Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease

Abstract

Background: In patients with autosomal dominant polycystic kidney disease (ADPKD), most of whom have a mutation in PKD1 or PKD2, abnormally large numbers of macrophages accumulate around kidney cysts and promote their growth. Research by us and others has suggested that monocyte chemoattractant protein-1 (Mcp1) may be a signal for macrophage-mediated cyst growth.

Methods: To define the role of Mcp1 and macrophages in promoting cyst growth, we used mice with inducible knockout of Pkd1 alone (single knockout) or knockout of both Pkd1 and Mcp1 (double knockout) in the murine renal tubule. Levels of Mcp1 RNA expression were measured in single-knockout mice and controls.

Results: In single-knockout mice, upregulation of Mcp1 precedes macrophage infiltration. Macrophages accumulating around nascent cysts (0-2 weeks after induction) are initially proinflammatory and induce tubular cell injury with morphologic flattening, oxidative DNA damage, and proliferation-independent cystic dilation. At 2-6 weeks after induction, macrophages switch to an alternative activation phenotype and promote further cyst growth because of an additional three-fold increase in tubular cell proliferative rates. In double-knockout mice, there is a marked reduction in Mcp1 expression and macrophage numbers, resulting in less initial tubular cell injury, slower cyst growth, and improved renal function. Treatment of single-knockout mice with an inhibitor to the Mcp1 receptor Ccr2 partially reproduced the morphologic and functional improvement seen with Mcp1 knockout.

Conclusions: Mcp1 is upregulated after knockout of Pkd1 and promotes macrophage accumulation and cyst growth via both proliferation-independent and proliferation-dependent mechanisms in this orthologous mouse model of ADPKD.

Keywords: ADPKD; Immunology and pathology; MCP-1; genetic renal disease; kidney tubule; polycystic kidney disease.

Figure 1.

Conditional knockout of Mcp1 in tubular epithelial cells prevents infiltration of macrophages. (A) Quantification of F4/80⁺ macrophages in kidney sections from 6 to 8 week old SKO compared with UI mice. **P<0.01 relative to UI. (B) Renal expression of the mRNA for Mcp1 and Sdf1 relative to Hprt1 in 8-week-old UI and SKO kidneys. ***P<0.001 relative to UI. (C) Expression levels of chemokine receptors Ccr1 and Ccr2 mRNA in flow-sorted CD45⁺F4/80⁺Cd11c⁻ macrophages from 12-week-old UI and SKO mice kidneys. *P<0.05 related to UI. (D) Quantitative real-time PCR of whole kidney RNA from UI (8 weeks old) and SKO (6 and 8 weeks old) mice for Mcp1 and Pkd1. *P<0.05 relative to UI Pkd1 levels; ***P<0.001 relative to UI Mcp1 levels. (E and F) Quantitative real-time PCR of Mcp1 (E) and F4/80 (F) in whole kidney RNA from UI, SKO, and DKO mice at the indicated ages (weeks). P<0.001 by two-way ANOVA for both genes comparing SKO with UI and SKO with DKO. (G) Quantitation of F4/80+ macrophages in sections from UI, SKO, and DKO kidneys at the indicated ages (weeks). P<0.001 by two-way ANOVA comparing SKO with UI and SKO with DKO. (H) Representative images for immunofluorescence staining for F4/80 in 8-week-old UI, SKO, and DKO kidneys (red, F4/80). The n listed under each bar represents the number of individual mice analyzed at that time point.

Figure 2.

Tubular epithelial deletion of Mcp1 slows cyst growth and improves renal function. (A and B) Kidney weight (A) and kidney weight-to-body weight ratio (B) of SKO and DKO mice compared with UI littermate controls at the indicated ages (weeks). P=0.009 by two-way ANOVA comparing SKO with DKO. (C) Representative images of sagittal kidney sections from 18-week-old UI, SKO, and DKO mice. Scale bar, 3 mm. (D) Cystic index in UI (n=4–6 per time point), SKO (n=5 per time point), and DKO (n=5–10 per time point) mice at the indicated ages (weeks). P=0.002 by two-way ANOVA comparing SKO with DKO. (E and F) BUN and creatinine values from UI, SKO, and DKO mice at the indicated time points. P<0.001 by two-way ANOVA for both BUN and creatinine comparing SKO with DKO. (G) Kaplan–Meier survival curve comparing SKO and DKO mice at 18 weeks of age (P=0.005). The n listed under each bar represents the number of individual mice analyzed at that time point.

Figure 3.

CCR2 inhibition reduces macrophage infiltration and cyst index in SKO mice. (A) Quantification of F4/80⁺ macrophages in kidney sections from 12-week-old SKO mice treated with either DMSO vehicle (SKO-V) or CCR2 antagonist INCB3344 (SKO-INCB). n=number of individual mice analyzed. ***P<0.001 versus vehicle treatment. (B) Kidney-to-body weight ratio of UI mice compared with SKO mice treated with vehicle (V) or INCB. **P<0.01 versus vehicle treatment. (C) Representative images of sagittal kidney sections from 12 weeks age SKO mice treated with either vehicle (V) or INCB3344 (INCB). Scale bar, 4 mm. (D) Cystic index of UI mice compared with SKO mice treated with vehicle (V) or INCB. **P<0.01 versus vehicle treatment. (E) BUN values of SKO mice treated with vehicle (V) or INCB. P=NS versus vehicle treatment. (F) Creatinine values of SKO mice treated with vehicle (V) or INCB. *P<0.05 versus vehicle treatment.

