Recombinant antithrombin attenuates acute kidney injury associated with rhabdomyolysis: an in vivo animal study

**Background:** Rhabdomyolysis, characterized by skeletal muscle necrosis, leads to acute kidney injury (AKI) primarily through myoglobin-induced tubular damage and inflammation. Antithrombin III (AT) has known anti-inflammatory and endothelial-protective properties beyond its anticoagulant role. This study hypothesized that recombinant antithrombin (rAT) could attenuate rhabdomyolysis-induced AKI by promoting tissue repair and protecting the vascular endothelium.

**Methods:** Ten-week-old male C57BL6 mice were fasted for 24 hours and then injected intramuscularly with 5 mL/kg of 50% glycerol in the left thigh to induce rhabdomyolysis. Sham mice received saline. At 48 and 72 hours post-glycerol, mice received intraperitoneal rAT (750 IU/kg) or saline. At 96 hours, mice were euthanized. Blood was collected for serum creatinine, blood urea nitrogen (BUN), and neutrophil gelatinase-associated lipocalin (NGAL) measurements. Kidneys were harvested for histology (H&E staining), immunohistochemistry (Ki-67, PCNA, CD31, CD15, wheat germ agglutinin lectin), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Histological injury was scored from 0 (no injury) to 6 (>75% tubular necrosis). Ki-67 and PCNA positive cells were counted in 10 high-power fields per section. Glycocalyx integrity was assessed by lanthanum nitrate staining and fluorescence intensity of WGA lectin in CD31-positive areas. Statistical comparisons used Mann–Whitney U test; P<0.05 was significant.

**Key Results:** - Serum BUN, creatinine, and NGAL were significantly elevated in saline-treated rhabdomyolysis mice compared to sham, and significantly reduced in rAT-treated mice (P<0.05 for all). - Histological injury scores were significantly higher in saline (mean ~4-5) vs sham (~0), and significantly lower in rAT-treated mice (P<0.05). - Ki-67-positive cells were significantly increased in rAT-treated kidneys compared to saline (P<0.05), and double staining with CD15 confirmed proliferation in proximal tubular cells. PCNA-positive cells were also significantly higher in the rAT group. - SEM showed that rhabdomyolysis caused shedding of microvilli from the brush border of proximal tubules; rAT treatment preserved microvilli coverage, though shorter than sham. - TEM revealed perivascular fibrosis and gaps between capillaries and tubules in saline-treated mice, which were reduced by rAT. Lanthanum nitrate staining showed loss of endothelial glycocalyx in saline, with restoration in rAT-treated mice. - Quantitative fluorescence analysis of WGA lectin in CD31-positive capillaries showed significantly lower intensity in saline vs sham, and significantly higher intensity in rAT vs saline (P<0.05).

**Clinical Implications:** This study provides preclinical evidence that rAT attenuates rhabdomyolysis-induced AKI through multiple mechanisms: reducing tubular necrosis, promoting tubular epithelial cell regeneration (increased Ki-67 and PCNA), protecting the brush border ultrastructure, and preserving the endothelial glycocalyx to maintain peritubular capillary integrity. Since rAT is already clinically available, these findings suggest a potential new therapeutic strategy for rhabdomyolysis-associated AKI, which currently lacks specific treatments beyond supportive care. Limitations include the use of only male mice, the need for quantitative glycocalyx shedding markers (e.g., syndecan-1), and the small area assessed by electron microscopy. Further studies are needed to confirm sex differences and the precise mechanisms of rAT's regenerative effects.