Photoacoustic imaging assessment of acute kidney injury associated with experimental necrotizing enterocolitis

Abstract

Background: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal condition increasingly recognized to cause systemic complications, including acute kidney injury (AKI). However, the underlying mechanisms and variations in renal impairment remain largely unknown, and there is a lack of non-invasive imaging methods for detecting early kidney injury.

Methods: We evaluated kidney injury through a multimodal approach, using a neonatal rat model of experimental NEC. Plasma and renal tissue were analyzed for pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) by ELISA. Histological analysis and immunofluorescence staining were used to identify renal abnormalities and expression of KIM-1 and NGAL. Photoacoustic imaging (PAI) was used to assess renal oxygenation and total hemoglobin as functional biomarkers of kidney injury.

Results: NEC pups showed significant elevations of systemic and renal cytokines, as well as increased expression of KIM-1 and NGAL in proximal tubules of the kidney. Histological examination further confirms the renal injury in the NEC pup kidneys. PAI demonstrated reduction in renal oxygen saturation, offering non-invasive physiological biomarker assessment of kidney damage.

Conclusion: This study introduces PAI as a promising imaging modality for non-invasive evaluation of renal injury in NEC and characterizes AKI associated with NEC as an inflammatory and hypoxic response in the neonatal rat NEC model.

Figure 1.. Assessments of body weight and clinical sickness score.

(A) Body weight of BF and NEC cohort (mean ± SEM); NEC animals showed progressive weight loss relative to BF. NEC pups were weighed daily from day 0 through day 3 and BF pups were weighed on day 0 and day 4, as they remained with the dam and could not be handled on intermediate days (B) Clinical sickness scores on a 0 – 3 scale in BF and NEC cohorts from day 0 to day 4 (mean ± SEM). NEC pups exhibited significantly higher sickness scores compared to BF.

Figure 2.. Plasma and kidney cytokine levels in BF and NEC cohort.

Concentrations of TNF-α, IL-6, and IL-1 β were quantified by ELISA in (A) plasma and (B) kidney tissue homogenates from BF and NEC pups. Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired t-test with Welch’s correction, with p values indicated on the graphs (*p < 0.05, **p < 0.01).

Figure 3.. Plasma biochemical markers in BF and NEC cohort.

(A) Plasma blood urea nitrogen (BUN) and (B) creatinine concentrations were measured in BF and NEC pups. Data are presented as mean ± SEM. Comparisons between groups were made using an unpaired t-test with Welch’s correction. ns = no statistically significant differences were observed among groups.

Figure 4.. Histological abnormalities in the kidneys of BF and NEC pups.

Representative images of H&E-stained kidney sections indicating tubular dilation (black arrow), interstitial edema (red arrow) and tubular epithelial inflammation (green arrow).

Figure 5.. Expression of KIM-1 and NGAL in BF and NEC kidney tissue.

(Left) Representative images of immunofluorescence staining of (A) NGAL and (B) KIM-1. BF kidney sections showing negative background staining for NGAL and KIM-1, NEC sections showing strong focal KIM-1 and NGAL expression. (Right) Quantitative analysis of NGAL and KIM-1 expression by ImageJ software. All data are presented as mean ± SD, **p < 0.01; ****p < 0.0001.

Figure 6.

(Left) US and PAI images of tissue oxygenation in representative BF and NEC 4-day old rat pups. Scale bar = 5mm. (Right) Renal tissue oxygenation in NEC pups demonstrated a significant reduction compared to BF controls. *p < 0.05.

Figure 7.

(Left) US and PAI images of total hemoglobin in representative BF and NEC 4-day old rat pups. Scale bar = 5 mm. (Right) Renal total hemoglobin in NEC pups demonstrated a significant increase compared to BF controls. *p < 0.05.