*Contributed equally
Chronic kidney disease (CKD) involves progressive and irreversible loss of renal function, often causing complications and comorbidities and impairing the function of various organs. In particular, lung injury is observed not only in advanced CKD but also in early-stage CKD. The present study investigated the potential involvement of mineralocorticoid receptors (MRs) and lymphatic vessels in lung injury using a 180-day unilateral ureteral obstruction (UUO) model for CKD. Changes in lung associated with lymphangiogenesis and inflammatory were analyzed in UUO rats. The pathology of the lung tissue was observed by hematoxylin and eosin and Masson's staining. Detection of the expression of lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1), Podoplanin, vascular endothelial growth factor receptor-3 (VEGFR-3) and VEGF C to investigate lymphangiogenesis. The mRNA and protein expression levels of IL-1β, monocyte chemotactic protein 1, tumor necrosis factor-α, nuclear factor κB, phosphorylated serum and glucocorticoid-induced protein kinase-1 and MR were evaluated using western blot, reverse transcription-quantitative PCR, immunohistochemical staining and immunofluorescence staining. In the present study, long-term UUO caused kidney damage, which also led to lung inflammation, accompanied by lymphangiogenesis. However, treatment with eplerenone, an MR blocker, significantly reduced the severity of lung injury and lymphangiogenesis. Therefore, lymphangiogenesis contributed to lung fibrosis in UUO rats due to activation of MRs. In addition, transdifferentiation of lymphatic epithelial cells into myofibroblasts may also be involved in lung fibrosis. Collectively, these findings provided a potential mechanism for lung fibrosis in CKD and suggested that the use of eplerenone decreased kidney damage and lung fibrosis.
Chronic kidney disease (CKD) is a key contributor to non-communicable disease morbidity and mortality. In 2017, there were 697.5 million CKD cases worldwide and the prevalence of CKD was estimated to be 9.1% (
Kidney damage activates the renin-angiotensin-aldosterone system (RAAS), which serves a key role in inflammation and fibrosis (
Animal care and experimental protocols were in accordance with the Regulations of the Ministry of Health of the People's Republic of China on Animal Management (documentation no. 55; 2001). All animals received humane care according to Hebei University of Chinese Medicine animal care guidelines (
Male Wistar rats (n=30; age, ~7 weeks; weight, 170±10 g; cat. no. SCXK 2018-004; Animal Center of Hebei Medical University) were used. Rats had free access to standard chow and tap water at a temperature of 23±2˚C, relative humidity of 60±5% and a 12/12-h light-dark cycle.
After 7 days of acclimatization, the rats were divided randomly into three groups (n=10/group) as follows: Sham, UUO and UUO + EPL (EPL), 10 rats per group. In the sham group, the left ureter was exposed without ligation. In the UUO group, the left ureter was ligated. Following UUO, the rats in the UUO + EPL group were orally administered mixed EPL (1.25 g/kg diet, equivalent to 100 mg eplerenone/kg/day; Pfizer, Inc.). After 180 days, lung tissue was collected for analysis, as described previously (
Lungs were harvested and fixed overnight in 4% paraformaldehyde, then dehydrated with 70, 85, 95, 100% anhydrous ethanol for 2 h each, xylene for 15 min, the above steps were all performed at room temperature, then paraffin embedded. Lung sections (thickness, 4 µm) were stained with hematoxylin and eosin (H&E) and Masson's stain. For H&E and Masson's staining, nuclei were stained with hematoxylin for 5 min at 37˚C, 0.5% eosin for 3 min and Ponceau and aniline blue for 5 min, respectively. H&E staining was evaluated using the Ashcroft score (
Total protein from lung tissue was extracted with RIPA lysis buffer (cat. no. BB-3201-100 ml; Bestbio) and protein concentration was determined using bicinchoninic acid kit. The concentration of each histone was adjusted to 5 mg/ml. A total of 8 µl (40 µg) of each histone sample per lane was loaded onto a 10% gel for western blotting and transferred to polyvinylidene difluoride (PVDF) membranes. Following blocking with 5% non-fat milk at room temperature for 2 h, PVDF membranes were incubated overnight at 4˚C with primary antibodies against NF-κB (p65), IL-1β, MCP-1, TNF-α, VEGFR-3, LYVE-1 (1:500; cat. no. A4352; Abclonal Biotech Co., Ltd.), podoplanin (1:500; cat. no. A13261; Abclonal Biotech Co., Ltd.), VEGF-C (1:500; cat. no. ab9546; Abcam), MR (1:500; cat. no. ab64457; Abcam), phosphorylated-SGK-1 (p-SGK-1; 1:500; cat. no. AF3001; Affinity Biosciences) and GAPDH (1:2,000; cat. no. 60004-1-1 g; ProteinTech, Group, Inc.). The PVDF membranes were incubated with fluorescein-conjugated secondary antibodies (1:20,000; cat. no. D10603-15; LI-COR) for 1 h at room temperature. Protein bands were visualized using Odyssey Infrared Imaging System (LI-COR Biosciences) and protein expression was quantified using ImageJ version 1.8.0 with GAPDH as loading control.
