Windows of susceptibility to neonatal acute kidney injury and nephron loss in a rabbit model

Abstract

Nephrogenesis is completed before term birth, but preterm infants continue this process postnatally. It is unknown if preterm neonates are more susceptible to acute kidney injury (AKI) immediately after birth (during nephrogenesis) or later in postnatal development, and if this AKI timing impacts nephron number. Rabbits were exposed to gentamicin (100 mg/kg intraperitoneal) and indomethacin (5 mg/kg orally) on postnatal day (P) 0-3 (early-exposed) or P6-9 (late-exposed). Animals were euthanized three hours after last nephrotoxin dose or at 6 weeks of life. Histologic injury was assessed, and Kidney injury marker 1 (Kim1) expression was quantified. At 6 weeks, blood urea nitrogen (BUN) and serum creatinine (SCr) levels were measured. Ex vivo MRI imaging was performed with automated quantitation of nephron numbers. AKI was induced in early and late-exposed rabbits. At 6 weeks, we identified a mean of 170,972 glomeruli in the controls (n = 5), 159,655 in the early-exposed (n = 4), and 145,748 glomeruli in the late-exposed group (n = 3, a 14.8% reduction compared to control, p = 0.01). Late-exposed rabbits had elevated BUN and SCr relative to early-exposed. This study suggests that exposure to nephrotoxins during early postnatal nephrogenesis causes AKI but may have less impact on long-term nephron number than exposure during nephron maturation.

Keywords: Chronic kidney disease; Neonatal acute kidney injury; Nephrogenesis; Nephrotoxins; Prematurity.

Fig. 1

Periodic Acid-Schiff staining confirms acute kidney injury in both early and late-exposed rabbits. A Healthy tubules are defined by bright pink-staining brush border. Both early and late exposed rabbits demonstrate injury to the proximal tubule including dilatation of the proximal tubule/flattening of the tubular epithelium (yellow arrow) and vacuolization of the proximal tubular lumen (blue arrow). B Quantification of proximal tubular injury identified increased injury in both early and late exposed rabbits compared to controls.

Fig. 2

Havcr1 (Kim1) is increased in the cortex of both early and late-exposed rabbits compared to saline controls, however injury is greater in those exposed early. A Kim1 qPCR fold change in nephrotoxin-exposed animals relative to age-matched control samples and normalized by changes in beta-actin levels. B Quantification of Kim1 in cortex of RNAScope demonstrates increased Kim1 in nephrotoxin-induced samples, with significantly increased Kim1 in early-exposed rabbits compared to controls. C Kim1 expression is localized to the cortico-medullary border/inner cortex in early-exposed rabbits while sparing the nephrogenic zone. D Late-exposed rabbits still favor injury in the inner cortex although Kim1 staining is more diffuse.

Fig. 3

Assessment of rabbits at 6 weeks of age demonstrates decreased nephron number, increased markers of CKD, and increased fibrosis in late-exposed rabbits compared to early-exposed rabbits. A A 15% reduction in nephron number was seen in rabbits exposed to nephrotoxins during tubular maturation (P6–9) compared to controls. B Although clinically small in difference, a statistically significant increase in serum creatinine and C blood urea nitrogen (BUN) was seen in the late-exposed rabbits compared to early-exposed rabbits. D–L Trichome staining was performed to assess for regions of fibrosis. Blue staining indicates fibrotic region of the kidney, with the most staining seen in late-exposed rabbits (J–L) compared to controls. Patches of fibrosis were also seen in early-exposed (G–I).

Fig. 4

Late-exposed rabbits at 6 weeks of age express overall higher percentage of Kim1, with overlapping staining of injury marker Krt8. A RNAScope imaging of early exposed rabbit and B late-exposed rabbit at lower magnification and higher magnification (C–D). Co-staining of Kim1 and Krt8 on tissue can be seen in both early and late samples (E–H). I Despite one outlier, overall Kim1 expression increased in late-exposed compared to controls at 6 weeks of age, although overall expression decreased from acute injury phase. J Evaluating the percent fold change from the median control value, we identified a larger percent change in Kim1 expression in the late-exposed compared to early-exposed rabbits.