Alpha-1 Antitrypsin Protects Against Cisplatin-Induced Acute Kidney Injury by Restoring Redox and Mitochondrial Homeostasis

Figures

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  Figure 1. Alpha-1 antitrypsin attenuates cisplatin-induced renal dysfunction and tubular damage. (A) Schematic representation of the in vivo experimental protocol. Mice were randomly assigned to four groups (control, alpha-1 antitrypsin (AAT), cisplatin, and cisplatin + AAT) and received AAT and/or cisplatin according to the indicated schedule, followed by the collection of blood and kidney tissues for biochemical and histological analyses. (B,C) Serum blood urea nitrogen (BUN) and creatinine levels in the four groups (n = 4 per group). Cisplatin markedly increased BUN and creatinine levels compared with the control, whereas AAT co-administration significantly attenuated these elevations. (D) Representative Western blots and densitometric analysis of neutrophil gelatinase-associated lipocalin (NGAL) expression in kidney tissue (n = 6 per group). Cisplatin induced a strong increase in NGAL expression, which AAT reduced. (E) Representative periodic acid–Schiff (PAS) staining of renal cortex sections (n = 6 per group), which indicates tubular necrosis, epithelial desquamation, and brush border loss in the cisplatin group, with preservation of tubular architecture in the cisplatin + AAT group.…

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  Figure 2. Alpha-1 antitrypsin reduces oxidative stress and modulates NADPH oxidase isoforms in cisplatin-treated kidneys. (A) Representative immunohistochemical staining for 8-hydroxy-2′-deoxyguanosine (8-OHdG) in renal cortex sections from the four groups (n = 6 per group). (B,C) Quantification and representative images of 8-OHdG and 4-hydroxynonenal (4-HNE) staining (n = 6/group; 10 fields/mouse). Cisplatin markedly increased oxidative stress markers, whereas alpha-1 antitrypsin (AAT) co-administration reduced the expression of these markers. (D) mRNA expression of NADPH oxidase isoforms Nox1, Nox2, and Nox4 in kidney tissue (n = 6 per group); cisplatin significantly upregulated Nox1 and suppressed Nox2 and Nox4, while AAT co-administration partially normalized these changes. (E) mRNA expression of the antioxidant enzymes Sod2 and Cat (n = 6 per group). Cisplatin induced downregulation, which was restored by AAT treatment. Each bar represents the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 3. Alpha-1 antitrypsin suppresses inflammatory responses and apoptotic signaling in cisplatin-induced kidney injury. (A) Representative Western blots and quantification of TNF-α and IL-1β expression in kidney tissue (n = 6 per group). Cisplatin slightly increased TNF-α and markedly increased IL-1β, whereas alpha-1 antitrypsin (AAT) co-treatment attenuated IL-1β induction. (B) Western blots and densitometric analysis of Bcl-2 and Bax expression and the Bax/Bcl-2 ratio (n = 6 per group). Cisplatin decreased Bcl-2 and increased Bax, resulting in an elevated Bax/Bcl-2 ratio, which AAT restored toward control levels. (C) Representative immunohistochemical staining for the macrophage marker F4/80 in the renal cortex (n = 6 per group). (D,E) Quantification and representative images of F4/80 and osteopontin (OPN) staining (n = 6 mice/group; 10 fields/mouse). Cisplatin increased macrophage infiltration and tubular inflammation, while AAT treatment reduced these processes. Each bar represents the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Figure S2 contains Original Western Blot of Figure 3A,B.

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  Figure 4. Alpha-1 antitrypsin preserves mitochondrial metabolic proteins and markers of mitochondrial dynamics. (A) Representative Western blots of mitochondrial fatty acid oxidation and dynamics-related proteins CPT1A, CPT2, PDK4, UCP3, PGC1α, and DRP1 in kidney tissue from the four groups (n = 6 per group). (B) Densitometric analysis of protein expression levels (n = 6 per group). Cisplatin significantly increased CPT1A and PDK4 expression and significantly decreased CPT2, UCP3, PGC1α, and DRP1; these changes were largely reversed by alpha-1 antitrypsin (AAT) co-administration. Each bar represents the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Figure S3 contains Original Western Blot of Figure 4A.

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  Figure 5. Alpha-1 antitrypsin attenuates renal fibrosis through epithelial–mesenchymal transition and extracellular matrix deposition. (A) Representative Western blots and densitometric analysis of fibronectin and α-smooth muscle actin (α-SMA) in kidney tissue from the four groups (n = 6 per group). Cisplatin markedly increased fibronectin and α-SMA, consistent with epithelial-to-mesenchymal transition (EMT), whereas alpha-1 antitrypsin (AAT) co-treatment reversed these changes. (B) Representative Masson’s trichrome staining of renal sections (n = 6 per group). Cisplatin-treated kidneys exhibited increased collagen deposition and interstitial fibrosis, whereas AAT led to a marked reduction in the fibrotic area. (C,D) Quantification of trichrome-positive fibrotic area and fibronectin immunohistochemical staining (n = 6 mice/group; 10 fields/mouse). Cisplatin exposure resulted in extracellular matrix accumulation, which AAT attenuated. Each bar represents the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Figure S4 contains Original Western Blot of Figure 5A.

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  Figure 6. Alpha-1 antitrypsin suppresses cisplatin-induced activation of mitogen-activated protein kinase signaling pathways. (A) Representative Western blots of phosphorylated and total ERK, JNK, and p38 MAPK in kidney tissue from four groups (n = 6 per group). (B) Densitometric analysis of the ratios of phosphorylated to total ERK, JNK, and p38 (n = 6 per group). Cisplatin markedly increased phosphorylation of ERK, JNK, and p38, whereas alpha-1 antitrypsin (AAT) co-treatment significantly reduced MAPK phosphorylation without altering total protein levels. Each bar represents the mean ± SEM. ** p < 0.01, *** p < 0.001. Figure S5 contains Original Western Blot of Figure 6A.

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  Figure 7. Proposed mechanisms of the renoprotective effects of alpha-1 antitrypsin in cisplatin-induced acute kidney injury. Schematic representation of the effects of cisplatin and AAT on renal tubular cells. Cisplatin activates NOX1 and alters NOX2/NOX4 expression, leading to increased oxidative stress (8-OHdG, 4-HNE) and reduced antioxidant defenses (SOD2, catalase). In parallel, cisplatin triggers inflammatory and apoptotic responses, characterized by elevated IL-1β, macrophage infiltration (F4/80, osteopontin), an increased Bax/Bcl-2 ratio, and activation of MAPK signaling (p-ERK, p-JNK, p-p38). Cisplatin also induces mitochondrial dysfunction, with decreased CPT2, UCP3, and PGC1α and a paradoxical pattern of increased CPT1A and decreased DRP1, resulting in impaired β-oxidation and altered mitochondrial dynamics. Finally, cisplatin promotes renal fibrosis and EMT, as reflected by increased fibronectin and α-SMA, and collagen deposition. AAT counteracts these pathways by reducing NOX1 activity and oxidative damage, suppressing IL-1β-driven inflammation and MAPK activation, restoring CPT2, UCP3, and PGC1α expression and mitochondrial homeostasis, and inhibiting EMT and extracellular…