CXCR4 inhibition suppresses Cd-induced renal oxidative stress, apoptosis, and fibrosis by inhibiting the TGF-β1/Smad pathway

2.1 Animals

This study used 30 adult male Wistar albino rats (CDC, Hubei, China) weighing 220 ± 15 g (10 weeks). The rats were housed in a control room at a temperature of 23 ± 2℃ and a humidity of 45 ± 15%. A 12 h light and 12 h dark cycle was maintained and the rats were fed a standard diet and water. After adapting to the surrounding environmental conditions, the rats were used for subsequent experiments.

2.2 Establishment of Cd-induced chronic renal injury model in rats and CXCR4 treatment

Thirty rats were randomly divided into five groups (six rats in each group), Con group the rats were first administered orally 3 ml isotonic NaCl solution kg⁻¹ day⁻¹ and after 2 h they intraperitoneally (i.p.) injected with 1 ml isotonic NaCl solution kg⁻¹ day⁻¹ over 30 successive days. Cd group the rats received CdCl2 (dissolved in an isotonic NaCl solution) at a dose of 3.5 mg kg⁻¹ bw day⁻¹ via i.p. injections for 30 successive days, at the same time 2 h before the CdCl2 injection, the rats were received orally 3 ml isotonic NaCl solution kg⁻¹ day⁻¹. Control animals received only the vehicle (saline). Intraperitoneal injection of 15 mg/kg CXCR4 inhibitor(AMD-3100; Sigma-aldridge) twice weekly in the Cd+si-CXCR4 group. In the Cd+enalapril group, enalapril was used as the positive control drug (intragastrically administered once daily at 10 mg/kg). TGF-β1 group was injected 6 ng/mL of TGF-β 1 through a caudal vein (Sigma Aldrich, USA). One-day after receiving the last treatment after 2 weeks of treatment rats were euthanized with pentobarbital (200 mg/kg bodyweight). Blood and tissue samples were collected from the inferior vena cava of each rat, clots were allowed to form, and then samples were centrifuged. Serum was harvested and used in renal functional tests. The kidney was immediately dissected and weighted. Renal ti ssue was placed in ice-cold 50 mM Tris-HCl buffer (pH 7.4) and homogenates were generated using a tissue homogenizer, producing a 10% (w/v) tissue homogenate. The homogenate was centrifuged 10 min at 4 °C and the supernatant was stored at −70 °C for biochemical tests.

The schematic diagram is as follows:

2.3 Western blot analysis

The kidney tissues were homogenized and centrifuged. The supernatant was discarded and 1000 μL of RIPA lysis solution containing l% phenylmethylsulfonyl fluoride was added (Beyotime Biotechnology, Shanghai, China). The total protein of the cells was collected. The protein concentration of each group was measured using a BCA (Rockford, IL, USA) assay. Samples of protein (20 μL of 30 mg) were used in SDS-PAGE(Sigma Aldrich, St. Louis, MO, USA) electrophoresis and electrotransferred to PVDF membranes (Millipore, Bedford, MA, USA). Connect CXCR41: (ab181020; 1:1000, Abcam, Shanghai, China）, TGF-β1(ab142139; 1:1000, Abcam, Shanghai, China）, TGF-βR1(ab14319; 1:1000, Abcam, Shanghai, China）, P- Smad2(ab280888; 1:1000, Abcam, Shanghai, China）, P- Smad 3(ab52903; 1:1000, Abcam, Shanghai, China）, Smad2 (ab40855; 1:1000, Abcam, Shanghai, China）, Smad3(ab40854; 1:1000, Abcam, Shanghai, China）, Bcl-2(ab; 1:1000, Abcam, Shanghai, China）, Bax (ab40855; 1:1000, Abcam, Shanghai, China）, Cleaved caspase-3(ab40854; 1:1000, Abcam, Shanghai, China）, Fibronectin (FN) (AB268200; 1:1000, Abcam, Shanghai, China) and collagen I(ab138492; 1:1000, Abcam, Shanghai, China) were incubated overnight with the membrane and then with the secondary antibody, GOAT anti-rabbit IgG H&L (AB150077, 1: 2000, Abcam Inc, Cambridge, UK). The protein is color imaged using an ECL chemiluminescent solution {Rhodes, 2011 #69}{Fellous, 2020 #70}.

