Fibrinogen β-derived Bβ(15-42) peptide protects against kidney ischemia/reperfusion injury

Abstract

Ischemia/reperfusion (I/R) injury in the kidney is a major cause of acute kidney injury (AKI) in humans and is associated with significantly high mortality. To identify genes that modulate kidney injury and repair, we conducted genome-wide expression analysis in the rat kidneys after I/R and found that the mRNA levels of fibrinogen (Fg)α, Fgβ, and Fgγ chains significantly increase in the kidney and remain elevated throughout the regeneration process. Cellular characterization of Fgα and Fgγ chain immunoreactive proteins shows a predominant expression in renal tubular cells and the localization of immunoreactive Fgβ chain protein is primarily in the renal interstitium in healthy and regenerating kidney. We also show that urinary excretion of Fg is massively increased after kidney damage and is capable of distinguishing human patients with acute or chronic kidney injury (n = 25) from healthy volunteers (n = 25) with high sensitivity and specificity (area under the receiver operating characteristic of 0.98). Furthermore, we demonstrate that Fgβ-derived Bβ(15-42) peptide administration protects mice from I/R-induced kidney injury by aiding in epithelial cell proliferation and tissue repair. Given that kidney regeneration is a major determinant of outcome for patients with kidney damage, these results provide new opportunities for the use of Fg in diagnosis, prevention, and therapeutic interventions in kidney disease.

Figure 1

Fg (Fgα, Fgβ, and Fgγ chains) is significantly up-regulated in kidney cortex and medulla of male Wistar rats after 20 minutes of bilateral renal I/R injury compared with sham surgery. (A) A heatmap shows the expression levels of the most variable 1571 genes that were selected from 22 523 transcripts (see “Whole genome expression profiling and hierarchical clustering”). Coexpressed genes are grouped by hierarchical clustering. The samples are presented in order of time after ischemic injury (0, 6, 24, and 120 hours; triplicate for each). (B) Real-time PCR analysis in kidney. (C) Liver tissues for Fgα, Fgβ, and Fgγ chains, normalized using a housekeeping gene (Gapdh) and fold change determined over sham group (n = 5/group). *P < .05 as determined by 1-way ANOVA in comparison with sham rats.

Figure 2

Fg (immunoreactive Fgα, Fgβ, and Fgγ chains) protein is expressed in the kidney of rats and humans. (A) Representative formalin-fixed paraffin-embedded kidney tissue sections of sham male Wistar rats and those from rats that had undergone 20 minutes of bilateral ischemia reperfusion stained for immunoreactive proteins to Fgα (i-iii), Fgβ (iv-vi), Fgγ (vii-ix), and Fg (x-xii) after 24 and 72 hours of reperfusion and compared with healthy (sham) kidneys. Bar represents 50μm. (B) Representative formalin-fixed human-biopsied kidney sections of AKI and non-AKI patients, stained for immunoreactive molecules recognizing Fgα (i-ii), Fgβ (iii-iv), Fgγ (v-vi), and Fg (vii-viii). Bar represents 50 μm. Arrowheads indicate respective immunoreactive molecules. Asterisks in panels Bvii and Bix mark tubules with similar expression pattern along the basolateral side of respective tubules.

Figure 3

A significant increase in urinary Fg after kidney injury in rats and humans. (A) Urinary Fg was compared with tubular injury biomarkers NAG and Kim-1 in rats subjected to 20 minutes of bilateral renal I/R injury (n = 5/group). *P < .05 as determined by Student t test in comparison with sham rats. (B) Urinary Fg was measured in a human cross-sectional study with clinically established multifactorial AKI and/or CKD (n = 25) versus healthy volunteers (n = 25). Magenta line and corresponding number marked by arrow indicate a threshold cutoff value at 95% specificity. (C) ROC comparing the sensitivity and specificity of urinary Fg, NAG, and KIM-1 to distinguish patients with AKI and/or CKD from healthy volunteers.

Figure 4

Bβ_15-42 peptide protects mice from renal I/R injury. Male C57Bl/6 mice were subjected to 27 minutes of bilateral renal I/R injury or sham surgery and 3.6 mg/kg Bβ_15-42 peptide or random peptide was administered intravenously 1 minute after reperfusion. (A) The medullary congestion after ischemia was significantly less in the Bβ_15-42 peptide–administered mice compared with mice administered random peptide. Outline of vascular congestion traced by white dots. (B) SCr and BUN as indicators of renal dysfunction and urinary levels of Fg and Kim-1 as indicators of kidney injury were measured at 24 and 48 hours in all the groups (n = 5/group of sham, n = 10/group at 24 hours, and n = 5/ group at 48 hours). *P < .05 as determined by 1-way ANOVA in comparison with sham mice. (C) Representative H&E stained kidney sections comparing sham, Bβ_15-42 and random peptide–administered groups of mice at 24 and 48 hours after ischemia. Bar represents 100 μm (20× magnification).

Figure 5

Bβ_15-42 peptide aids in the resolution of injury by decreasing necrosis/apoptosis and inducing rapid tissue regeneration. Paraffin-embedded kidneys of mice subjected to 27 minutes of bilateral I/R that were administered either Bβ_15-42 or random peptides were compared at 24 and 48 hours for number of apoptotic cells (green; A) by TUNEL assay (bar represents 100 μm, 40× magnification). Arrowheads indicate TUNEL-positive nuclei. (B) Number of proliferative cells determined by Ki67-positive staining cells (red; bar represents 50 μm, 60× magnification). The numbers of positive staining TUNEL and Ki67 nuclei are represented graphically on the right of respective photomicrographs. Arrowheads indicate Ki67 positively stained nuclei. *P < .05 as determined by Student t test between the 2 groups within the same time point.