Axl−/− mice have significantly reduced body weight and survival following sub-total nephrectomy and high phosphate diet.

<p>Weight in grams of (A) females and (B) males expressed as means +/− standard error of the mean (SEM). Statistical test is a 2-way ANOVA with a Sidak compensation for multiple comparison, § = p≤0.005 for difference between genotypes and * = p≤0.05 for individual time points, n number (surviving animals) in brackets. (C–F) Kaplan-Meier survival plots of Axl+/+ and Axl−/− mice following nephrectomy and high phosphate diet. (C) All mice, (D) males, (E) females, (F) Axl−/− mice alone.</p

Loss of Axl results in elevated renal tubulo-interstitial apoptosis.

<p>(A) Representative TUNEL- and DAPI-stained kidney sections post-sub-total nephrectomy and high phosphate diet. Tubulo-interstitial areas of the kidney are shown, bar  = 50 microns. (B) Quantification of TUNEL staining expressed as mean +/− SEM, n = 10 per group. Statistical test is Student's <i>t</i>-test; ns  =  not significant, * = p≤0.05.</p

Axl and its ligand Gas6 are up-regulated following sub-total nephrectomy and high phosphate diet.

<p>(A) Western blots of kidney lysates for Axl, pAkt, total Akt and α-tubulin; 4 individual animals per group. The groups are Axl+/+ animals at baseline (11 weeks of age), 25 week old Axl+/+ animals with no nephrectomy (age-matched controls; no Nx), and 25 week old Axl+/+ animals that had a sub-total nephrectomy (Nx) at 11 weeks (Nx + phosphate). (B, C) Plasma Gas6 levels determined by ELISA; (B) Gas6 levels by genotype, (C) Gas6 levels by gender. A minimum of 4 animals were analysed per group. Results expressed as individual data points with means +/− Std Dev. Statistical test is a Kruskal-Wallis test with Dunn's compensation for multiple comparisons, * = p≤0.05, ** = p≤0.01, *** = p≤0.005.</p

Axl−/− mice develop elevated uraemia following sub-total nephrectomy and high phosphate diet.

<p>(A) Blood Urea Nitrogen (BUN) levels expressed as individual data points with means +/− standard deviation (Std Dev). Statistical test is a 1-way ANOVA with Sidak compensation for multiple comparisons. (B–G) BUN levels of Axl+/+ and Axl−/− mice expressed as means +/− SEM; (B) all mice, (C) Axl−/− sub-populations (survived/deceased), (D) females analysed by genotype, (E) males analysed by genotype, (F) Axl+/+ mice analysed by gender, (G) Axl−/− mice analysed by gender. Statistical test is a 2-way ANOVA with Sidak compensation for multiple comparisons. § = p≤0.05 for difference between genotypes, * = p≤0.05, ** = p≤0.01, *** = p≤0.005.</p

Loss of Axl did not affect the extent of renal calcification following sub-total nephrectomy and high phosphate diet.

<p>Histological analysis of kidney calcification (A) Representative scans of von Kossa stained sections from Axl+/+ and Axl−/− kidneys with and without nephrectomy and high phosphate diet. Bar  = 1000 microns. (B) Magnified images of kidney calcification 14 weeks post-initial surgery. Bar  = 200 microns. (C) Quantification of von Kossa staining, Axl+/+ n = 13, Axl−/− n = 15. Statistical test is Student's <i>t</i>-test. ns  =  not significant. Nx  =  nephrectomy.</p

Kidney mass pre- and post-surgery.

<p>Kidney mass at baseline and the kidney remnant mass are expressed as a percentage of the animal's weight at these time points. When calculating kidney mass at baseline and the percentage kidney mass removed, total kidney mass was assumed to be twice that of the right kidney upon removal. The n number and standard deviation are in brackets. There is no statistically significant difference between genotypes.</p

Plasma phosphate and calcium concentrations.