Total RNA from whole tissue samples was isolated using the EZNA™ Total RNA kit II (Omega Bio-Tek, Inc.). Total RNA was reverse transcribed into cDNA using MonAmp™ ChemoHS qPCR Mix (Monad Biotech Co., Ltd.) according to the manufacturer's instruction. qPCR was performed on the 7500 Fast Real-Time PCR System (Applied Biosystems; ThermoFisher Scientific, Inc.) using SYBR-Green as the detection fluorophore according to the manufacturer's instruction. The thermocycling conditions were as follows: Initial denaturation for 10 min at 95˚C, followed by 40 cycles of 95˚C for 10 sec, 60˚C for 10 sec and 72˚C for 30 sec. The mRNA expression of IL-1β, MCP-1, TNF-α, SGK-1, NF-κB and GAPDH was measured by 7500 Fast Real-Time PCR System using the following primers: IL-1β forward, 5'-GGCAACTGTCCCTGAACTCAAC-3' and reverse, 5'-AAGCTCCACGGGCAAGACATA-3'; MCP-1 forward, 5'-GCTTGGATGACAGAGGCTTGGAG-3' and reverse, 5'-ATTCACAGGTGGCTTGGCTATGAG-3'; TNF-α forward, 5'-CCAGGTTCTCTTCAAGGGACAA-3' and reverse, 5'-GGTATGAAATGGCAAATCGGCT-3'; SGK-1 forward, 5'-CTTCTGTGGCACGCCTGAGTATC-3' and reverse, 5'-AGCCTCTTGGTCCGGTCCTTC-3'; NF-κB forward 5'-TTTTCAGCACTGATTATAGCAGGTT-3' and reverse, 5'-AAGGTATCGCAGTCCCCACC-3' and GAPDH forward, 5'-GTCCATGCCATCACTGCCACTC-3' and reverse, 5'-CGCCTGCTTCACCACCTTCTTG-3'. The primers for MCP-1, NF-κB, SGK-1 and GAPDH were supplied by Sangon Biotech Co., Ltd. The primers for IL-1β and TNF-α were supplied by Wuhan Servicebio Technology Co., Ltd. The mRNA levels were quantified using the 2-ΔΔCq method and normalized to the internal reference gene GAPDH (
Each experiment was repeated 6 times. Data are presented as the mean ± standard deviation. One-way ANOVA followed by Tukey's post hoc test was used for multiple groups. Tests were performed to determine whether data were normally distributed. Data were analyzed using SPSS version 26.0 (IBM Corp.) and Prism 8.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
The present study focused on lung fibrosis in rats that underwent UUO for 180 days. H&E staining showed that UUO caused thickening of alveolar walls and increased infiltration of inflammatory cells, which suggested that long-term UUO induced lung injury (
To investigate if lymphangiogenesis was involved in the lung fibrosis induced by UUO, tissue sections were immunostained with antibodies against lymphatic endothelial cell-specific proteins (e.g. LYVE-1, podoplanin and VEGFR-3). In Sham rats, lymphatic vessels in the lung were present around the airways and large blood vessels, whereas few lymphatic vessels were observed in the lung parenchyma; in UUO rats, lymphatic vessels close to the airways and large blood vessels were enlarged and more lymphatic vessels were present in the lung parenchyma, most of which were in the area infiltrated by inflammatory cells; moreover, lymphangiogenesis was inhibited by eplerenone (
These results indicated the underlying pathogenesis of fibrosis in the lungs of UUO rats. Lymphangiogenesis was associated with lung fibrosis and the MR-specific blocker eplerenone mitigated UUO-induced lymphangiogenesis. Hence, MRs may be involved in lymphangiogenesis and lung fibrosis.
