Post-Ischemic Renal Fibrosis Progression Is Halted by Delayed Contralateral Nephrectomy: The Involvement of Macrophage Activation

Abstract

(1) Background: Successful treatment of acute kidney injury (AKI)-induced chronic kidney disease (CKD) is unresolved. We aimed to characterize the time-course of changes after contralateral nephrectomy (Nx) in a model of unilateral ischemic AKI-induced CKD with good translational utility. (2) Methods: Severe (30 min) left renal ischemia-reperfusion injury (IRI) or sham operation (S) was performed in male Naval Medical Research Institute (NMRI) mice followed by Nx or S one week later. Expression of proinflammatory, oxidative stress, injury and fibrotic markers was evaluated by RT-qPCR. (3) Results: Upon Nx, the injured kidney hardly functioned for three days, but it gradually regained function until day 14 to 21, as demonstrated by the plasma urea. Functional recovery led to a drastic reduction in inflammatory infiltration by macrophages and by decreases in macrophage chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) mRNA and most injury markers. However, without Nx, a marked upregulation of proinflammatory (TNF-α, IL-6, MCP-1 and complement-3 (C3)); oxidative stress (nuclear factor erythroid 2-related factor 2, NRF2) and fibrosis (collagen-1a1 (Col1a1) and fibronectin-1 (FN1)) genes perpetuated, and the injured kidney became completely fibrotic. Contralateral Nx delayed the development of renal failure up to 20 weeks. (4) Conclusion: Our results suggest that macrophage activation is involved in postischemic renal fibrosis, and it is drastically suppressed by contralateral nephrectomy ameliorating progression.

Keywords: MCP-1; TNF-α; acute kidney injury; chronic kidney disease; inflammation; mice.

Figure 1

Effects of nephrectomy (Nx) on plasma urea concentrations in mice with left renal ischemia-reperfusion injury (IRI). (a) Blood samples were taken on days -1, 1, 6, 8, 9 and 10 in the 10-day study. (b) Blood samples were taken on days -1, 1, 6, 8, 14, 21 and 28 in the 28-day study. Data are expressed as mean ± SEM; two-way ANOVA with Tukey’s post hoc test; *: p < 0.05 and ***: p < 0.001; IR-S (sham) vs. IR-Nx.

Figure 2

Plasma urea of mice in the long-term study. Samples were taken on days -1, 1, 6, 8, 9, 14 and once a week thereafter. Data are expressed as mean ± SEM; two-way ANOVA with Tukey’s post hoc test; **: p < 0.01 and ***: p < 0.001; IR-Nx (green) vs. all other groups.

Figure 3

Plasma Lcn-2 of mice in the IR-Nx group in the long-term study. Data are expressed as mean ± SEM; one-way ANOVA with Tukey’s post hoc test; *: p < 0.05, **: p < 0.01 and ***: p < 0.001; day -1 vs. all other time points.

Figure 4

Kidney morphology. Representative pictures of nonischemic right (left column), postischemic left kidneys in the IR-S (middle histology column) and IR-Nx (right histology column) groups and the histology scores (right column). (a) Macroscopic picture of a nonischemic right and a pale, fibrotic/atrophic (postischemic) left kidney in the IR-S group on day 28 in situ. (b) PAS-stained sections (800×, scale bar: 50 μm) and tubular injury (TI) scores (0–4) (c) Masson’s trichrome-stained sections (800×, scale bar: 50 μm) and the tubulointerstitial (TI) fibrosis scores (0–4). Data are expressed as mean ± SEM; unpaired, nonparametrical Mann-Whitney t-test. *: p < 0.05, **: p < 0.01 and ***: p < 0.001 between the IR-S (red) vs. IR-Nx (green) groups.

Figure 4

Kidney morphology. Representative pictures of nonischemic right (left column), postischemic left kidneys in the IR-S (middle histology column) and IR-Nx (right histology column) groups and the histology scores (right column). (a) Macroscopic picture of a nonischemic right and a pale, fibrotic/atrophic (postischemic) left kidney in the IR-S group on day 28 in situ. (b) PAS-stained sections (800×, scale bar: 50 μm) and tubular injury (TI) scores (0–4) (c) Masson’s trichrome-stained sections (800×, scale bar: 50 μm) and the tubulointerstitial (TI) fibrosis scores (0–4). Data are expressed as mean ± SEM; unpaired, nonparametrical Mann-Whitney t-test. *: p < 0.05, **: p < 0.01 and ***: p < 0.001 between the IR-S (red) vs. IR-Nx (green) groups.

Figure 5

Macrophage-specific (F4/80) immunostaining of the kidney. Representative pictures of nonischemic right (left column) and postischemic left kidneys in the IR-S (middle column) and IR-Nx (right column) groups. (a) F4/80 staining (800x, scale bar: 50 μm). (b) Positive staining per whole kidney area ratio. Two-way ANOVA with Tukey’s post hoc test. ***: p < 0.001 IR-S (red) vs. nonischemic right kidneys (blue); +++: p < 0.001 IR-Nx (green) vs. nonischemic right kidneys (blue), #: p < 0.05, ##: p < 0.01 and ###: p < 0.001 IR-S (red) vs. IR-Nx (green).

Figure 6

Fold changes in inflammation- and immune system-related mRNAs normalized to 18S. (a) TNF-α, (b) IL-6, (c) MCP-1 and (d) C3. Data are expressed as mean ± SEM; two-way ANOVA with Tukey’s post hoc test. ***: p < 0.001 IR-S (red) vs. nonischemic right kidneys (blue); +++: p < 0.001 IR-Nx (green) vs. nonischemic right kidneys (blue), #: p < 0.05, ##: p < 0.01 and ###: p < 0.001 IR-S (red) vs. IR-Nx (green).

Figure 7

Fold changes in hypoxia- and oxidative stress-related mRNAs normalized to 18S. (a) HIF-1α, (b) HIF-2α and (c) nuclear factor erythroid 2-related factor 2 (NRF2). Data are expressed as mean ± SEM and two-way ANOVA with Tukey’s post hoc test. *: p < 0.05, **: p < 0.01 and ***: p < 0.001 IR-S (red) vs. nonischemic right kidneys (blue); +: p < 0.05, ++: p < 0.01, +++: p < 0.001 IR-Nx (green) vs. nonischemic right kidneys (blue), #: p < 0.05, ##: p < 0.01 and ###: p < 0.001 IR-S (red) vs. IR-Nx (green).

Figure 8

Fold changes in lipocalin-2 (Lcn-2) and fibrotic mRNAs normalized to 18S. (a) Lcn-2, (b) transforming growth factor-beta (TGF-β), (c) alpha-smooth muscle actin (α-SMA), (d) collagen-1a1 (Col1a1) and (e) fibronectin-1 (FN1). Data are expressed as mean ± SEM and two-way ANOVA with Tukey’s post hoc test. ***: p < 0.001 IR-S (red) vs. nonischemic right kidneys (blue); +++: p < 0.001 IR-Nx (green) vs. nonischemic right kidneys (blue), #: p < 0.05, ##: p < 0.01 and ###: p < 0.001 IR-S (red) vs. IR-Nx (green).

Figure 9

Overview of the interventions in each experiment of various durations. All major interventions are marked with arrow on the timeline. The time when blood and urine samples were taken after day 14 are marked as major and minor ticks, respectively.