Ciclopirox olamine induces ferritinophagy and reduces cyst burden in polycystic kidney disease

Abstract

Despite the recent launch of tolvaptan, the search for safer polycystic kidney disease (PKD) drugs continues. Ciclopirox (CPX) or its olamine salt (CPX-O) is contained in a number of commercially available antifungal agents. CPX is also reported to possess anticancer activity. Several mechanisms of action have been proposed, including chelation of iron and inhibition of iron-dependent enzymes. Here, we show that CPX-O inhibited in vitro cystogenesis of primary human PKD cyst-lining epithelial cells cultured in a 3D collagen matrix. To assess the in vivo role of CPX-O, we treated PKD mice with CPX-O. CPX-O reduced the kidney-to-body weight ratios of PKD mice. The CPX-O treatment was also associated with decreased cell proliferation, decreased cystic area, and improved renal function. Ferritin levels were markedly elevated in cystic kidneys of PKD mice, and CPX-O treatment reduced renal ferritin levels. The reduction in ferritin was associated with increased ferritinophagy marker nuclear receptor coactivator 4, which reversed upon CPX-O treatment in PKD mice. Interestingly, these effects on ferritin appeared independent of iron. These data suggest that CPX-O can induce ferritin degradation via ferritinophagy, which is associated with decreased cyst growth progression in PKD mice. Most importantly these data indicate that CPX-O has the potential to treat autosomal dominant PKD.

Keywords: Chronic kidney disease; Nephrology.

Figure 1. CPX-O inhibits cyst formation of ADPKD cells.

ADPKD cells were grown to form cysts on a 3D system using collagen matrix in the presence of FSK and EGF for 3–5 days followed by treatment with vehicle, CPX-O, or N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) for 6 additional days. (A) Cysts were fixed, imaged, and measured. Original magnification, ×10. (B) Average fold change in cyst size with vehicle cyst size set at 1 (from 4 patients). Within each patient 6 replicates/treatment were used and data were averaged. (C) Cyst size is represented as surface area ± SEM from a single ADPKD patient (K298) (6 replicates/treatment). (D) Primary cells from normal human kidney (NHK) (n = 3) and ADPKD kidney (n = 3) cells were cultured in the presence of CPX-O for 6 days followed by viability assays. Data presented as percentage viability ± SEM. Statistical significance was determined using 1-way ANOVA followed by Tukey’s honestly significant difference (HSD) test (*P < 0.05, **P < 0.01).

Figure 2. CPX-O ameliorates disease progression in a mouse model of ADPKD.

(A) Experimental timeline where 22-day-old PKD or WT mice were intraperitoneally injected with vehicle or CPX-O (10 mg/kg body weight) for 27 consecutive days until P49. At P50, mice were euthanized and samples were collected. (B) H&E staining of kidney sections. Representative images of each treatment group are shown. (C) Renal cystic index of vehicle-treated (n = 7) and CPX-O–treated (n = 8) PKD mice, presented as percentage cystic area ± SEM. (D) Kidney-to-body weight ratio from vehicle- or CPX-O–treated WT mice (n = 5 each) and from vehicle-treated (n = 7) or CPX-treated (n = 8) PKD mice. (E) Blood urea nitrogen values measured as mg/dL ± SEM from hemolysis-free serum samples of vehicle- and CPX-O–treated WT mice (n = 5 each) or from vehicle- and CPX-O–treated PKD mice (n = 5 and n = 8, respectively). Statistical significance was determined using unpaired Student’s 2-tailed t test (C) or 1-way ANOVA followed by Tukey’s HSD test (D and E) (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Scale bar: 1 mm.

Figure 3. CPX-O slows down cell proliferation in PKD.

(A) Immunohistochemistry (IHC) for cell proliferation assessed by Ki67 staining on 7-week-old kidneys from PKD mice treated with vehicle or CPX-O. Arrow points to a highly proliferative area on a vehicle-treated (Veh) PKD mouse kidney section. Hematoxylin staining shows nuclei in blue, and Ki67-positive nuclei are shown in dark brown. (B) Cells were counted and expressed as percentage Ki67-positive cells ± SEM from at least 14 sections per treatment from 3 mice per group. Unpaired Student’s 2-tailed t test used for statistical analysis (*P < 0.05). Scale bar: 100 μm.

Figure 4. CPX-O inhibits ferritin accumulation in the cystic and interstitial cells in ADPKD kidneys.

(A) IHC for ferritin in 7-week-old WT and PKD mouse sections treated with vehicle (Veh) or CPX-O. Note (arrowheads) accumulation of ferritin-positive cells near cystic areas in PKD mice. Ferritin-positive cells were reduced in kidneys of CPX-O–treated PKD mice. Scale bar: 100 μm. (B) Western blot (WB) of kidney lysates for ferritin (top) and quantification of ferritin expression relative to the Ponceau S expression (lower) in vehicle- and CPX-O–treated PKD mice in contrast to vehicle- and CPX-O–treated WT mice (n = 3 per group). (C) IHC was performed in NHK and ADPKD kidney for ferritin expression. Arrowheads indicate high ferritin in both cyst epithelium and interstitial cells. Original magnification, ×20. (D) WB for ferritin using lysates of primary cells from normal (n = 3) or ADPKD (n = 3). Quantification of ferritin expression normalized to Ponceau S is shown in the right panel. Data presented as relative fold change in ferritin ± SEM. (Unpaired Student’s 2-tailed t test, *P < 0.05.)

Figure 5. Ferritin is expressed in collecting duct cells and macrophages in patients with ADPKD.

(A) NHK and ADPKD sections were colabeled for ferritin (red) and DBA (a collecting duct marker, green). DAPI (a nuclear stain, blue) was used as a counterstain. Arrowheads show same cells with coexpression. Merged image from lower panels shows ferritin colocalization with DBA (arrowhead) in cyst-lining cells. Thin arrow shows interstitial cells with ferritin expression. Scale bar: 100 μm. (B) NHK and ADPKD sections were colabeled with ferritin (green) and CD68 (macrophage marker) (red). Arrowheads show coexpression of CD68 with ferritin. The area with arrowhead is amplified in the inset (bottom, right panel). Scale bar: 75 μm.

Figure 6. CPX-O induces ferritinophagy in primary cyst epithelial cells from patients with ADPKD.

(A) WBs for LC3B-II, and NCOA4, on kidney lysates of 7-week-old WT (n = 3) and PKD (n = 4) mice treated with vehicle (veh) or CPX-O. (B) Quantification of LC3B-II from A and expression normalized to Ponceau S and expressed as relative expression ± SEM. (C) Quantification of NCOA4 expression from A normalized to Ponceau S and expressed as relative expression ± SEM. (D) Total intracellular iron fold change in ADPKD primary cyst epithelial cells (n = 4) relative to NHK primary cells (n = 3) ± SEM. (E) Cyst-lining epithelial cells from a patient with ADPKD were grown to form cysts on a 3D system using collagen matrix in the presence of FSK and EGF for 5 days followed by treatment with vehicle, CPX-O (5 μM), holoferritin (50 μg/mL), apoferritin (50 μg/mL), holoferritin + CPX-O, and apoferritin + CPX-O for 6 additional days. Cysts were fixed, imaged, and measured. Cyst size is represented as surface area/cm² (n = 6/treatment). One-way ANOVA followed by Tukey’s HSD test was used for statistical analyses of all graphs except D, where unpaired Student’s 2-tailed t test was used (*P < 0.05, **P < 0.01, ****P < 0.0001).