The molecular mechanisms that lead to tubular atrophy capillary loss and

The molecular mechanisms that lead to tubular atrophy capillary loss and fibrosis following acute kidney injury are not very clear but may involve cell cycle inhibition by increased expression of cyclin kinase inhibitors. and levels of p16INK4a apoptosis cell proliferation and capillary rarefaction. KO mice displayed decreased tubular cell apoptosis improved cell proliferation and lower creatinine AZD3514 levels after injury. KO mice experienced significantly higher capillary denseness compared with WT mice at 14-42 days after IRI. Plasma granulocyte colony-stimulating element (G-CSF) improved after ischemia in both WT and KO mice and was elevated markedly in KO compared with WT mice. KO kidney digests contained higher counts of Gr-1+/Cd11b+ myeloid cells by circulation cytometry. KO mice treated having a Gr-1-depleting antibody displayed reduced vascular endothelial growth element mRNA plasma G-CSF and capillary denseness and an increase in serum creatinine and medullary myofibroblasts compared with untreated KO mice 14 days after ischemia. The anti-angiogenic effect of Gr-1 depletion in KO mice was confirmed by Matrigel angiogenesis assays. These results suggest that the absence of p16INK4a and p19ARF following IRI has a protective effect on the kidney through improved epithelial and microvascular restoration in part by enhancing the mobilization of myeloid cells into the kidney. = 5 mice/group). Western blotting. Cells ethnicities were washed with PBS and lysed in RIPA buffer (1% Nonidet P-40 0.5% sodium deoxycholate 0.1% SDS 1 mM EDTA and 0.5% Triton X-100 in PBS) containing Complete Mini protease inhibitor (Roche) and 2 mM phenylmethylsulfonyl fluoride. Protein concentrations were measured using a Bradford assay (Bio-Rad Hercules CA). The protein samples were denatured by adding SDS sample buffer and heating at 95°C for 5 min. Twenty to forty micrograms of sample containing β-mercaptoethanol were resolved on 4-20% Tris glycine polyacrylamide gels. Gels were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Bedford MA) and clogged with 5% nonfat dry milk in 0.1% Tween 20 in PBS. The membranes were then incubated over night at 4°C with main antibody in obstructing buffer. This was followed by washing and 1 h incubation with an appropriate horseradish peroxidase-conjugated secondary antibody. Protein bands were recognized by chemiluminescence using a commercial kit (Thermo Scientific Rockford IL) according to the manufacturer’s instructions. For quantification of protein levels autoradiographs were scanned with the Scion Image densitometry program and results were corrected for variations in the amount of protein loaded on each lane using corresponding POLB β-tubulin levels. Matrigel assay. Eight- to ten-week-old WT and INK4a/ARF KO male mice were anesthetized with ketamine and xylazine. Three hundred microliters of growth factor-reduced Matrigel (BD Biosciences) diluted to 10 mg/ml with minimal essential medium were injected subcutaneously. Mice were injected with 100 μg of intravenous 500 0 mol wt dextran (Invitrogen) 5 min before death. Matrigel plugs were removed after 10 days and fixed in 4% paraformaldehyde. Plugs were embedded in paraffin for hematoxylin and eosin staining or OCT for cryostat sections for immunofluorescence staining. For Gr-1 depletion KO mice were injected intrperitoneally with 100 μg of anti-mouse Ly-6G 1 and 4 days after plug implantation. Aortic ring angiogenesis assay. Thoracic aortas were removed from WT and INK4a/ARF KO mice using sterile technique. Surrounding connective tissue was removed completely followed by washes in EGM-2 medium (Lonza Walkersville MD). One-millimeter slices were placed between two layers of growth factor-reduced Matrigel (10 mg/ml) and incubated with EGM-2 medium. Capillary outgrowth was measured after 2 wk using Metamorph software. Flow cytometry. Kidneys were removed aseptically and digested using a modification of the method of Vielhauer et al. (30). The minced kidney was treated with 1 mg/ml collagenase in Hanks’ balanced salt answer (HBSS) for 20 min at 37°C with shaking. After being washed with HBSS the kidney digest was treated with HBSS with 2 mM EDTA for 20 min AZD3514 at 37°C with shaking. The kidneys were centrifuged at 250 AZD3514 AZD3514 for 5 min. The supernatant was reserved and kept on ice. The pellet was resuspended in 1 mg/ml collagenase in HBSS and incubated for 20 min at 37°C with shaking. These cells were exceeded through a 20-gauge needle three times. The digest was then placed on ice for 5 min after which the.