<p>Plasma phosphate and calcium levels in mM +/− Std Dev, n number in brackets. Statistical test is Student's <i>t</i>-test, * = p≤0.05. Nx  =  nephrectomy.</p

FTI-277 inhibits smooth muscle cell calcification by up-regulating PI3K/Akt signaling and inhibiting apoptosis

<div><p>Background</p><p>Vascular calcification is associated with increased cardiovascular morbidity and mortality in patients with atherosclerosis, diabetes and chronic kidney disease. However, no viable treatments for this condition have been identified. This study aimed to determine whether farnesyl transferase inhibitors (FTIs) can reduce vascular calcification and the mechanism by which this reduction occurs.</p><p>Results</p><p>We demonstrate that FTI-277 significantly inhibits phosphate-induced mineral deposition by vascular smooth muscle cells (VSMC) <i>in vitro</i>, prevents VSMC osteogenic differentiation, and increases mRNA expression of matrix Gla protein (MGP), an inhibitor of mineralization. FTI-277 increases Akt signaling in VSMC in short-term serum-stimulation assays and in long-term mineralization assays. In contrast, manumycin A has no effect on Akt signaling or mineralization. Co-incubation of VSMC with FTI-277 and SH6 (an Akt inhibitor) significantly reduces the inhibitory effect of FTI-277 on mineralization, demonstrating that FTI-277 inhibits calcification by activating Akt signaling. Over-expression of the constitutively active p110 sub-unit of PI3K in VSMC using adenovirus activates Akt, inhibits mineralization, suppresses VSMC differentiation and significantly enhances MGP mRNA expression. FTI-277 also inhibits phosphate-induced activation of caspase 3 and apoptosis of VSMC, and these effects are negated by co-incubation with SH6. Finally, using an <i>ex vivo</i> model of vascular calcification, we demonstrate that FTI-277 inhibits high phosphate-induced mineralization in aortic rings derived from rats with end-stage renal failure.</p><p>Conclusions</p><p>Together, these results demonstrate that FTI-277 inhibits VSMC mineral deposition by up-regulating PI3K/Akt signaling and preventing apoptosis, suggesting that targeting farnesylation, or Akt specifically, may have therapeutic potential for the prevention of vascular calcification.</p></div

FTI-277 inhibits phosphate-induced apoptosis.

<p><b>A</b>) Human VSMCs were incubated in serum-free medium containing elevated phosphate (2.6 mM) ± FTI-277 (10 μM) or vehicle control. Cell lysates were collected 2 and 4 hours later and analysed by western blotting. (<b>B</b>) Human VSMCs incubated in serum-free medium containing phosphate (2.6 mM) ± FTI-277 (10 μM) ± SH6 (10 μM) for 12 hours were fixed and stained with DAPI. Apoptotic cells are expressed as a percentage of total cells (mean ± SEM); >750 cells were counted per variable. Data were analyzed by one-way ANOVA and Tukey post-hoc tests; *p<0.05.</p

Over-expression of constitutively activated p110 sub-unit of PI3K inhibits mineralization.

<p>Constitutively active p110 sub-unit of PI3K (p110) or empty virus (Empty) were over-expressed in VSMC using adenovirus and cells were incubated in 10% FCS-DMEM containing βGP (5 mM) for 9 days. Controls were incubated without BGP. (<b>A</b>) Representative western blots of cell lysates for pAkt and total Akt. (<b>B</b>) Representative phase contrast images of alizarin red-stained cells (bar = 500 μm). (<b>C</b>) Quantification of mineralization (mean ± SEM; n = 6). Data were normalized using log₁₀ and analyzed using one-way ANOVA and Tukey post-hoc tests; *p<0.05 compared to βGP. (<b>D</b>) qPCR for Runx2 and matrix Gla protein (MGP) was performed. Relative mRNA expression of Runx2 and MGP are shown as fold-change to cells treated with empty virus and βGP (mean ± SEM; n = 6). Data were analyzed using one-way ANOVA and Tukey post-hoc tests; *p<0.05.</p