). On the second day, the membrane was incubated with a secondary antibody (1:10,000) at room temperature for 1 h. Odyssey imaging system (LI-COR system, Lincoln, NE, USA) or e-BLOT system (Shanghai e-BLOT Photoelectrics Technology Co., Ltd., Shanghai, China) was used to detect the protein bands, determination of target protein bands with ImageJ software (National Institutes of Health) relative to the housekeeping protein such as GAPDH (1:1000, Affinity, Cat# T0004), $\beta$-actin (1:1000, Affinity, Cat# T0022), $\beta$-Tubulin (1:1000, Affinity, Cat# T0023), or PCNA bands (1:1000, Proteintech, Cat# 10205-2-AP).

RAW264.7 (Cat# CL-0190, Procell Life Science and Technology Co., Ltd., Wuhan, China) was cultured as previously described [10]. HLECs (Cat# HUM-CELL-0053, PriCells, Wuhan, China) were cultured in endothelial cell medium (ECM, ScienCell, Carlsbad, CA, USA) supplemented with 10% FBS, 1% endothelial cell growth supplement (ECGS) and 1% penicillin and streptomycin. The cells were maintained at 37 ℃ in a humidified atmosphere of 5% CO₂. All the cell lines were validated by STR profiling and tested negative for mycoplasma.

RAW264.7 cells were divided into 3 groups: the CON group, ALD group (treated with 10^-7 mol/L ALD for 24 h), and ESA group (treated with 10^-6 mol/L esaxerenone 2 h before aldosterone treatment). HLECs were divided into 6 groups: the CON group, the ALD group (treated with 10^-7 mol/L aldosterone for 24 h), the ESA group (treated with 10^-6 mol/L esaxerenone 2 h before aldosterone treatment), the TGF-$\beta$1 group (10 ng/mL administered for 24 h; MCE, Shanghai, China, Cat# HY-P70543), the TGF-$\beta$1+LY2109761 group (TGF-$\beta$1 inhibitor; cells were treated with 2 $\times$ 10^-6 mol/L LY2109761 2 h before TGF-$\beta$1 treatment, MCE, Shanghai, China, Cat# HY-12075/CS-0571), and the ALD+LY2109761 group (pretreated with 2 $\times$ 10^-6 mol/L LY2109761 2 h before aldosterone treatment). In these experiments, the cells were divided into the CON group, the ALD group, the ESA group, the VEGFC group (MCE, Shanghai, China, Cat#: HY-P7313, 100 ng/mL was administered for 24 h), the VEGFC+VEGFR-3-IN-1 group (MCE, Shanghai, China, Cat#: HY-132305/CS-0200376, VEGFC receptor blocker, cells were treated with 10^-7 mol/L VEGFR-3-IN-1 2 h prior to VEGFC treatment), and the ALD+VEGFR-3-IN-1 group (cells were pretreated with 10^-7 mol/L VEGFR-3-IN-1 2 h before aldosterone treatment). In addition, RAW264.7, rat kidney fibroblasts (RKF; Cat#: RAT-iCELL-u014, Cellverse Bioscience Technology Co., Ltd., Shanghai, China), distal convoluted tubule cells (DCT; Cat#: CRL-3250, American Type Culture Collection, Manassas, VA, USA), and Human Kidney-2 cell (HK-2; Cat#: CL-0109, Procell Life Science & Technology, Wuhan, China) cells were stimulated with 10^-7 mol/L aldosterone for 24 hours, after which VEGFC expression was detected by Western blot.

Statistical analyses were conducted using SPSS statistics software (version 26.0, IBM, Armonk, NY, USA). After normality assessment of the data via the Shapiro-Wilk test, one-way ANOVA was performed followed by a least significant difference test for multiple groups. The date are presented as the means $\pm$ standard deviations (SDs). Statistical significance was defined as a p-value 0.05.

LYVE-1, Podoplanin, and VEGFR3 are markers of lymphatic endothelial cells. Immunohistochemical staining of the kidneys of mice treated with aldosterone at 12 weeks revealed that renal interstitial lymphatic vessel markers were significantly increased and that lymphangiogenesis was significantly reduced after esaxerenone treatment (Fig. 1A). The levels of VEGFR3, LYVE-1, and Podoplanin in renal tissues (determined by Western blot of the whole kidney) reinforced the results of immunohistochemical staining (Fig. 1B). In addition, in the 12-week aldosterone-treated mouse model, H&E staining showed pathologic changes in the kidneys of aldosterone-treated mice, and Masson staining showed that the aldosterone-treated mice had significantly more renal collagen deposition than the control mice (Supplementary Fig. 1).

