Is Hemopexin a Nephrotoxin or a Marker of Kidney Injury in Renal Ischemia-Reperfusion?

Abstract

Destabilization of heme proteins is recognized to play a role in acute kidney injury (AKI). Hemopexin (Hpx), known for its role in binding heme, mitigates free heme toxicity. Despite this, the potential adverse effects of Hpx deposition in kidney tissues and its impact on kidney function are not fully understood. Deferoxamine (DFO) chelates iron released from heme and mitigates associated kidney damage. Therefore, this study aimed to evaluate whether Hpx contributes to kidney injury in an ischemia-reperfusion injury (IRI) induced AKI model and to investigate if DFO could alleviate this damage. Mice were categorized into five groups: Sham-Vehicle, Sham-Hpx, IRI-Vehicle, IRI-Hpx, and IRI-Hpx-DFO. Decline in kidney function was observed exclusively in the IRI group, independent of Hpx injection. Serum Hpx levels remained comparable across all groups, and administration of Hpx did not alter serum Hpx levels or kidney function after 24 hours. Although increased Hpx deposition in kidneys was noted in both the IRI and Hpx groups, this accumulation did not correlate with impaired kidney function. Additionally, DFO did not exhibit a protective effect against kidney injury. In summary, Hpx does not directly induce kidney injury and cannot be considered a biomarker for kidney damage caused by IRI.

Keywords: acute kidney injury; biomarker; hemopexin; ischemia-reperfusion injury.

Figure 1

Kidney function was decreased in ischemia-reperfusion injury (IRI) groups. (A) Serum blood urea nitrogen levels in each group; (B) serum creatinine levels in each group; (C) representative microscopic images of kidney tissues stained with periodic acid-Schiff (PAS) (×400). Scale bar = 50 µm; (D) Renal injury scores in each group. **** p < 0.0001 versus SH group. #### p < 0.0001, ### p < 0.001 versus SV group.

Figure 2

IRI increased Neutrophil Gelatinase-Associated Lipocalin (NGAL) expression in kidneys irrespective of hemopexin (Hpx) infusion. (A) Western blot analysis of kidney injury-related markers. Protein levels of (B) kidney injury molecule (KIM)-1, (C) NGAL, (D) fibronectin, and (E) Heme oxygenase (HO)-1 determined using Western blot. Red markers: target protein molecular weight, Black markers: protein standards marker. *** p < 0.001, ** p < 0.01, * p < 0.05 versus SV group, ## p < 0.01, # p < 0.05 versus SH group. Original images can be found in Figure S1, Supplementary Materials.

Figure 3

Serum NGAL and Hpx levels exhibit different responses to IRI. Serum levels of (A) NGAL and (B) Hpx in the SV (n = 4), SH (n = 2), IV (n = 4), IH (n = 4), and IHD groups (n = 4). **** p < 0.0001 versus SV group. ### p < 0.001 versus SH group.

Figure 4

Collagen deposition was observed exclusively in the IRI groups. (A) Representative images of kidney tissue stained with Masson’s trichrome (×400), Scale bar = 50 µm; (B) Semi-quantitative analysis of collagen deposition area. *** p < 0.001, ** p < 0.01 versus SV group. ### p < 0.001, ## p < 0.01 versus SH group.

Figure 5

Hpx deposition in response to Hpx infusion and IRI. (A) Representative images of Hpx staining in the kidney tissues (×400), Scale bar = 50 µm; (B) Semi-quantitative analysis of Hpx positive areas in each group. **** p < 0.0001 versus SV group. ### p < 0.001, ## p < 0.01 versus SH group. $ p < 0.05 versus IV group.