Immunostaining showed significant inflammatory cell infiltration in the lung of UUO rats. Compared with sham group, the staining intensity of F4/80 and CD68 in the UUO group was significantly increased, F4/80-positive macrophages and CD68-positive T cells were more widespread in the lung of UUO rats and eplerenone treatment mitigated this change (
To investigate the role of MRs in lung injury, immunohistochemical staining, western blotting and RT-qPCR were performed to identify the downstream molecules of MRs, such as SGK-1, p-SGK-1 and NF-κB. The results showed that compared with the Sham group, the activation of MR enhanced the expression of MR, p-SGK-1/SGK-1 and NF-κB in the UUO group and NF-κB was significantly expressed in the nucleus of the lungs of UUO rats, while EPL significantly decreased expression (
To ascertain if lymphatic endothelial cells were involved in UUO-induced lung fibrosis, immunofluorescent co-staining was performed using specific antibodies against the lymphatic endothelial cell-specific marker LYVE-1 and myofibroblast marker α-SMA. Most lymphatic endothelial cells in the lung of UUO rats co-expressed LYVE-1 and α-SMA, indicating endothelial-myofibroblast transition; this co-staining was attenuated by eplerenone (
CKD is an important risk factor for development of end-stage renal and lung disease (
A previous study showed that inflammatory cell infiltration occurs in UUO-induced lung fibrosis, macrophages participate in the development of lung fibrosis via macrophage-myofibroblast transition and MR blockers protect the lung from chronic damage and fibrosis (
Lymphatic vessels contribute to human diseases such as cardiovascular disease (
Lymphangiogenesis has been studied in airway and interstitial lung disease and is associated with tissue repair (
The present study showed that MRs participate directly in lymphangiogenesis in the lung tissue of UUO rats. Long-term UUO in rats leads to increased levels of aldosterone in plasma. This leads to MR activation, accumulation of inflammatory cells and release of proinflammatory factors in the lung (
Myofibroblasts are key effector cells involved in organ fibrosis by continuously accumulating and contracting scar tissue beyond normal repair. Myofibroblasts are the primary collagen-producing cell type during fibrosis, but their origin remains unclear. Evidence suggests that myofibroblasts have multiple cellular origins, including bone marrow-derived fibrocytes and fibroblasts (
One limitation to this study is the lack of observation at multiple time points. The present study only tested indicators after 180 days of modeling; future studies should investigate the dynamic changes in the lung during this process in UUO models of different durations, for example 90 and 120 days. Moreover, future studies should investigate changes in density and contractility of lymphatic vessels and examine whether organelles of lymphatic endothelial cells are damaged to verify the function of lymphatic vessels. Further studies are needed to clarify the mechanism by which eplerenone attenuates long-term UUO-induced lung fibrosis by inhibiting inflammation. The present immunostaining results revealed higher macrophage and T cell accumulation in the lung. However, the role of these cells in release of proinflammatory factors remains unclear. Therefore, further investigation is required to determine the precise molecular mechanisms by which MR promotes lymphangiogenesis using both
In conclusion, the present results suggested a novel mechanism by which lymphangiogenesis participates in the pathogenesis of UUO-induced lung fibrosis. UUO-induced kidney injury increased aldosterone release, which led to activation of MR in the lung and enhancement of inflammation and lymphangiogenesis, resulting in lung damage and fibrosis. The mechanism identified in the present study not only adds to understanding of the pathogenesis of lung injury in patients with CKD but also provides a potential novel therapeutic target for anti-lung fibrosis medications based on crosstalk between the kidney and lung.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
ZL, CZ, JH, GC and LL performed the experiments, collected data and wrote the manuscript. YX, YC, HL, FY, TS and QX analyzed the data and wrote the manuscript. FY, TS and QX confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
The present study protocol was approved by the Ethics Committee of Hebei University of Chinese Medicine (approval no. DWLL2021063).
Not applicable.
The authors declare that they have no competing interests.