2.4 Renal function tests

Before the rats were euthanized at week 12, 24-h urine was collected and the levels of protein (24 h-Pro) were measured. Blood was obtained from the abdominal aorta and the serum was centrifuged at 3000g for 10 min at 4 °C. The insoluble material was discarded and the serum was collected. Levels of blood urea nitrogen (BUN) and serum creatinine (sCr) were measured with BUN detection kit and serum creatinine detection kit (StressNarq Bioscience, British Columbia, Canada).

2.5 Detection of oxidative stress factor levels

Kidney samples were weighed to 0.1 g and used to prepare a 10% tissue homogenate. Kidney malondialdehyde (MDA) content was measured by using thiobarbituric acid, superoxide dismutase (SOD) activity was measured by biphenyl triol autoxidation and glutathione peroxidase (GSH-Px) by using a colorimetric method.

2.6 TUNEL staining

Paraffin-embedded tissue sections were dewaxed and hydrated routinely, rinsed twice with PBS, Proteinase K working solution was added, and left to react at 37 °C for 15 min. The samples were blotted with absorbent paper. TdT enzyme reaction solution (50 μL) was added dropwise to each sample and incubated with coverslips at 37 °C for 60 min. The samples were rinsed with PBS three times. Nuclei were stained with DAPI and then rinsed three times with PBS. A quenching agent was added, sealed, protected from light, and then examined on a Leica (DM2500) fluorescence microscope and photographed. Ten high magnification fields were randomly selected for each sample and the number of positive cells was counted.

2.7 Immunohistochemical (IHC) staining

The rabbit anti-rat caspase 3, Bax, and Bcl-2 immunohistochemistry kits (Xin'aosheng, Shanghai, China) were used to observe the expression of the above proteins. The manufacturer’s procedure was followed. After IHC staining, kidney tissue sections were observed under a microscope. The total number of positive cells was calculated using the HPIAS-1000 image analysis system (In Situ Cell Death Detection Kit; Roche Diagnostics, San Francisco, CA, USA).

2.8 PAS staining

Sections were routinely dewaxed, oxidized in periodate solution for 10 min, and rinsed in distilled water for 1 min. The samples were then stained in Schiff's reagent for 20 min and rinsed in running water for 1 min. Then hematoxylin was used to stain the nuclei for 5 min. The samples were then rinsed in running water for 1 min and routinely dehydrated. The transparent samples were sealed in neutral glue.

2.9 Masson's trichrome staining

Masson's trichrome staining was used to visualize the extent of glomerulosclerosis and interstitial fibrosis. The dewaxed and dehydrated sections were then stained with a mild alkaline magenta solution for 3 min, rinsed with deionized water, and treated with 1% phosphomolybdic acid solution for 1 min. Deionized water was used to remove the residual phosphomolybdic acid solution, the samples were stained with 2% blue aniline solution for 2 min, rinsed and dehydrated with 95% ethanol, dried, and embedded. After staining, the collagen fibers of the kidney tissue appeared blue-green under light microscopy and the kidney tissue appeared red.

2.10 Cell culture and Cd treatment

Human renal proximal tubular epithelial (HK-2) cells from Sino Biological (Beijing, China) were stored in RPMI-1640 medium (Manassas, VA, USA) containing 10% FBS and 1% penicillin (Gibco, Carlsbad, CA, USA)/streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C and 5% CO2. Cells were treated with CdCl2 for 24 h, and then Cd (2–10 µM) was added to serum-free medium for 12 h. A micromolar concentration (1–10 µM) of Cd was selected. The schematic diagram is as follows:

2.11 Cell transfection

After cells had grown to 50% confluence, 100 pmol of targeting CXCR4 (si-CXCR4) or TGF-β or negative control siRNA (NC) (Ribobio, Guangdong, China) was transfected with HK-2 cells using Lipo6000 transfection reagent ((Invitrogen, Carlsbad, CA, USA)) in conformity with the manufacturer's instructions. After 4 h of transfection, the original medium was replaced with fresh medium for 24 h until treatment with CdCl₂.