Fig. 1.

Aldosterone induces renal lymphangiogenesis in mice. (A) Immunohistochemical staining was performed with anti-VEGFR3, LYVE-1, and Podoplanin antibodies to label lymphatic vessels (scale bar = 50 µm). (B) Western blotting was performed to analyze VEGFR3, LYVE-1, and Podoplanin in renal tissues. The data are expressed as the mean $\pm$ standard deviation (n = 6). ^*p 0.05 vs. the CON group, ^#p 0.05 vs. the ALD group. VEGFR3, vascular endothelial growth factor receptor-3; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; CON, control; ALD, aldosterone.

The classical pathway for lymphangiogenesis is the VEGFC/VEGFR3 signaling pathway, and VEGFC is produced primarily by renal tubular cells and macrophages [11, 12]. Aldosterone-treated mice exhibited abundant infiltration of macrophages labeled with F4/80 or CD68 which was inhibited by esaxerenone (Fig. 2A). Aldosterone can induce inflammatory macrophage infiltration and the secretion of inflammatory cytokines and growth factors, which mediate the inflammatory response and lead to tissue damage [13]. Immunohistochemical staining and western blot for TNF-$\alpha$, IL-1$\beta$, TGF-$\beta$1 and VEGFC revealed that the levels of these cytokines were significantly greater in the ALD group than in the CON group, and these effects were mitigated by esaxerenone treatment. The expression of VEGFC was predominantly observed in the distal tubules in the CON group and was significantly upregulated following aldosterone treatment, particularly in the tubulointerstitial infiltrating cells (Fig. 2A,B). Next, we studied the origin of VEGFC in relation to macrophages. Immunofluorescence labeling of renal tissue for F4/80 and VEGFC revealed that the renal mesenchyme in the ALD group had significantly more F4/80⁺VEGFC⁺ cells compared with the other groups, suggesting that aldosterone promotes the secretion of VEGFC by macrophages. The number of double-positive cells was markably reduced by treatment with esaxerenone (Fig. 2C). In addition, western blot was also used to detect the expression of VEGFC in RAW264.7, RKF, DCT and HK-2 cells treated with aldosterone (10^-7 mol/L aldosterone for 24 h), and the results revealed that the expression of VEGFC in these cells was upregulated after induction with aldosterone (Supplementary Fig. 2). These results indicated that VEGFC could be secreted by a variety of cells, such as renal tubular epithelial cells, fibroblasts, and especially macrophages, in aldosterone-treated mice.

Fig. 2.

Aldosterone stimulates the infiltration of macrophage and the secretion of inflammatory cytokines, VEGFC and TGF-$\beta$1 in mice. (A) Immunohistochemical staining was performed to detect macrophages, inflammatory cytokines, and TGF-$\beta$1 using anti-CD68, anti-F4/80, anti-TNF-$\alpha$, anti-IL-1$\beta$, anti-TGF-$\beta$1, and anti-VEGFC antibodies (scale bar = 50 µm). (B) Western blotting was performed to analyze the protein levels of IL-1$\beta$, TNF-$\alpha$, TGF-$\beta$1, and VEGFC in renal tissue. (C) Immunofluorescence staining of kidney sections with anti-F4/80 antibody (green) and anti-VEGFC antibody (red) was performed to study the relationship between macrophages and VEGFC. The cell nuclei (blue) were stained with DAPI (scale bar = 50 µm). Enlarged images showed a 5× magnification of the areas within the white rectangles in the merged images. The data are expressed as the mean $\pm$ standard deviation (n = 6). ^*p 0.05 vs. the CON group, ^#p 0.05 vs. the ALD group.

To further investigate the roles of aldosterone and VEGFC in lymphangiogenesis, HLECs were cultured for 24 h with aldosterone and the inhibitor esaxerenone, VEGFC and the inhibitor VEGFR-3-IN-1, or aldosterone+VEGFR-3-IN-1. Cell proliferation was assessed with a CCK8 assay. The proliferation of HLECs in the aldosterone and VEGFC treated groups was markedly greater than that in CON group (Fig. 3A). To further investigate whether aldosterone could promote the secretion of VEGFC by HLECs, we then detected the expression of VEGFC in HLECs stimulated with aldosterone through western blot. The results showed that aldosterone could upregulate the expression of VEGFC in HLECs, and this process was inhibited by esaxerenone (Fig. 3B), further confirming that aldosterone regulated the secretion of VEGFC by HLECs through the activation of MR. In addition, aldosterone and VEGFC directly promoted the migration rate and tube formation capacity of HLECs, whereas the VEGFC inhibitors VEGFR-3-IN-1 and esaxerenone attenuated these effects (Fig. 3C,D). These findings indicated that MR/VEGFC might play a regulatory role in lymphangiogenesis.

