Macrophages promote cyst growth in polycystic kidney disease

Abstract

Polycystic kidney disease (PKD) exhibits an inflammatory component, but the contribution of inflammation to cyst progression is unknown. Macrophages promote the proliferation of tubular cells following ischemic injury, suggesting that they may have a role in cystogenesis. Furthermore, cultured Pkd1-deficient cells express the macrophage chemoattractants Mcp1 and Cxcl16 and stimulate macrophage migration. Here, in orthologous models of both PKD1 and PKD2, abnormally large numbers of alternatively activated macrophages surrounded the cysts. To determine whether pericystic macrophages contribute to the proliferation of cyst-lining cells, we depleted phagocytic cells from Pkd1(fl/fl);Pkhd1-Cre mice by treating with liposomal clodronate from postnatal day 10 until day 24. Compared with vehicle-treated controls, macrophage-depleted mice had a significantly lower cystic index, reduced proliferation of cyst-lining cells, better-preserved renal parenchyma, and improved renal function. In conclusion, these data suggest that macrophages home to cystic areas and contribute to cyst growth. Interruption of these homing and proliferative signals could have therapeutic potential for PKD.

Figure 1.

Macrophages surround cysts in murine models of PKD. Kidneys were perfusion fixed with 4% PFA and cryosections stained for macrophage marker F4/80. (A) Cyst-lining cells showing closely apposed macrophages stained green. Green, F4/80; Red, DBA. (B) Quantitative analysis of FACS-sorted macrophages defined as F4/80⁺CD45⁺CD11c^- cells and further analyzed for Ly6c expression; ***P < 0.0001. (C) Cystic area from Pkd2^WS25/- kidney showing several F4/80⁺ cells (green). Inset shows magnified view of area marked with an asterisk. (D) Noncystic area from Pkd2^ws25/+ kidney showing very few F4/80⁺ cells. Inset shows magnified view of selected area indicated by the asterisk. (E) Quantitative analysis of macrophages in kidneys from Pkd2^WS25/- and Pkd2^ws25/+ (control) mice. (F) Transwell migration of macrophages toward DMEM-F12, DMEM-F12 + 10%FBS, DMEM-F12 conditioned medium from Pkd1^fl/- cells, and DMEM-F12 conditioned media from Pkd1^-/- cells, respectively, n = 3. Inset: qRT-PCR for Mcp-1 and Cxcl16 was performed on RNA from Pkd1^fl/- and Pkd1^-/- cells and reported as the fold increase of Pkd1^-/- as compared with Pkd1^fl/-; n = 3.

Figure 2.

Clodronate treatment reduces macrophage load and improves renal function. Cystic kidneys from Pkd1^fl/fl;Pkhd1-Cre mice treated with LC show (B) fewer F4/80⁺ macrophages (arrows) along the cyst-lining cells compared with LV-treated mouse kidneys (A, arrows). (C) Scan of a sagittal section of a representative kidney from LV- and LC-treated mice, respectively, shows (D) better-preserved parenchyma in LC-treated mice and lowered cystic index, (E) improved kidney/body weight ratio, and (F) significantly improved renal BUN values. A, B: Green, F4/80; Red, DBA. *P < 0.01; **P < 0.001.

Figure 3.

LC administration reduces cystic-lining cell proliferation. Representative kidney sections are shown from (A) LV-treated and (B) LC-treated mice stained for Ki67 (arrows). Green, DBA, Red/purple-Ki67; Blue, DAPI. (C) Ki67 positive nuclei quantitated within DBA positive tubules from kidneys of LV- and LC-treated mice. Data pooled from eight individual mice is shown. ***P < 0.0001.