2.12 Measurement of ROS production

To detect changes in intracellular ROS, DCFH-DA fluorescent dye was diluted to 10 μmol/L using serum-free culture medium. Each well was incubated with DCFH-DA 200 μL (Sigma, USA) for 60 min. The appropriate field of view was photographed under a microscope. The fluorescence in images was measured by optical density using Image J software, and the fluorescence intensity of each group was analyzed to indirectly reflect the changes of ROS in cells.

2.13 Immunofluorescence (IF)

HK-2 cells and cells derived from rat tissues were digested with trypsin and re-inoculated in 24-well plates. After 48 h of incubation at 37 °C and 5% CO₂, they were then fixed in 4% paraformaldehyde for 20 min at room temperature. The fixed cells were blocked for 1 h in 5% goat serum and then incubated in primary antibody FN and collagen I (1:500 dilution) overnight at 4 °C. The secondary antibody was incubated for 1 h at room temperature. Diaminobenzidine solution was used for color development. Nuclei were stained with hematoxylin and then cells were observed under a light microscope and images were collected.

2.14 Statistical analysis

SPSS 22.0 software was used in the analysis and all data were expressed as the mean ± SD. One-way ANOVA was used to compare measured indicators between multiple groups and the LSD test was used for two-way comparison between groups. A P-value <0.05 was considered statistically different.

3.1 CXCR4 inhibition suppressed the TGF-β1/Smad pathway in renal tissues of Cd-treated rats.

To assess the role of CXCR4 in renal tissue, CXCR4 inhibition was used in a chronic kidney injury model constructed by using Cd-treated rats. Western blotting results demonstrated that CXCR4 expression was upregulated in the Cd-induced chronic kidney injury model and downregulated after CXCR4 inhibition (Fig. 1A and B). Subsequently, the role of the TGF-β1/Smad signaling pathway in CXCR4 regulation of Cd-induced chronic kidney injury was assessed. The results showed that TGF-β1, TGF-βR1, p-Smad2/3, and Smad2/Smad3 expression was upregulated in the Cd-treated group compared with the control group. Compared with the Cd-treated si-control group, the Cd-treated si-CXCR4 group exhibited decreased expression, In addition, the same was true for the cadmium-treated enalapril group (Fig. 1A, C–F). This suggested that si-CXCR4 could inhibit Cd-induced chronic kidney injury by suppressing the TGF-β1/Smad signaling pathway.

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Fig. 1

Inhibition of CXCR4 suppressed the TGF-β1/Smad pathway in renal tissues of cadmium-treated rats. (A) Western blotting was used to detect CXCR4, TGF-β1, TGF-βR1, p-Smad2/Smad2, and p-Smad3/Smad3 levels. (B–F) Western blotting-based statistical plots of CXCR4, TGF-β1, TGF-βR1, p-Smad2/Smad2, and p-Smad3/Smad3 protein expression. N = 6/per group. *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con.

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3.2 Effect of CXCR4 inhibition on renal function and oxidative stress in Cd-treated rats

Subsequently, the regulation of renal function by si-CXCR4 was assessed in a Cd-induced chronic kidney injury model. The results showed that the renal function-related renal weight index (KW/DW), urinary protein, Scr, and BUN were prominently elevated in the Cd-treated group compared to the control group. Yet si-CXCR4 downregulated and enalapril KW/DW, urinary protein, Scr, and BUN levels. This suggested that CXCR4 downregulation could promote the recovery of renal function in Cd-induced chronic kidney injury rats, the same was true for the cadmium-treated enalapril group (Fig. 2A–D). Previous studies suggest that treatment against oxidative stress can effectively protect renal function and alleviate renal injury (Liu et al., 2019; Wu et al., 2018). Therefore, the role of CXCR4-regulated oxidative stress in Cd-induced chronic kidney injury was assessed. The results showed that oxidative stress factor MDA expression was increased and levels of SOD and GSH-Px expression were lowered in the Cd-treated group. However, si-CXCR4 and enalapril intervention could downregulate MDA and upregulate SOD and GSH-Px levels (Fig. 2E–G), suggesting that CXCR4 inhibition was able to inhibit oxidative stress to improve renal function in a Cd-induced kidney injury rat model.

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Fig. 2

Effect of CXCR4 inhibition on renal function and oxidative stress in cadmium-treated rats. (A–D) Tests for renal function indicators KW/BW, urine protein, creatinine, and BUN. (E–G) Expression of stress-related markers MDA, SOD, and GSH-Px. *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con.

