Cyclophilin Inhibition Protects Against Experimental Acute Kidney Injury and Renal Interstitial Fibrosis

Figures

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  Figure 1. GS-642362 inhibits oxidant-induced cell death. Primary kidney tubular epithelial cells had varying concentrations of GS-642362 added prior to stimulation with 1 mM H2O2 to induce cell death in primary cultures of tubular epithelial cells. Data shows groups of 6 replicate samples. N.S., not significant.

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  Figure 2. Cyclophilin expression, renal function and tubular damage in renal ischemia/reperfusion injury (IRI). Groups of mice (n = 10) were treated with vehicle, 10 or 30 mg/kg/BID GS-642462 and compared to sham controls (n = 6). RT-PCR for mRNA levels of (A) CypA and (B) CypD. (C) Plasma creatinine levels. (D–G) Periodic-acid-Schiff-stained kidney sections. (D) Sham operated showing normal kidney structure. (E) Vehicle-treated IRI showing severe tubular damage in the outer medulla featuring tubular cell loss/sloughing and cast formation. (F) IRI treated with 10 mg/kg GS-642362 shows extensive tubular damage. (G) IRI treated with 30 mg/kg GS-642362 shows lesser, but still significant, tubular damage. Bar = 200 µm. (H) Graph of tubular damage. (I) RT-PCR for mRNA levels of the tubular damage marker, NGAL/LCN2.

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  Figure 3. Cell death in renal ischemia/reperfusion injury (IRI). Groups of mice (n = 10) were treated with vehicle, 10 or 30 mg/kg/BID GS-642462 and compared to sham controls (n = 6). (A–C) TUNEL staining. (A) Sham control lacks TUNEL+ cells. (B) Vehicle-treated IRI showing many TUNEL+ tubular cells in the outer medulla. (C) IRI treated with 30 mg/kg GS-642362 shows a marked reduction in the number of TUNEL+ cells. Bar = 100 µm. (D) Graph of TUNEL+ cells.

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  Figure 4. Neutrophil and macrophage infiltration in renal ischemia/reperfusion injury (IRI). Groups of mice (n = 10) were treated with vehicle, 10 or 30 mg/kg/BID GS-642462 and compared to sham controls (n = 6). (A–C) Immunostaining of Ly6G+ peroxidases. (A) Sham control lacks neutrophils. (B) Vehicle-treated IRI shows many infiltrating neutrophils in an area of tubular damage. (C) IRI treated with 30 mg/kg GS-642362 shows a dramatic reduction in neutrophil infiltration. Bar = 100 µm. (D) Graph of Ly6G+ neutrophils. RT-PCR analysis of mRNA levels for (E) CD68, (F) CCL2, (G) TNF, and (H) IL-2. N.S., not significant.

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  Figure 5. Cyclophilin expression and tubular damage in the day 7 unilateral ureteric obstruction (UUO) model (n = 8 to 10/group). RT-PCR for mRNA levels of (A) CypA and (B) CypD. (C–E) Periodic-acid-Schiff-stained kidney sections. (C) Normal mouse kidney. (D) Vehicle-treated UUO showing marked tubular dilation. (E) UUO treated with 30 mg/kg GS-642362 also shows marked tubular dilation. Bar = 200 µm. (F) Graph of tubular damage. (G) RT-PCR for mRNA levels of the tubular damage marker, NGAL/LCN2.

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  Figure 6. Cell death in the day 7 unilateral ureteric obstruction (UUO) model (n = 8 to 10/group). (A–C) TUNEL staining. (A) Normal mouse kidney. (B) Vehicle-treated UUO showing small numbers of TUNEL+ tubular cells in the cortex. (C) UUO treated with 30 mg/kg GS-642362 shows a clear reduction in the number of TUNEL+ cells. Bar = 100 µm. (D) Graph of TUNEL+ cells. N.S., not significant.

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  Figure 7. Macrophage infiltration in the day 7 unilateral ureteric obstruction (UUO) model (n = 8 to 10/group). (A–D) Immunostaining for F4/80+ macrophages. (A) Normal mouse kidney showing resident F4/80+ macrophages. (B) Vehicle-treated UUO showing a significant increase in macrophages in the cortex. (C) UUO treated with 30 mg/kg GS-642362 shows a partial in macrophage infiltration. Bar = 100 µm. (D) Graph of the area of F4/80 macrophage staining. RT-PCR for mRNA levels of (E) CD68, (F) CCL2, and (G) IL-2. N.S., not significant.

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  Figure 8. Renal fibrosis in the day 7 unilateral ureteric obstruction (UUO) model (n = 8 to 10/group). (A) Hydroxyproline assay of total collagen content. (B–D) Immunostaining for collagen IV. (B) Normal kidney shows fine staining of collagen IV in the glomerular and tubular basement membranes. (C) Vehicle-treated UUO shows a substantial increase in interstitial collagen IV staining. (D) UUO treated with 30 mg/kg GS-642362 shows a partial in interstitial collagen IV staining. Bar = 100 µm. (E) Graph of the area of interstitial collagen IV staining. (F) Graph of the area of interstitial staining for α-SMA. RT-PCR for mRNA levels of (G) collagen IV, (H) α-SMA/Acta2, (I) TGF-β1, and (J) PDGF-B. N.S., not significant.

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  Figure 9. Peritubular capillary loss in the day 7 unilateral ureteric obstruction (UUO) model (n = 8 to 10/group). (A–C) Immunostaining for CD31+ endothelial cells. (A) Normal kidney shows a dense network of peritubular capillaries and glomerular capillary tufts. (B) Vehicle-treated UUO shows a partial loss of peritubular capillaries. (C) UUO treated with 30 mg/kg GS-642362 also shows a partial loss of peritubular capillaries. Bar = 100 µm. (D) Graph of the area of interstitial CD31 staining. N.S., not significant.

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  Figure 10. Stress-activated protein kinases in the day 7 unilateral ureteric obstruction (UUO) model (n = 8 to 10/group). Western blots for (A) phospho-p38 MAPK and (B) phospho-JNK, with a tubulin loading control. Graphs show quantification for (C) phospho-p38 MAPK and (D) phospho-JNK. N.S., not significant.