Fig. 3.

Aldosterone and VEGFC promote lymphangiogenesis through mediating the proliferation, migration, and tube formation of HLECs. (A) VEGFC-induced proliferation of HLECs was determined with a CCK8 assay. (B) Western blotting was performed to analyze VEGFC expression in HLECs. (C) VEGFC promotes lymphatic vessel endothelial cell migration (scale bar = 100 µm) and (D) tube formation (scale bar = 100 µm). The data are expressed as the mean $\pm$ standard deviation (n = 6). ^*p 0.05 vs. the CON group, ^#p 0.05 vs. the ALD/VEGFC group. HLECs, human lymphatic endothelial cells; CCK8, cell counting kit-8.

To confirm the in vivo findings, an in vitro study was performed on aldosterone-treated RAW264.7 cells. Western blots revealed that the expression of VEGFC, TGF-$\beta$1, TNF-$\alpha$, and IL-1$\beta$ was elevated in aldosterone-treated RAW264.7 cells compared with CON (Fig. 4A). Moreover, the ELISA results for aldosterone-treated RAW264.7 cell supernatants were consistent with the above results (Fig. 4B). Aldosterone acts through MR activation [14]. Western blot analysis demonstrated that total MR expression levels were comparable among the CON, ALD, and ESA groups. However, a significant difference was observed in the nuclear expression of MR, with higher levels detected in the ALD group than in the CON group. whereas nuclear accumulation of MR was inhibited by esaxerenone (Fig. 4A).

Fig. 4.

Aldosterone activates MR and induces RAW264.7 to secrete inflammatory cytokines, TGF-$\beta$1 and VEGFC. (A) Western blots for VEGFC, TGF-$\beta$1, TNF-$\alpha$, IL-1$\beta$, and MR in the total and nuclear fractions of RAW264.7 cells. (B) ELISA for serum VEGFC, TGF-$\beta$1, TNF-$\alpha$, and IL-1$\beta$ protein levels in aldosterone-treated RAW264.7 cells. The data are expressed as the mean $\pm$ standard deviation (n = 6). ^*p 0.05 vs. the CON group, ^#p 0.05 vs. the ALD group.

EndMT is a recognized cell transdifferentiation type that has emerged as an alternative source of tissue myofibroblasts. TGF-$\beta$1 can induce EndMT. Numerous studies have shown that EndMT is involved in the development of renal fibrosis [15]. In this study, we conducted immunofluorescence staining for LYVE-1 and vimentin in renal tissues. The number of LYVE-1⁺ vimentin⁺ cells was meaningfully increased in the kidneys of aldosterone-treated mice, and esaxerenone remarkably decreased the number of these double-positive cells (Fig. 5A). In the CON and ESA groups, we selected the locations in which LYVE-1⁺ and vimentin⁺ cells were adjacent to each other, indicating that little lymphatic endothelial cell transformation into myofibroblasts occurred in both CON group and the ESA group (Fig. 5A). We also performed immunofluorescence labeling of kidney tissues with LYVE-1 and $\alpha$-SMA antibodies, and the results were consistent with those described above (Fig. 5B). Immunohistochemical staining for vimentin, $\alpha$-SMA and collagen III revealed significantly greater collagen deposition in aldosterone-treated mice than in control mice (Fig. 5C). Using western blot, we semiquantified renal tissue vimentin and $\alpha$-SMA expression, and the results were consistent with those of the histochemical analysis (Fig. 5D). Moreover, to elucidate the mechanism by which aldosterone promotes EndMT in HLECs, we incubated HLECs with aldosterone in the presence or absence of esaxerenone for 24 hours in vitro. Flow cytometry of HLECs cultured with aldosterone revealed that vimentin and $\alpha$-SMA expression was significantly greater in ALD treated cells than in control cells, while the addition of the MR antagonist esaxerenone inhibited the transformation of the lymphatic endothelium into myofibroblasts (Fig. 5E). These results suggested that aldosterone promoted the transformation of lymphatic endothelial cells into myofibroblasts and that esaxerenone inhibited EndMT.

Fig. 5.

