Linagliptin Protects against Endotoxin-Induced Acute Kidney Injury in Rats by Decreasing Inflammatory Cytokines and Reactive Oxygen Species

Abstract

Septic shock can increase pro-inflammatory cytokines, reactive oxygen species (ROS), and multiple organ dysfunction syndrome (MODs) and even lead to death. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been proven to exert potential antioxidant and anti-inflammatory effects. We investigated the effects of linagliptin on endotoxic shock and acute kidney injury (AKI) in animal and cell models. In the cell model, linagliptin attenuated ROS by activating the AMP-activated protein kinase (AMPK) pathway, restoring nuclear-factor-erythroid-2-related factor (Nrf2) and heme oxygenase 1 (HO-1) protein, and decreasing pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β)). In the animal model, 14-week-old conscious Wistar-Kyoto rats were randomly divided into three groups (n = 8 in each group). Endotoxin shock with MODs was induced by the intravenous injection of Klebsiella pneumoniae lipopolysaccharide (LPS, 20 mg/kg). Linagliptin improved animal survival without affecting hemodynamic profiles. In the histopathology and immunohistochemistry examinations of the rat kidneys, linagliptin (10 mg/kg) suppressed nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inducible nitric oxide synthase (iNOS), decreased injury scores, and preserved E-cadherin expression from LPS damage. In conclusion, linagliptin ameliorated endotoxin-shock-induced AKI by reducing ROS via AMPK pathway activation and suppressing the release of TNF-α and IL-1β in conscious rats.

Keywords: AMPK pathway; NF-κB; acute kidney injury; conscious rats; dipeptidyl peptidase-4 inhibitors; endotoxic shock; linagliptin; pro-inflammatory cytokines; reactive oxygen species.

Figure 1

Reactive oxygen species (ROS) assay of NRK-52E cells after 12 or 24 h incubation with/without 10 μg/mL lipopolysaccharide (LPS) or 0.1 µg/mL linagliptin (LGP), using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) as a fluorescent probe and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) to stain the nuclear acid. (A) 0.1 µg/mL linagliptin attenuated ROS induced by 10 μg/mL LPS in NRK-52E cells. (B) Semi-quantitative results of the ROS production by software ImageJ 1.47t.

Figure 2

Quantitative real-time polymerase chain reaction (RT-qPCR) of NRK-52E cells after 12 or 24 h incubation with/without 10 μg/mL lipopolysaccharide (LPS) or 0.1 µg/mL linagliptin (LGP). The quantification results of RT-qPCR for (A) nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), (B) chemokine ligand 2 (CCL2), (C) Interleukin -1β (IL-1β), and (D) IL-6. The relative normalized expression of target genes [2^−ΔΔCq] were calculated using the reference gene β-actin as the internal control, sample “12 h control” for the rest of the groups in 12 h, and sample “24 h control” for the rest of the groups in 24 h. The error bar represents the standard error of the mean (SEM). * p < 0.05 for the LPS group compared with the Control group. # p < 0.05 for the LPS + Linagliptin group compared with the LPS group.

Figure 3

Western blotting of NRK-52E cells 12 or 24 h after incubation with/without 10 μg/mL LPS or 0.1 µg/mL linagliptin (LGP). Representative image and quantification results for inflammatory mediators include (A) extracellular-signal-regulated kinases (ERK) and phospho-ERK (p-ERK at tyrosine 204), (B) Protein kinase B (Akt) and phospho-Akt (serine 473), (C) NF-κB p65 and phospho-NF-κB p65 (at serine 536), (D) AMP-activated protein kinase (AMPK) ⍺1/2 and phospho-AMPK ⍺1/2 (at threonine 183 and threonine 172, respectively), (E) Nrf2, phosphor-Nrf2 (at serine 42), and (F) HO-1. The band of each sample was quantified by software ImageJ 1.47t, and all results were normalized to the value of the control β-actin. * p < 0.05 for the LPS group compared with the Vehicle group. # p < 0.05 for the LPS + Linagliptin group compared with the LPS group.

Figure 4

Changes in (A) survival curves, (B) mean arterial pressure, (C) heart rate, and (D) glucose after endotoxic shock in conscious rats. * p < 0.05 for the LPS group compared with the Vehicle group. Vehicle (n = 8), LPS (n = 4), and LPS + Linagliptin (n = 6) groups at 48 h after LPS administration.

Figure 5

Changes in serum (A) glutamic oxaloacetic transaminase (GOT), (B) glutamic pyruvic transaminase (GPT), (C) blood urea nitrogen (BUN), (D) creatinine (Cre), (E) lactic dehydrogenase (LDH), and (F) creatine phosphokinase (CPK) after endotoxic shock in conscious rats. * p < 0.05 for the LPS group compared with the Vehicle group. # p < 0.05 for the LPS + Linagliptin group compared with the LPS group. Vehicle (n = 8), LPS (n = 4), and LPS + Linagliptin (n = 6) groups at 48 h after LPS administration.

Figure 6

Changes in serum (A) tumor necrosis factor-α (TNF-α) and (B) interleukin-1β (IL-1β) after endotoxic shock in conscious rats. * p < 0.05 for the LPS group compared with the Vehicle group. # p < 0.05 for the LPS + Linagliptin group compared with the LPS group. Vehicle (n = 8), LPS (n = 4), and LPS + Linagliptin (n = 6) groups at 48 h after LPS administration.

Figure 7

Post-treatment with linagliptin affected the histopathologic changes in the kidneys. Histologic sections stained with hematoxylin and eosin (magnification ×200) from the (A) LPS group, (B) LPS + Linagliptin group, and (C) Vehicle group. Renal tissue injury score after endotoxic-shock-induced acute renal failure in rats (D). Immunohistochemical staining for E-cadherin in kidneys (magnification ×200) from the (E) LPS group, (F) LPS + Linagliptin group, and (G) Vehicle group. E-cadherin-positive tubule score after endotoxic-shock-induced acute renal failure in rats (H). Immunohistochemical staining for iNOS in kidneys (magnification ×200) from the (I) LPS group, (J) LPS + Linagliptin group, and (K) Vehicle group. Inducible nitric oxide synthase (iNOS)-positive tubule score after endotoxic-shock-induced acute renal failure in rats (L). Immunohistochemical staining for NF-κB in kidneys (magnification ×200) from the (M) LPS group, (N) LPS + Linagliptin group, and (O) Vehicle group. NF-κB-positive tubule score after endotoxic-shock-induced acute kidney injury in rats (P). * p < 0.05 for the LPS group compared with the Vehicle group. # p < 0.05 for the LPS + Linagliptin group compared with the LPS group. Vehicle (n = 8), LPS (n = 4), and LPS + Linagliptin (n = 6) groups at 48 h after LPS administration.

Figure 8

Schematic summary graph of the protective mechanism of linagliptin against lipopolysaccharide (LPS)-induced kidney injury by restoring the AMP-activated protein kinase (AMPK) pathway, nuclear factor erythroid 2-related Figure 2. and heme oxygenase 1 (HO-1) protein, and decreasing pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β, IL-6)), attenuating reactive oxidative species (ROS) overproduction. NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; AKT, protein kinase B; ERK, extracellular signal-regulated kinases.