Attenuation of renal ischemia and reperfusion injury by human adrenomedullin and its binding protein

Abstract

Background: Acute renal failure secondary to ischemia and reperfusion (I/R) injury poses a significant burden on both surgeons and patients. It carries a high morbidity and mortality rate and no specific treatment currently exists. Major causes of renal I/R injury include trauma, sepsis, hypoperfusion, and various surgical procedures. We have demonstrated that adrenomedullin (AM), a novel vasoactive peptide, combined with AM binding protein-1 (AMBP-1), which augments the activity of AM, is beneficial in various disease conditions. However, it remains unknown whether human AM/AMBP-1 provides any beneficial effects in renal I/R injury. The objective of our study therefore was to determine whether administration of human AM/AMBP-1 can prevent and/or minimize damage in a rat model of renal I/R injury.

Methods: Male adult rats were subjected to renal I/R injury by bilateral renal pedicle clamping with microvascular clips for 60 min followed by reperfusion. Human AM (12 microg/kg BW) and human AMBP-1 (40 microg/kg BW) or vehicle (52 microg/kg BW human albumin) were given intravenously over 30 min immediately following the clip removal (i.e., reperfusion). Rats were allowed to recover for 24 h post-treatment, and blood and renal tissue samples were collected. Plasma levels of AM were measured using a radioimmunoassay specific for rat AM. Plasma AMBP-1 was measured by Western analysis. Renal water content and serum levels of systemic markers of tissue injury were measured. Serum and renal TNF-alpha levels were also assessed.

Results: At 24 h after renal I/R injury, plasma levels of AM were significantly increased while plasma AMBP-1 was markedly decreased. Renal water content and systemic markers of tissue injury (e.g., creatinine, BUN, AST, and ALT) were significantly increased following renal I/R injury. Serum and renal TNF-alpha levels were also increased post injury. Administration of human AM/AMBP-1 decreased renal water content, and plasma levels of creatinine, BUN, AST, and ALT. Serum and renal TNF-alpha levels were also significantly decreased after AM/AMBP-1 treatment.

Conclusion: Treatment with human AM/AMBP-1 in renal I/R injury significantly attenuated organ injury and the inflammatory response. Thus, human AM combined with human AMBP-1 may be developed as a novel treatment for patients with acute renal I/R injury.

Figure 1. Alterations in serum levels of AM and AMBP-1 after renal I/R injury

A. Plasma samples from sham and renal I/R rats at 24 h post injury were assessed for AM using specific RIA kit. Results are shown as pg/ml estimated from known standards (n=6). B. Plasma samples were subjected to Western blotting using human anti-AMBP-1 antibody. Results are shown as arbitrary densitometric units (n=4). Data are presented as means ± SE and compared by Student’s t- test: *P < 0.05 versus Sham group.

Figure 2. Alterations in renal water content after renal I/R injury

Kidneys from sham and renal I/R rats (vehicle or human AM/AMBP-1 treatment) were collected at 24 h post I/R injury. Data are presented as means ± SE (n=7–8) and compared by one-way analysis of variance (ANOVA) and Student–Newman–Keuls method: *P < 0.05 versus Sham group, #P < 0.05 versus Vehicle group.

Figure 3. Alterations in serum levels of renal injury markers after renal I/R injury

Serum samples from sham and renal I/R rats (vehicle or human AM/AMBP-1 treatment) at 24 h post I/R injury were assessed for creatinine (A) and BUN (B). Data are presented as means ± SE (n=5–7) and compared by one-way analysis of variance (ANOVA) and Student–Newman–Keuls method: *P < 0.05 versus Sham group, #P < 0.05 versus Vehicle group.

Figure 4. Alterations in serum levels of systemic injury indicators after renal I/R injury

Serum samples from sham and renal I/R rats (vehicle or human AM/AMBP-1 treatment) at 24 h post I/R injury were measured for AST (A) and ALT (B). Data are presented as means ± SE (n=5–6) and compared by one-way analysis of variance (ANOVA) and Student–Newman–Keuls method: *P < 0.05 versus Sham group, #P < 0.05 versus Vehicle group.

Figure 5. Alterations in TNF-α levels after renal I/R injury

TNF-α from serum (A) and renal tissue (B) samples of sham and renal I/R rats (vehicle or human AM/AMBP-1 treatment) at 24 h post I/R injury were measured. Data are presented as means ± SE (n=6–8) and compared by one-way analysis of variance (ANOVA) and Student–Newman–Keuls method: *P < 0.05 versus Sham group, #P < 0.05 versus Vehicle group.