Aldosterone induces EndMT in mouse kidneys and HLECs. (A) Kidney sections were immunofluorescently stained with antibodies against the lymphatic endothelial cell marker LYVE-1 (green) and the myofibroblast marker vimentin (red) to identify EndMT (cells expressing both markers indicate EndMT; nuclei (blue) were stained with DAPI) (scale bar = 50 µm). Enlarged images showed a 5× magnification of the areas within the white rectangles in the merged images. (B) Kidney sections were subjected to immunofluorescence staining with LYVE-1 antibody (green) and $\alpha$-SMA antibody (red) to identify EndMT (cells expressing both markers indicate EndMT; cell nuclei (blue) were stained with DAPI) (scale bar = 50 µm). Enlarged images showed a 5× magnification of the areas within the white rectangles in the merged images. (C) Myofibroblastic renal infiltration and collagen deposition were examined using immunohistochemical staining for $\alpha$-SMA, vimentin and collagen III (scale bar = 50 µm). (D) Western blots for vimentin and $\alpha$-SMA in kidney tissues (n = 6). (E) Flow cytometry analysis of vimentin and $\alpha$-SMA expression in HLECs treated with ALD. Q2 indicates the percentage of vimentin⁺/$\alpha$-SMA⁺ and VEGFR3⁺ EndMT cells (n = 3). The data are expressed as the mean $\pm$ standard deviation. ^*p 0.05 vs. the CON group, ^#p 0.05 vs. the ALD group. EndMT, endothelial-to-mesenchymal transition.

EndMT contributes to fibrosis by driving collagen production, as observed in the analysis of aldosterone-infused mouse kidneys via trichromatic confocal microscopy and Z-stacks, which revealed an increase in the coexpression of LYVE-1⁺ vimentin⁺ type III collagen cells. In contrast, the ESA group had significantly fewer coexpressing cells and significantly less fibrosis (Fig. 6A,B; Supplementary Video). These results suggested that collagen secretion by lymphatic endothelial cells undergoing phenotypic transformation was involved in renal fibrosis.

Fig. 6.

EndMT produces collagen to participate in renal fibrosis. (A) Three-color confocal microscopy images of cells coexpressing LYVE-1 (green), vimentin (red), and collagen III (blue) (scale bar = 50 µm). Enlarged images showed a 5× magnification of the areas within the white rectangles in the merged images. (B) Z-stack images showing the coexpression of LYVE-1 (green), vimentin (red), and collagen III (blue) in the ALD groups (scale bar = 25 µm) (also shown in the Supplementary Video).

To further investigate the effects of aldosterone and TGF-$\beta$1 on EndMT, we cultured HLECs with aldosterone and the antagonist esaxerenone or with TGF-$\beta$1 and the receptor blocker LY2109761 for 24 h. Flow cytometry demonstrated that the expression of vimentin and $\alpha$-SMA was upregulated in the ALD group and the TGF-$\beta$1 group compared with the CON group. The addition of esaxerenone or LY2109761 inhibited the transformation of the lymphatic endothelium to myofibroblasts, and the number of $\alpha$-SMA or vimentin positive cells in the ALD+LY2109761 group was lower than that in the ALD group (Fig. 7A), indicating that the TGF-$\beta$1 receptor blocker antagonized the aldosterone-induced EndMT. Semiquantitative analysis of Vimentin and $\alpha$-SMA expression by western blot was performed, and the results supported the above conclusions (Fig. 7B). To further investigate whether aldosterone regulated the expression of TGF-$\beta$1 in HLECs via MR, we detected TGF-$\beta$1 expression in HLECs stimulated with aldosterone using western blot. The results showed that aldosterone upregulated TGF-$\beta$1 expression in HLECs, and this effect was inhibited by esaxerenone (Supplementary Fig. 3), further confirming that aldosterone regulated TGF-$\beta$1 expression in HLECs by activating MR.

Fig. 7.

Aldosterone and TGF-$\beta$1 induce EndMT in HLECs. (A) Flow cytometry analysis of vimentin and $\alpha$-SMA expression in HLECs treated with ALD and TGF-$\beta$1; Q2 indicates the percentage of vimentin⁺/$\alpha$-SMA⁺ and VEGFR3⁺EndMT cells (n = 3). (B) Western blots showing vimentin and $\alpha$-SMA expression in HLECs (n = 3) (B). The data are expressed as the mean $\pm$ standard deviation. ^*p 0.05 vs. the CON group, ^#p 0.05 vs. the ALD/TGF-$\beta$1 group.