Hemodynamic changes in the kidney in a pediatric rat model of sepsis-induced acute kidney injury

Abstract

Sepsis is a leading cause of acute kidney injury (AKI) and mortality in children. Understanding the development of pediatric sepsis and its effects on the kidney are critical in uncovering new therapies. The goal of this study was to characterize the development of sepsis-induced AKI in the clinically relevant cecal ligation and puncture (CLP) model of peritonitis in rat pups 17-18 days old. CLP produced severe sepsis demonstrated by time-dependent increase in serum cytokines, NO, markers of multiorgan injury, and renal microcirculatory hypoperfusion. Although blood pressure and heart rate remained unchanged after CLP, renal blood flow (RBF) was decreased 61% by 6 h. Renal microcirculatory analysis showed the number of continuously flowing cortical capillaries decreased significantly from 69 to 48% by 6 h with a 66% decrease in red blood cell velocity and a 57% decline in volumetric flow. The progression of renal microcirculatory hypoperfusion was associated with peritubular capillary leakage and reactive nitrogen species generation. Sham adults had higher mean arterial pressure (118 vs. 69 mmHg), RBF (4.2 vs. 1.1 ml·min(-1)·g(-1)), and peritubular capillary velocity (78% continuous flowing capillaries vs. 69%) compared with pups. CLP produced a greater decrease in renal microcirculation in pups, supporting the notion that adult models may not be the most appropriate for studying pediatric sepsis-induced AKI. Lower RBF and reduced peritubular capillary perfusion in the pup suggest the pediatric kidney may be more susceptible to AKI than would be predicted using adults models.

Fig. 1.

Cecal ligation and puncture (CLP) produces an inflammatory response in rat pups. Serum levels of the cytokines tumor necrosis factor (TNF)-α (A) and interleukin (IL)-Iβ (B) were elevated at 4 and 10 h after sepsis (n = 6–8). Because preliminary studies indicated there were no differences in cytokine levels in sham animals over the time course, sham values plotted were pooled from the various time points. Appearance of cytokines was associated with a decrease in core temperature (C) (n = 6–11). Serum nitrate + nitrite (NO_x) concentration (D) increased by 10 h post-CLP (n = 7–12). Data are expressed as means ± SE. *P < 0.05 compared with sham.

Fig. 2.

CLP produced multiorgan injury in rat pups. Serum alanine aminotransferase (ALT, A), creatinine (B), and blood urea nitrogen (BUN, C) in rat pups were increased at 18 h following CLP. Capillary leakage measured by the presence of Evans blue dye (EBD) in kidney homogenates was elevated by 10 h post-CLP (D). Data are means ± SE (n = 4–13 animals/group). *P < 0.05 compared with sham.

Fig. 3.

Reactive nitrogen species (RNS) generation in the pup kidney. Representative images (from 3 to 4 animals/group) of immunoreactive inducible nitric oxide synthase (iNOS) protein are shown in A (sham) and B (6 h post-CLP). Staining for iNOS was weak and diffuse in sham but more intense in both proximal and distal tubules of the cortex following CLP. Representative images (from 3 to 4 animals/group) of immunoreactive nitrated protein (a marker of peroxynitrite generation) are show in C (sham), D (6 h post-CLP), and E (18 h post-CLP). Specific staining for nitrotyrosine adducts was absent in sham but weak and diffuse at 6 h post-CLP. In contrast, staining for nitrotyrosine adducts was more intense at 18 h post-CLP. The specificity of the antinitrotyrosine antibody was determined by preincubation of the antibody with 10 mM 3-nitrotyrosine (data not shown). Oxidation of 1,2,3-dihydrorhodamine (DHR123) to rhodamine was used as a second indicator of RNS generation. Representative images of rhodamine fluorescence in sham and 18 h CLP are shown in G and H, respectively. Analysis of images of rhodamine fluorescence captured during the intravital videomicroscopy (IVVM) procedure showed an increase in rhodamine fluorescence at 18 h but not at 6 h following CLP (F). Data are means ± SE (n = 5–8 animals/group). *P < 0.05 compared with sham.

Fig. 4.

Renal peritubular capillary perfusion status. CLP produced a time-dependent change in the percentage of capillaries with continuous, intermittent, and no flow in pups. Data are means ± SE (n = 6–8 pups/group). *P < 0.05 compared with sham.

Fig. 5.

Time course of peritubular capillary flow in pups. CLP produced a time-dependent change in red blood cell (RBC) velocity (A) and volumetric flow (B) in continuously flowing capillaries. Data are means ± SE (n = 4 pups/group). *P < 0.05 compared with sham.

Fig. 6.

Comparison of systemic and renal hemodynamics between adults and pups. Early changes in mean arterial pressure (MAP), heart rate, and renal blood flow (RBF) are presented for comparisons in A, C, and E, respectively. The full time course for MAP and heart rate is presented in B and D. A comparison of peritubular capillary perfusion at 6 h is presented in F. Data are means ± SE (n = 5–8 pups/group and n = 4–7 adults/group). In A, *P < 0.05 by one-way ANOVA compared with 2 h. In B and E, *P < 0.05 using 2-way ANOVA. In F, *P < 0.05 by 1-way ANOVA compared with sham and #P < 0.05 when adult is compared with pup.

Fig. 7.

Frequency distribution of renal cortical capillary RBC velocities at 6 h. CLP shifted the distribution of capillary RBC velocities in pups (A) and adults (B) toward lower velocities (n = 4 animals/group). *P < 0.05, sham vs. CLP.