Effect of 180-day UUO and treatment with EPL on lung histology and fibrosis. (A) H&E staining of histological changes in the lung. (B) Masson's staining of fibrosis. (C) Immunohistochemistry staining for α-SMA. Magnification, x200. (D) Immunohistochemistry staining for collagen I. Magnification, x400. (E) Ashcroft score for each group. (F) Collagen volume fraction for each group. (G) α-SMA and (H) collagen I positive expression area. Data are presented as the mean ± standard deviation (n=6). #P<0.05 vs. Sham; *P<0.05 vs. UUO. α-SMA, α-smooth muscle actin; UUO, unilateral ureteral obstruction; H&E, hematoxylin and eosin; EPL, eplerenone.
Lymphangiogenesis in the lung of UUO rats. (A) Immunohistochemical staining of lymphatic vessel marker LYVE-1. Magnification, x200. Immunohistochemical staining for (B) podoplanin and (C) VEGFR-3. Magnification, x400. (D) LYVE-1, (E) podoplanin and (F) VEGFR-3 positive expression. (G) Protein expression of podoplanin, LYVE-1, VEGF-C and VEGFR-3 in lung tissue of UUO rats as detected by western blotting. (H) podoplanin, (I) LYVE-1, (J) VEGF-C and (K) VEGFR-3 protein expression. Data are presented as the mean ± standard deviation (n=6). #P<0.05 vs. Sham; *P<0.05 vs. UUO. UUO, unilateral ureteral obstruction; LYVE-1, lymphatic vessel endothelial receptor-1; VEGFR, VEGF receptor; EPL, eplerenone.
Effect of UUO and EPL on expression of F4/80, CD68, MCP-1, IL-1β and TNF-α in the lung of UUO rats. Immunohistochemical staining of (A) F4/80, (B) CD68, (C) MCP-1, (D) IL-1β and (E) TNF-α. Magnification, x400. (F) F4/80, (G) CD68, (H) MCP-1, (I) IL-1β and (J) TNF-α positive expression area. Expression of (K) MCP-1, (L) IL-1β and (M) TNF-α mRNA as detected by RT-qPCR. (N) Protein expression of IL-1β, TNF-α and MCP-1 as detected by western blotting. (O) IL-1β, (P) TNF-α and (Q) MCP-1 protein expression. Data are presented as the mean ± standard deviation (n=6). #P<0.05 vs. Sham; *P<0.05 vs. UUO. MCP-1, monocyte chemotactic protein 1; UUO, unilateral ureteral obstruction; RT-q, reverse transcription-quantitative; TNF, tumor necrosis factor; EPL, eplerenone.
Effect of UUO and EPL on expression of downstream molecules of MRs in lung tissue. Immunohistochemical staining for (A) SGK-1 and (B) NF-κB. Magnification, x400. (C) Protein expression of p-SGK-1, SGK-1, MR and NF-κB as detected by western blotting. (D) SGK-1 and (E) NF-κB positive expression. (F) p-SGK-1 and (G) SGK-1 protein expression in each group. (H) Ratio of p-SGK-1 to SGK-1. (I) MR and (J) NF-κB protein expression. mRNA expression of (K) SGK-1 and (L) NF-κB as detected by RT-qPCR. Data are presented as the mean ± standard deviation (n=6). #P<0.05 vs. Sham; *P<0.05 vs. UUO. UUO, unilateral ureteral obstruction; SGK-1, serum- and glucocorticoid-inducible kinase-1; RT-q, reverse transcription-quantitative; p-, phosphorylated; EPL, eplerenone; MR, mineralocorticoid receptor.
EndMT in the lung of UUO rats. Co-expression of lymphatic vessel (LYVE-1, green) and myofibroblast (α-SMA, red) markers was detected by two-color immunofluorescence staining and indicated EndMT. Magnification, x400. Representative image of LYVE-1 +/α-SMA + EndMT cells is magnification, x8. Nuclei were stained blue with DAPI. UUO, unilateral ureteral obstruction; EndMT, endothelial-mesenchymal transition; LYVE-1, lymphatic vessel endothelial receptor-1; α-SMA, α-smooth muscle actin; EPL, eplerenone.
Cells undergoing endothelial-mesenchymal transition produce collagen in the lung tissue of UUO rats. Three-color confocal laser scanning microscopy of cells co-expressing LYVE-1 (green), α-SMA (red) and collagen I (blue). Lymphatic vessels co-expressing LYVE-1 and α-SMA are surrounded by collagen I. Magnification, x400. LYVE-1, lymphatic vessel endothelial receptor-1; α-SMA, α-smooth muscle actin; UUO, unilateral ureteral obstruction; EPL, eplerenone.