Figure 4.

Tubule cell proliferation after Pc1 loss involves a sustained macrophage-independent component along with late acceleration dependent on a switch to alternative macrophage activation. (A) Representative images of 8-week-old UI, SKO, and DKO kidneys showing cortical Ki-67⁺ tubular cells (arrows). Scale bar, 200 µm. (B) Quantification of Ki-67⁺ tubular cells as shown in (A). UI (n=6 mice per time point), SKO (n=6 mice per time point), and DKO (n=5 mice per time point). P=0.004 for SKO versus DKO mice by two-way ANOVA. (C–I) Quantitative real-time PCR of Nos2 (C), Tnfα (D), IL12 (E), Arg1 (F), Mrc1 (G), Csf1 (H), and Csf2 (I) using whole kidney RNA from UI, SKO, and DKO mice at the indicated ages (weeks). P<0.05 for Tnfα, Mrc1, and Csf1, and P<0.01 for Nos2, IL12, Arg1, and Csf2 using two-way ANOVA comparing SKO with DKO. The n listed under each bar represents the number of individual mice analyzed at that time point.

Figure 5.

Proinflammatory macrophages induce early oxidative DNA damage and single-strand DNA breaks. (A) Quantitative real-time PCR of Kim1 using whole kidney RNA from UI, SKO, and DKO mice at 8 weeks age. **P<0.01 for SKO versus UI and SKO versus DKO mice. (B) Representative images of immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG) in cortex from 8-week-old kidneys of the indicated genotypes. Arrows depict the 8-OHdG⁺ cells from cystic and dilated tubules. Scale bar, 50 µm. (C) Quantification of 8-OHdG⁺ tubular cells in renal cortex as in (B). **P<0.01 for SKO versus UI and SKO versus DKO mice. (D) Representative images of cortical TUNEL staining in 8-week-old mice of the indicated genotypes. Arrows depict the TUNEL-positive cells. (E) Quantification of TUNEL-positive tubular cells in renal cortex as shown in (D). ***P<0.001 for SKO versus UI and SKO versus DKO mice. (F) Representative electron microscopy image of cortex from an 8-week-old SKO mouse kidney demonstrating normal morphology of both tubular nuclei (tn) and adjacent macrophage nucleus (mn). (G) Western blotting for cleaved caspase-3 (CC3, upper panel) and γH2AX (lower panel) in lysates from 8-week-old mouse kidneys. Each lane represents a separate kidney. GAPDH was used as the loading control. (H and I) Quantification of CC3 and γH2AX Western blotting analysis normalized to GAPDH. P=NS between all groups. (J) Representative images of alkaline comet assay using freshly isolated cells from 8-week-old UI, SKO, and DKO mouse kidneys. Arrows indicate nuclei with a comet tail. Scale bar, 400 µm. (K) Quantification of comet tail moment from freshly isolated tubular cells [at isolation, as shown in (J)] and after 7 days in culture [cultured, as shown in (M)]. **P<0.01 for SKO versus UI mice; P<0.05 for SKO versus DKO mice. (L) PCR analysis of genomic DNA from cultured epithelial cells isolated from UI, SKO, and DKO mice. Each lane represents a separate culture. (M) Representative images of alkaline comet assay using cultured cells from 8-week-old UI, SKO, and DKO mouse kidneys. Scale bar, 400 µm. The n listed under each bar represents the mice analyzed at that time point.

Figure 6.

Proinflammatory macrophages promote proliferation-independent tubule dilation. (A–C) Representative multiphoton microscopic images of the indicated genotypes. (D–F) Graphs showing calculated tubular and epithelial area from 100 randomly analyzed S1 segments from kidneys of 8-week-old mice of the indicated genotypes. n=3 separate mice for the UI analysis, and four separate mice for the SKO and DKO analyses. Each bar represents an individual tubule area, with the overlying dot representing the epithelial area for that same tubule cross section. Drop down arrows and the red bar identify the specific isolated tubule cross section shown in the accompanying images. The ten tubule sections with smallest epithelial areas (S) and largest epithelial areas (L) were used for absolute tubule and epithelial area quantification. (G) Average tubular and epithelial area determined by absolute quantification of optically isolated tubule cross sections from 8-week-old kidneys of the indicated genotypes (n=10 per group). (H) Ratio of tubular-to-epithelial area obtained from absolute quantification shown in (G). ***P<0.001 for SKO “S” versus UI “S,” P<0.001 for SKO “S” versus DKO “S.” (I) Quantification of nuclei in isolated tubule cross-sections from the same sections analyzed in (G). **P<0.01 for SKO “L” versus UI “L” and DKO “L” versus UI “L.”