Astaxanthin- Enriched Fraction from the Microalga Haematococcus pluvialis Mitigates Kidney Injury via Modulation of Nrf2/Keap1 and MAPK/ERK Pathways in Rats

Gentamicin injection induced a significant increase in MAPK and ERK tissue levels by 3.4-fold and 2.6-fold, respectively, as compared with the normal control values as a sign of kidney inflammation. On the other hand, administration of astaxanthin and astaxanthin esters showed a significant decrease in the level of MAPK by 55 and 77% and ERK by 46 and 65%, respectively, as compared with the gentamicin group (Fig. 2a,b).

Fig. 2figure 2

Effect of astaxanthin (1)–enriched fraction and astaxanthin ester–enriched fraction on a MAPK, b ERK, and c RAS kidney contents. Data are presented as mean ± SE for each group (n = 8). Statistical analysis was carried out by one-way analysis of variance followed by Tukey’s multiple-comparison tests. a, statistically significant from the normal control group. b, statistically significant from the gentamicin group. c, statistically significant from the astaxanthin group at p < 0.05

RAS Kidney Content

Gentamicin injection induced a significant increase in RAS tissue levels by twofold as compared with the normal control value as a sign of kidney inflammation. On the other hand, treatment with astaxanthin and astaxanthin esters decreased the level of RAS by 20% and 60% as compared with the gentamicin group (Fig. 2c).

Histopathological Results

In the control group, the kidney section showed no histopathological alterations and showed ordinary histological architecture of average glomeruli with average Bowman’s spaces, average proximal tubules with preserved brush borders, and average distal tubules (Fig. 3a The gentamicin treatment revealed remarkable histopathological alterations in the kidney section, including markedly atrophied glomeruli with widened Bowman’s spaces, proximal tubules with markedly apoptotic epithelial lining, and mild interstitial inflammatory infiltrate as shown in Fig. 3c, d). The control group also showed normal histopathological structure in the renal medulla, with average collecting tubules with average epithelial lining and average interstitium (Fig. 3e). The gentamicin group showed remarkable histopathological changes in the renal medulla, including collecting tubules with mildly apoptotic epithelial lining, intra-tubular hyaline casts, and mildly congested peri-tubular capillaries (Fig. 3f). The treatment with astaxanthin and astaxanthin esters also improved the gentamicin histopathological changes in the renal medulla and showed average collecting tubules with average epithelial lining and average interstitium (Fig. 3g, h).

Fig. 3figure 3

Kidney section of a normal rats showing the normal architecture of average glomeruli (G) with average Bowman’s spaces (BS), average proximal tubules (P) with preserved brush borders (black arrow), and average distal tubules (D). b Gentamicin-treated rats showing markedly atrophied glomeruli (G) with widened Bowman’s spaces (BS), proximal tubules (P) with markedly apoptotic epithelial lining (black arrow), and mild interstitial inflammatory infiltrate (yellow arrow). c Kidney section of astaxanthin-enriched-fraction-treated rats showing mildly congested glomeruli (G) with average Bowman’s spaces (BS), proximal tubules (P) with markedly edematous epithelial lining (black arrow), and average distal tubules (D). d Kidney section of astaxanthin ester–enriched fraction–treated rats showing mildly congested glomeruli (G) with average Bowman’s spaces (BS), proximal tubules (P) with mildly edematous epithelial lining (black arrow), and average distal tubules (D) (H&E × 400)Kidney section in the renal medulla of e normal rats showing the normal architecture of average collecting tubules (CT) with average epithelial lining (black arrow) and average interstitium (blue arrow). f Gentamicin-treated rats showing collecting tubules (CT) with mildly apoptotic epithelial lining (black arrow), intra-tubular hyaline casts (blue arrow), and mildly congested peri-tubular capillaries (yellow arrow). g Astaxanthin-enriched fraction–treated rats showing average collecting tubules (CT) with average epithelial lining (black arrow) and average interstitium (blue arrow). h Astaxanthin ester–enriched fraction–treated rats showing average collecting tubules (CT) with average epithelial lining (black arrow) and average interstitium (blue arrow) (H&E × 400)

In the gentamicin animal model, its accumulation in the renal tubules induces oxidative stress in the kidney, which causes nephrotoxicity. In the current study, the histopathological results showed that gentamicin-treated rats showed markedly atrophied glomeruli and apoptotic epithelial lining of proximal tubules with inflammatory infiltrate. These results are in accord with earlier reports, which reported that gentamicin causes kidney histopathological injuries such as proximal tubule epithelial edema, desquamation, and tubular fibrosis and necrosis (Xu et al. 2008). Our results showed that the administration of astaxanthin and astaxanthin esters mitigated all kidney and renal medulla histopathological alterations caused by gentamicin in rats. Administration of gentamicin increased serum creatinine and urea levels in our study, which is in consensus with the results of Xu et al. (2008), who reported that gentamycin elevated kidney function levels. Astaxanthin and astaxanthin ester administration in this study alleviated kidney dysfunction by decreasing the serum levels of creatinine and urea. Administration of astaxanthin attenuated elevated kidney function, oxidative stress, and apoptosis in acute kidney injury (Guo et al. 2015).

