Ketamine suppresses hypoxia-induced inflammatory responses in the late-gestation ovine fetal kidney cortex

Abstract

Acute fetal hypoxia is a form of fetal stress that stimulates renal vasoconstriction and ischaemia as a consequence of the physiological redistribution of combined ventricular output. Because of the potential ischaemia-reperfusion injury to the kidney, we hypothesized that it would respond to hypoxia with an increase in the expression of inflammatory genes, and that ketamine (an N-methyl-D-aspartate receptor antagonist) would reduce or block this response. Hypoxia was induced for 30 min in chronically catheterized fetal sheep (125 ± 3 days), with or without ketamine (3 mg kg(-1)) administered intravenously to the fetus 10 min prior to hypoxia. Gene expression in fetal kidney cortex collected 24 h after the onset of hypoxia was analysed using ovine Agilent 15.5k array and validated with qPCR and immunohistochemistry in four groups of ewes: normoxic control, normoxia + ketamine, hypoxic control and hypoxia + ketamine (n = 3-4 per group). Significant differences in gene expression between groups were determined with t-statistics using the limma package for R (P ≤ 0.05). Enriched biological processes for the 427 upregulated genes were immune and inflammatory responses and for the 946 downregulated genes were metabolic processes. Ketamine countered the effects of hypoxia on upregulated immune/inflammatory responses as well as the downregulated metabolic responses. We conclude that our transcriptomics modelling predicts that hypoxia activates inflammatory pathways and reduces metabolism in the fetal kidney cortex, and ketamine blocks or ameliorates this response. The results suggest that ketamine may have therapeutic potential for protection from ischaemic renal damage.

Figure 1. Volcano plot illustrating the relationship of gene expression in fetal kidney cortex measured by log₂ of fold‐change

Significantly upregulated (blue) and downregulated (red) genes under hypoxia (A), hypoxia + ketamine (B), and normoxia + ketamine (C). The dashed line indicates whether the gene is statistically significant (above), P ≤ 0.05, or not significant (below). Non‐significant genes are indicated by black circles, and the scale for the y‐axis varies between plots.

Figure 2. Venn diagram and network analysis of significant gene expression upregulated by hypoxia, but downregulated by ketamine during hypoxia

Venn and network inference analyses showing the number of significant genes that was upregulated by hypoxia (blue), downregulated with hypoxia and ketamine (red), and the common genes involved in both groups (yellow). Hypoxia (HC‐NC) and hypoxia + ketamine (HK‐HC).

Figure 3. Venn diagram and network analysis of significant gene expression downregulated by hypoxia, but upregulated by ketamine during hypoxia

Venn and network inference analyses showing the number of significant genes that was downregulated by hypoxia (blue), upregulated with hypoxia and ketamine (red), and the common genes involved in both groups (yellow). Hypoxia (HC‐NC) and hypoxia + ketamine (HK‐HC).

Figure 4. Ketamine ameliorates the increases in gene expression of inflammatory and immune related genes caused by hypoxia

RNA gene expression was measured by microarray and validated by real‐time qPCR for inflammatory and immune related genes. All values are represented as a fold‐change from normoxic control group (NC, normoxia control; NK, normoxia + ketamine; HC, hypoxia control; HK, hypoxia + ketamine). Statistical significance between groups was accepted at P ≤ 0.05 (interaction term in two‐way ANOVA), and Duncan's test was performed to determine differences between groups (for results of Duncan's test, bars with different letters are statistically significantly different from each other, P < 0.05). For gene expression with P > 0.05 between groups (interaction term in two‐way ANOVA), the P values listed here are for real‐time qPCR validation: CXCL10 (P = 0.06); and for the arrays: CXCL10 (P = 0.07), CCL5 (P = 0.08), IL‐6 (P = 0.099), NFκB (P = 0.13). Data are presented as means ± SEM, and the y‐axis scale varies between plots.

Figure 5. Local macrophages expressing Iba‐1 immunoreactivity in the kidney cortex of NC, NK, HC and HK fetuses

A and E, NC fetuses; B and F, NK fetuses; C and G, HC fetuses; D and H, HK fetuses. Sections were counterstained with methyl green. Macrophage counts per field at ×40 in the kidney cortex (J). Data are expressed as means ± SEM. Different letters indicate statistically significant difference (HC vs. HK P < 0.0001; HC vs. NC P < 0.0001; HC vs. NK P < 0.0001; HK vs. NC P = 0.018; HK vs. NK P = 0.005). Scale bars 200 μm (A–D), 50 μm (E–H). Abbreviations: NC, normoxia control; NK, normoxia + ketamine; HC, hypoxia control; HK, hypoxia + ketamine.