A role in the accumulation of bone Fasudil HCl web marrow-derived fibroblasts in the kidneys, WT and IL-6 KO mice were subjected to obstructive injury for 5 or 7 days. Kidney sections were stained for CD11b and procollagen I and examined with a fluorescent microscope. Our results showed that both WT and IL-6 KO mice 1326631 exhibited a marked increase in the number ofThe Role of IL-6 in Renal FibrosisTGF-b1 mRNA in the kidney following obstructive injury (Figure 4B). These data suggest that IL-6 signaling does not play a major role in the regulation of TGF-b1 gene expression in the kidney in response to obstructive injury. Since Th2 cytokines ?IL-4 and IL-13 have been shown to promote monocyte-to-fibroblast transition [14,19], we then examined if IL-6 deficiency affects these cytokine expression in the kidney. The results of real time RT-PCR showed that IL-6 deficiency did not influence mRNA levels of IL-4 and IL-13 in the kidney following obstructive injury (Figure 4 C ). These data indicate that IL-6 signaling does not modulate the gene expression of IL-4 and IL-13 in the kidney in response to obstructive injury.IL-6 Deficiency Has no Effect on Renal FibrosisSince IL-6 does not regulate the accumulation and activation of bone marrow-derived fibroblasts in the kidney in response to obstructive injury, we then examined if IL-6 deficiency has an effect on the development of renal fibrosis. WT and IL-6 KO mice were subjected to UUO for 14 days. Both WT and IL-6 KO mice developed similar degree of collagen deposition in obstructed kidneys as demonstrated by MedChemExpress FTY720 picrosirius red staining (Figure 5). These data indicate that IL-6 does not play a role in the pathogenesis of renal fibrosis.IL-6 Deficiency Does not Affect ECM Protein ExpressionWe next investigated the effect of targeted disruption of IL-6 on the expression and accumulation of collagen I and fibronectin, two major components of ECM. Both WT and IL-6 KO mice displayed a marked increase in the protein expression levels of collagen I and fibronectin in the kidneys following obstructive injury, which was not statistically different between these two groups (Figure 6 and 7). These data indicate that IL-6 does not regulate the production and deposition of ECM proteins in the kidney following obstructive injury.Figure 5. Effect of IL-6 deficiency on renal fibrosis and extracellular matrix deposition in the kidney. A. Representative photomicrographs show kidney sections stained with picrosirius red for assessment of total collagen deposition. B. Bar graph shows quantitative analysis of renal interstitial collagen in different groups as indicated. ** P,0.01 vs WT control, P.0.05 vs WT UUO, and ++ P,0.01 vs KO UUO. n = 5 per group. doi:10.1371/journal.pone.0052415.gDiscussionIn this study, we demonstrate that (1) IL-6 is induced in the kidney in response to obstructive injury; (2) WT and IL-6 KO mice accumulate similar number of bone marrow-derived fibroblast precursors in the kidney following obstructive injury; (3) Targeted disruption of IL-6 has no significant effect on myofibroblast formation and a-SMA expression; (4) Targeted disruption of IL-6 does not influence gene expression of profibrotic chemokines and cytokines; (5) Targeted disruption of IL-6 does not alter the severity of renal fibrosis and the expression of ECM proteins. These results indicate that IL-6 does not play an important role in the recruitment of bone marrow-derived fibroblasts and the development of renal fibrosis induced by obstructive i.A role in the accumulation of bone marrow-derived fibroblasts in the kidneys, WT and IL-6 KO mice were subjected to obstructive injury for 5 or 7 days. Kidney sections were stained for CD11b and procollagen I and examined with a fluorescent microscope. Our results showed that both WT and IL-6 KO mice 1326631 exhibited a marked increase in the number ofThe Role of IL-6 in Renal FibrosisTGF-b1 mRNA in the kidney following obstructive injury (Figure 4B). These data suggest that IL-6 signaling does not play a major role in the regulation of TGF-b1 gene expression in the kidney in response to obstructive injury. Since Th2 cytokines ?IL-4 and IL-13 have been shown to promote monocyte-to-fibroblast transition [14,19], we then examined if IL-6 deficiency affects these cytokine expression in the kidney. The results of real time RT-PCR showed that IL-6 deficiency did not influence mRNA levels of IL-4 and IL-13 in the kidney following obstructive injury (Figure 4 C ). These data indicate that IL-6 signaling does not modulate the gene expression of IL-4 and IL-13 in the kidney in response to obstructive injury.IL-6 Deficiency Has no Effect on Renal FibrosisSince IL-6 does not regulate the accumulation and activation of bone marrow-derived fibroblasts in the kidney in response to obstructive injury, we then examined if IL-6 deficiency has an effect on the development of renal fibrosis. WT and IL-6 KO mice were subjected to UUO for 14 days. Both WT and IL-6 KO mice developed similar degree of collagen deposition in obstructed kidneys as demonstrated by picrosirius red staining (Figure 5). These data indicate that IL-6 does not play a role in the pathogenesis of renal fibrosis.IL-6 Deficiency Does not Affect ECM Protein ExpressionWe next investigated the effect of targeted disruption of IL-6 on the expression and accumulation of collagen I and fibronectin, two major components of ECM. Both WT and IL-6 KO mice displayed a marked increase in the protein expression levels of collagen I and fibronectin in the kidneys following obstructive injury, which was not statistically different between these two groups (Figure 6 and 7). These data indicate that IL-6 does not regulate the production and deposition of ECM proteins in the kidney following obstructive injury.Figure 5. Effect of IL-6 deficiency on renal fibrosis and extracellular matrix deposition in the kidney. A. Representative photomicrographs show kidney sections stained with picrosirius red for assessment of total collagen deposition. B. Bar graph shows quantitative analysis of renal interstitial collagen in different groups as indicated. ** P,0.01 vs WT control, P.0.05 vs WT UUO, and ++ P,0.01 vs KO UUO. n = 5 per group. doi:10.1371/journal.pone.0052415.gDiscussionIn this study, we demonstrate that (1) IL-6 is induced in the kidney in response to obstructive injury; (2) WT and IL-6 KO mice accumulate similar number of bone marrow-derived fibroblast precursors in the kidney following obstructive injury; (3) Targeted disruption of IL-6 has no significant effect on myofibroblast formation and a-SMA expression; (4) Targeted disruption of IL-6 does not influence gene expression of profibrotic chemokines and cytokines; (5) Targeted disruption of IL-6 does not alter the severity of renal fibrosis and the expression of ECM proteins. These results indicate that IL-6 does not play an important role in the recruitment of bone marrow-derived fibroblasts and the development of renal fibrosis induced by obstructive i.