Nrf2 is a leading transcription factor that controls the endogenous antioxidant defense system via controlling different antioxidant-responsive elements (ARE) genes. Gentamicin treatment suppresses renal Nrf2 translocation and activation and prevents ARE gene expression. This result may be related to the impact of gentamicin on Keap1 expression or dissociation (Ali et al. 2021). Our findings showed that astaxanthin and astaxanthin esters increased the levels of Nrf2 and decreased the levels of Keap1 in the kidney tissues. Thus, they can alleviate the kidney damage induced by the oxidative stress of gentamicin in rats by regulating the Nrf2/Keap1 pathway. Astaxanthin regulated oxidative stress and kidney injury–related Nrf2/Keap1 pathway induced by ochratoxin in mice (Ruíz-Salinas et al. 2020). The positive role of astaxanthin and astaxanthin esters in regulating the Nrf2/Keap1 pathway in the present study is due to their potent antioxidant properties. Due to its special structure, astaxanthin exhibits higher antioxidant effects than other antioxidants because of its high ability to bind to the cell membrane from the inside to the outside. The astaxanthin end ring can capture free radicals on the surface and inside the cell membrane, while the double-single bond chain can capture free radicals only inside the cell membrane (Pashkow et al. 2008).

Upregulation of a regulatory defense factor against hypoxia-inducible factor-alpha (HIF-α) plays a vital role in acute or chronic kidney disease against podocyte injury with suppression of oxidative stress (Weidemann et al. 2008). HIF has an antioxidant effect also in diabetic nephropathy and renal ischemia–reperfusion injury and has a renal tubular cell antiapoptotic effect in cisplatin-induced nephrotoxicity (Weidemann et al. 2008). Gentamicin-induced nephrotoxicity elevated renal HIF-1α mRNA levels (Xu et al. 2008). Therefore, HIF-α may be involved in gentamicin-induced renal injury. This study showed that astaxanthin and astaxanthin esters have a therapeutic effect against gentamicin-induced renal tubule cell injuries through modulation of HIF-α activity. In oxidative stress, the activation of different pro-inflammatory mediators including nuclear factor-kappa beta (NF)-κB, TGF-β1, and MAPKs leads to the progression of kidney damage. MAPK engages in cellular growth, differentiation, proliferation, and apoptosis regulation. MAPK promotes the activation of NF-κB that is present in every cell cytoplasm; upon stimulation, it enhances TNF-α and IL-6 expressions aggravating the cellular damage (Qiao et al. 2012). In this study, gentamicin elevated kidney contents of MAPK and ERK. These results explain the vital role of proximal tubular epithelial cells in producing cytokines and cell adhesion molecules, provoking infiltrating cell recruitment, and activation at the inflammatory sites. Our findings showed that astaxanthin and astaxanthin esters alleviated MAPK/ERK signaling pathways. Thus, they may be considered a potential therapy for the renal disease caused by gentamicin through the inactivation of MAPK/ERK pathways; consequently, they can inhibit the inflammatory process and apoptosis in the kidney cells, as shown in the histopathological results. In a previous study, astaxanthin reduced acetaldehyde-induced apoptosis and inhibited MAPK signaling pathway activation in diabetic cardiomyopathy (Zhang and Xu 2018).

The kidney is a unique organ containing a renin-angiotensin system (RAS) component as angiotensin II. Oxidative stress also affects the proinflammatory effect of angiotensin II produced in the distal and proximal tubules, the collecting ducts, and renal endothelial cells. RAS disturbance is one of the chief pathways implicated in both glomerular and tubulointerstitial injuries resulting in renal injury through direct effects of angiotensin II type 1 receptor and also due to inflammatory cytokine activation (Al-Kuraishy et al. 2019). In the current study, astaxanthin and astaxanthin esters ameliorated the elevated levels of RAS induced by gentamicin in the kidney. Consequently, they can inhibit inflammation caused by RAS disturbance. In another study, astaxanthin modulated RAS and decreased TGF-β1 levels and calcium oxalate crystal deposition in the kidney (Alex et al. 2014). The therapeutic role of the astaxanthin and astaxanthin ester fractions in the present study might be due to the high contents of different isomers of free astaxanthin and astaxanthin mono- and di-esters; isomers of astaxanthin may be of 9-cis, 13-cis, and 15-cis isomers not only all-trans which exert high therapeutic effects against gentamicin-induced kidney damage in rats. The isomers of 9-cis and 13-cis-astaxanthin exhibited a higher antioxidant effect than all-trans-astaxanthin against oxidative stress in the human intestinal epithelial Caco-2 cells (Yang et al. 2017).

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