Complement inhibition attenuates acute kidney injury after ischemia-reperfusion and limits progression to renal fibrosis in mice.

The complement system is an essential component of innate immunity and plays a major role in the pathogenesis of ischemia-reperfusion injury (IRI). In this study, we investigated the impact of human C1-inhibitor (C1INH) on the early inflammatory response to IRI and the subsequent progression to fibrosis in mice. We evaluated structural damage, renal function, acute inflammatory response, progression to fibrosis and overall survival at 90-days post-injury. Animals receiving C1INH prior to reperfusion had a significant improvement in survival rate along with superior renal function when compared to vehicle (PBS) treated counterparts. Pre-treatment with C1INH also prevented acute IL-6, CXCL1 and MCP-1 up-regulation, C5a release, C3b deposition and infiltration by neutrophils and macrophages into renal tissue. This anti-inflammatory effect correlated with a significant reduction in the expression of markers of fibrosis alpha smooth muscle actin, desmin and picrosirius red at 30 and 90 days post-IRI and reduced renal levels of TGF-β1 when compared to untreated controls. Our findings indicate that intravenous delivery of C1INH prior to ischemic injury protects kidneys from inflammatory injury and subsequent progression to fibrosis. We conclude that early complement blockade in the context of IRI constitutes an effective strategy in the prevention of fibrosis after ischemic acute kidney injury

Protective renal effect of complement blockade after ischemia-reperfusion injury.

<p>Mice were assigned to four groups: 1) Sham + PBS (n = 6), 2) Sham + C1INH (n = 6), 3) IRI + PBS (n = 9), IRI + C1INH (n = 15). Ischemia was induced by clamping of the renal hilum for 60 minutes and followed by contra-lateral nephrectomy. Sterile PBS or C1INH (750 U/kg) were given intravenously via tail vein injection 1 hour prior to surgery. Serum was collected at 24 and 72 hours after injury and survival was monitored for 90 days after reperfusion. (A) Pharmacological targeting of complement activation using C1INH significantly improves animal survival after IRI (Log-rank test, <i>P</i> = 0.0015). (B) Serum creatinine level at 24 and 72 hours after IRI. Groups: 1) Sham + PBS (n = 8), 2) Sham + C1INH (n = 8), 3) IRI + PBS (n = 10), IRI + C1INH (n = 10). (C) Histopathological analysis of kidneys at 24 hours after surgery. Representative light microscopy images of hematoxylin-eosin (H&E) staining of the cortex and medulla of kidneys (200X magnification). Arrows indicate necrotic tubules, and asterisks indicate tubular casts. (D) Renal tubular injury scores (0–4, arbitrary units). All data presented are mean ± SD. Survival data was analyzed by the Kaplan–Meier survival method and the log-rank test. Statistical comparison for creatinine values and tubular injury scoring was performed by one-way ANOVA followed by Bonferroni’s post-hoc correction. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Guidelines for the management of a brain death donor in the rhesus macaque: A translational transplant model.

The development of a translatable brain death animal model has significant potential to advance not only transplant research, but also the understanding of the pathophysiologic changes that occur in brain death and severe traumatic brain injury. The aim of this paper is to describe a rhesus macaque model of brain death designed to simulate the average time and medical management described in the human literature.Following approval by the Institutional Animal Care and Use Committee, a brain death model was developed. Non-human primates were monitored and maintained for 20 hours after brain death induction. Vasoactive agents and fluid boluses were administered to maintain hemodynamic stability. Endocrine derangements, particularly diabetes insipidus, were aggressively managed.A total of 9 rhesus macaque animals were included in the study. The expected hemodynamic instability of brain death in a rostral to caudal fashion was documented in terms of blood pressure and heart rate changes. During the maintenance phase of brain death, the animal's temperature and hemodynamics were maintained with goals of mean arterial pressure greater than 60mmHg and heart rate within 20 beats per minute of baseline. Resuscitation protocols are described so that future investigators may reproduce this model.We have developed a reproducible large animal primate model of brain death which simulates clinical scenarios and treatment. Our model offers the opportunity for researchers to have translational model to test the efficacy of therapeutic strategies prior to human clinical trials

Treatment with C1INH prevented the progression to fibrosis in kidneys subjected to ischemia-reperfusion injury.

<p>(A) Immunohistochemical staining for alpha smooth muscle actin (α-SMA), desmin and picrosirius red staining in renal tissue recovered at 30 and 90-days post-ischemic injury from sham + PBS (n = 6), sham + C1INH (n = 6), IRI + PBS (n = 6) and IRI + C1INH (n = 6) mice. Representative light microscopy images (200X magnification) from each group are depicted. Semi-automated quantification of (B) α-SMA(+), (C) Desmin(+) and (D) Picrosirius Red(+) area per HPF. Stained area is calculated and shown as a percentage relative to total tissue area per field. Data are mean ± SD. Statistical comparison was performed by one-way ANOVA followed by Bonferroni’s post-hoc correction. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Renal mRNA expression and circulating level of inflammatory cytokines and chemokines at 24 and 72-hours post-injury.

<p>RT-PCR analysis was performed on renal tissue recovered at the specified time points from mice in all groups (A) mRNA expression of monocyte chemoattractant protein-1 (MCP-1, n = 6), Interleukin-6 (IL-6, n = 6) and CXCL1 (KC, n = 4). Expression was normalized to baseline expression of native controls and GAPDH was used as the endogenous control. (B) Plasma KC and CCL2 levels post-injury. Cytokine levels determined by ELISA on plasma recovered at the specified time points from mice in all groups (n = 3 for each group). Data are mean ± SD. Statistical comparison was performed by Kruskal-Wallis and Dunn’s post-hoc correction. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

C1INH significantly decreased renal C5a cleavage and limited C3b deposition in kidneys subjected to IRI.

<p>(A) Immunoblotting analysis for C5a in renal tissue at 24 and 72 hours post-IRI. (B) Quantification of C5a band density from western blots normalized to β-tubulin. Results are expressed as mean ± SD fold changes (n = 3 for each group) compared with native controls. (C) Immunoblotting analysis of phosphorylated ERK1/2 normalized to total ERK1/2 (p44/42 MAPK) in renal tissue at 24 and 72 hours post-IRI in renal tissue. (D) Quantification of phosphorylated ERK band density from western blots normalized total ERK and β-tubulin. Results are expressed as mean ± SD fold changes (n = 3 for each group) compared with native controls. (E) Representative images of immunofluorescent microscopy performed on renal tissue at 72 hours post-injury targeting C3b deposition (200X). (F) mRNA expression of bradykinin 1 (BR1) and BR2 receptor in renal tissue. Expression was normalized to baseline expression of native controls and GAPDH was used as the endogenous control. Results are expressed as mean ± SD fold changes (n = 3 for each group).</p

Algorithm for management of a hemodynamically unstable primate.

<p>An algorithm for vasoactive infusions and isotonic IV fluid boluses needed to augment the animal’s blood pressure is outlined above.</p

Histological analysis of brain dead vs. naïve primates.

<p>Histological analysis of brain dead vs. naïve primates.</p

Kidney H&E analysis.

<p>Biopsy A shows Kidney H&E of naïve rhesus, age 18 years, diagnosed as normal. Biopsy B shows Kidney H&E of naïve rhesus, age 15 years, diagnosed with mild acute tubular injury, focal mild arteriosclerosis. Biospy C shows Kidney H&E of brain dead rhesus, age 16 years, diagnosed normal. Biopsy D shows Kidney H&E of naïve rhesus, age 19 years, diagnosed with mild acute tubular injury, focal mild arteriosclerosis.</p