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3.3 CXCR4 inhibition inhibited renal apoptosis in the Cd-treated rats

Kidney injury will produce a large amount of oxygen free radicals. At the same time, the ability of the body to scavenge oxygen free radicals is reduced, leading to a considerable accumulation of oxygen free radicals, which induces apoptosis and ultimately leads to tissue dysfunction (Chen et al., 2019; Shahzad et al., 2016). Subsequently, the effect of CXCR4 on renal apoptosis in Cd-treated rats was evaluated. In the rats with Cd-induced kidney injury, TUNEL staining showed increased apoptosis but apoptosis was reduced by the si-CXCR4 and enalapril intervention (Fig. 3A and B). Likewise, IHC staining (Fig. 3A, C–E) and western blot (Fig. 4A–D) results indicated that Bax and Cleaved-caspase-3 levels were increased, whereas, Bcl-2 levels decreased in the Cd-treated group. However, levels of Bax and Cleaved-caspase-3 decreased and Bcl-2 levels increased in the si-CXCR4 and enalapril treated group. This suggests that si-CXCR4 can inhibit apoptosis through inhibition in Cd-treated rats.

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Fig. 3

CXCR4 inhibition inhibited renal apoptosis in cadmium-treated rats. (A) TUNEL and immunohistochemical staining to detect apoptosis. (B) Statistical plots of TUNEL staining. (C–E) The statistical plots of immunohistochemical detection of apoptosis-associated protein expression. (F–I) Western blot analysis to detect the expression of apoptosis-related proteins Bcl-2, Bax, and Cleaved-caspase-3. *P < 0.05 vs Control (Con); *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con.

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Fig. 4

CXCR4 inhibition inhibited renal apoptosis in cadmium-treated rats. (A–D) Western blot analysis to detect the expression of apoptosis-related proteins Bcl-2, Bax, and Cleaved-caspase-3. *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con.

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3.4 CXCR4 inhibition inhibited renal fibrosis in the Cd-treated rats

The regulation of CXCR4 inhibition on renal fibrosis was further assessed in Cd-treated rats. PAS staining indicated that si-CXCR4 intervention reduced glomerular basement membrane thickening in Cd-treated rats. Masson's trichrome staining showed that glomerular collagen fibers increased in the model group and si-CXCR4 and enalapril intervention reduced collagen fibrils in the model rats (Fig. 5A). In addition, si-CXCR4 and enalapril alleviated glomerulosclerosis and interstitial fibrosis in Cd-treated rats (Fig. 5B and C). Furthermore, IF (Fig. 6A–C) and western blotting (Fig. 6D–F) indicated that the expression of fibrosis marker proteins FN and collagen I were upregulated in Cd-treated rats. In addition, FN and collagen I expression was downregulated in the Cd-treated si-CXCR4 and enalapril group compared to the Cd-treated si-control. These results suggest that si-CXCR4 inhibits fibrosis in rats with Cd-induced kidney injury.

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Fig. 5

Inhibition of CXCR4 suppressed renal fibrosis in cadmium-treated rats. (A) PAS and Masson's trichrome staining. (B, C) Glomerulosclerosis and interstitial fibrosis. *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con.

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Fig. 6

Inhibition of CXCR4 suppressed renal fibrosis in cadmium-treated rats. (A-B) Statistical plots of fibronectin (FN) and collagen I protein expression detected by immunofluorescence. (D–F) Statistical plots of FN and collagen I protein expression detected by western blotting. *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con.

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3.5 TGF-β weakened the effects of CXCR4 inhibition of the TGF-β1/Smad pathway in Cd-treated cells

Previous studies suggest that TGF-β exacerbates renal injury (Chung et al., 2018). Subsequently, we assessed the role of TGF-β in the si-CXCR4 protection of Cd-induced cells. The results of western blotting showed that the TGF-β1/Smad pathway proteins were downregulated in the Cd-treated si-CXCR4 and enalapril group compared to the Cd-treated group. However, TGF-β interfered with TGF-β1, TGF-βR1, p-Smad2/Smad2, and p-Smad3/Smad3 expression that was upregulated in Cd-treated cells (Fig. 7A–E). This suggests that TGF-β attenuated the inhibitory effect of CXCR4 inhibition on the TGF-β1/Smad pathway in Cd-treated cells.

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Fig. 7

TGF-β weakened the effects of CXCR4 inhibition on the TGF-β1/Smad pathway in cadmium-treated cells. (A) Western blot detection of TGF-β1, TGF-βR1, p-Smad2/Smad2, and p-Smad3/Smad3 levels. (B–E) Western blot analysis gave statistical plots of TGF-β1, TGF-βR1, p-Smad2/Smad2, and p-Smad3/Smad3 protein expression. *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con; @P < 0.05 vs Cd + si-CXCR4.

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3.6 TGF-β weakened the effects of CXCR4 inhibition on oxidative stress in Cd-treated cells

Subsequently, the role of TGF-β in the inhibition of oxidative stress by si-CXCR4 in Cd-treated cells was assessed. The results showed that levels of the oxidative stress factors MDA and ROS were increased and SOD and GSH-Px expression was downregulated in the Cd-treated group. However, si-CXCR4 and enalapril intervention in Cd-treated cells was able to downregulate MDA and ROS levels and upregulate SOD and GSH-Px levels. Compared with the Cd-treated si-CXCR4 group, the Cd-treated si-CXCR4 + TGF-β group increased MDA, ROS expression, and the downregulation of SOD and GSH-Px expression (Fig. 8A–E).

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Fig. 8

TGF-β weakened the effects of CXCR4 inhibition on apoptosis in cadmium-treated cells. (A–C) Expression of stress-related indicators MDA, SOD, and GSH-Px were detected. (D, E) ROS levels were detected by ROS staining. *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con; @P < 0.05 vs Cd + si-CXCR4.

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3.7 TGF-β weakened the effects of CXCR4 inhibition on apoptosis in Cd-treated cells

Next, the role of TGF-β in si-CXCR4 inhibited apoptosis in Cd-treated cells was assessed. TUNEL staining (Fig. 9A–B) showed that apoptosis was remarkably increased in Cd-treated cells and si-CXCR4 and enalapril could inhibit the apoptosis rate. However, the apoptosis rate was remarkably increased in the TGF-β group of cells. Similarly, western blot results (Fig. 9C–F) showed that levels of Bax and Cleaved-caspase-3 levels were increased and Bcl-2 levels were downregulated in Cd-treated cells. However, the opposite trend occurred in the si-CXCR4 and enalapril-treated group. The Cd-treated si-CXCR4 + TGF-β group partially increased Bax and Cleaved-caspase-3 and downregulated Bcl-2 levels. This suggests that TGF-β partially reverses the ability of si-CXCR4 to inhibit apoptosis in Cd-treated cells.

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Fig. 9

TGF-β weakened the effects of CXCR4 inhibition on apoptosis in cadmium-treated cells. (A, B) TUNEL staining to detect apoptosis. (C–F) Western blot analysis for expression of apoptosis-associated proteins Bcl-2, Bax, and Cleaved-caspase-3. *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con; @P < 0.05 vs Cd + si-CXCR4.

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3.8 TGF-β weakened the effects of CXCR4 inhibition of fibrosis in Cd-treated cells

In addition, the role of TGF-β and si-CXCR4 was assessed in the inhibition of fibrosis in Cd-treated cells. Western blot (Fig. 10A–C) and IF (Fig. 10D–F) results showed that fibrosis marker proteins FN and collagen I expression were upregulated in Cd-treated rats, compared to the control group. Furthermore, FN and collagen I expression were downregulated in the Cd-treated si-CXCR4 and enalapril group compared to the Cd-treated si-control group. However, TGF-β intervention could upregulate FN and collagen I expression in the Cd-treated si-CXCR4 and enalapril group. These results suggest that TGF-β was able to attenuate the effect of CXCR4 inhibition on fibrosis in Cd-treated cells.

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Fig. 10

TGF-β weakened the effects of CXCR4 inhibition on fibrosis in cadmium-treated cells. (A–C) Western blot analysis of fibronectin (FN) and collagen I expression. (D-F) Immunofluorescence detection of FN and collagen I expression. *P < 0.05 vs Control (Con); #P < 0.05 vs Cd + si-con; @P < 0.05 vs Cd + si-CXCR4.

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