We subsequent assessed the expression of CD44 by Q-PCR and immunostaining in WT and CD44 KO kidneys. 1252003-15-8Q-PCR assessment showed an improve at 2, four and 24 hours in CD44 transcripts in WT kidneys (Determine 2A). Immunohistochemistry staining verified an increase in CD44-constructive cells in WT kidneys right after LPS administration (Figure 2B). CD44 expressing cells ended up found generally in the interstitium and some in the glomeruli. In distinction to what is observed in other renal pathological ailments [9,24,25], CD44 was absent on tubular epithelial cells (TEC) at all time-factors. Hyaluronan (HA) is a significant part of the extracellular matrix (ECM) and a major ligand of CD44. Fragments of HA are produced in the course of irritation or harm, and they can provide as endogenous triggers of TLR signaling and market cytokine generation [26]. LPS induced an accumulation of renal interstitial HA, which picked at 24 hrs in WT kidneys and at four hours in CD44 KO kidneys (Determine 2C). Collectively with CD44 and HA, we calculated the renal expression of the LPS/HA receptor TLR-4 [two,26], which confirmed no discrepancies amongst the strains in manage circumstance and was improved as early as 2 hrs article-injection in WT kidneys and later, at 4 several hours, in CD44 KO kidneys (data not shown).Cells had been incubated at 4 for 30 minutes in RIPA buffer containing 20mM Tris-HCl pH7.five, 150mM NaCl, 5mM EDTA, 1% NaDOC, one% NP-forty, 200 Na3VO4, 50mM NaF, 1% protease inhibitor cocktail (P8340, Sigma), .one% SDS, 10% glycerol. Following centrifuging at 14000 g, the supernatants ended up gathered and nuclei had been incubated with RIPA buffer that contains two% SDS and twenty% glycerol and afterwards sonicated for twenty seconds. Lysates were being subjected to western-blot (WB) examination working with anti-phospho-IB, anti-NF-B p65, anti-MD-2 (Enzo Lifetime Sciences), anti-MyD88 (Cell Signaling), anti-Trif (Abcam), anti-TRAF6 (Santa Cruz Biotechnology). HRPconjugated secondary antibodies (DAKO) ended up utilized and HRP action was visualized with ECL-reagent (GE Healthcare). actin (Abcam) and lamin A/C (Mobile Signaling) had been utilised as loading controls for cytoplasmic and nuclear fractions, respectively. Densitometric quantification examination was done on imagines of scanned movies employing the ImageJ software package.Subsequent erythrocytes disruption with lysis buffer (160mM NH4Cl, 10mM KHCO3 and .1mM EDTA, pH seven.four), blood cells ended up stained with monoclonal anti-Ly6-FITC-conjugated (BD Pharmingen), anti-CD11b-APC-conjugated (BD Pharmingen) or anti-F4/eighty-FITC-conjugated (Serotec). Prior to evaluation, cells renal functionality was assessed two, 4 and 24 hrs immediately after LPS administration by measuring plasma urea levels (Determine 3A). Blood urea amounts picked at 24 hrs in the two mice strains, but WT mice showed a compromised renal operate as early as 2 hrs right after LPS injection, whilst CD44 KO mice taken care of systemic irritation. Focus (pg/ml) of (A) MCP-1, (B) IL-one, (C) TNF-, (D) IL-6 in plasma of WT (white bars) and CD44 KO (black bars) mice assessed by ELISA. Facts expressed as imply + SEM, n=eight, =p<0.05 renal function at 2 and 4 hours as compared to saline-treated animals. LPS caused relatively mild morphological changes at 24 hours in renal tissues, including loss of brush border, tubular vacuolization and dilatation, but no necrosis or cast formation [6] (Figure 3B). These morphological alteration were similar in both mice groups, and so was the gene expression of the early markers of tubular injury KIM-1 and NGAL [27], which gradually increased in time after LPS administration in a similar manner in WT and CD44 KO kidneys. (Figure S1A,B). Upon LPS challenge, the apoptosis rate of tubular cells was low and comparable between the mice strains (Figure S1C) after LPS injection in both strains and were significantly higher in CD44 KO kidneys at 24 hours in respect to WT kidneys (Figure 4E).Considering the differences in cytokine expression between WT and CD44 KO mice, we wondered whether the recruitment of inflammatory cells would be affected in absence of CD44. The presence of granulocytes, macrophages and T lymphocytes in the kidneys was detected by immunohistochemistry (Figure 5A-C). The CD44 KO kidneys showed less granulocytes at 24 hours, less macrophages at 4 and 24 hours, and less T lymphocytes at 2 hours as compared to WT kidneys.Next, we assessed the inflammatory state in the kidneys by measuring local cytokine levels. LPS administration led to a rapid increase (2 hours) in renal production of pro-inflammatory cytokines (MCP-1, IL-1, TNF-, IL-6), which were generally higher at 2 and 4 hours in WT kidneys as compared to CD44 KO kidneys (Figure 4A-D). As a mechanism to counteract excessive inflammation, anti-inflammatory cytokines are produced after endotoxin stimulation [21]. Indeed, the renal levels of the anti-inflammatory IL-10 greatly increased at 4h the dissimilarities in renal influx of inflammatory cells could be attributed to the disparity in cytokine levels, but also to a differential endothelial inflammatory/activation state. Endotoxic shock is known to enhance the levels of VCAM-1 and ICAM-1, which indicate endothelial activation, and to induce NO synthesis, which decreases the systemic vascular resistance [6]. In WT mice, LPS administration rapidly (at 2 hours) renal CD44 and HA expression. (A) Expression of CD44 in kidneys in saline- and LPS-treated WT mice assessed by Q-PCR. Data normalized for TBP transcript expression. Mean + SEM, n=8, =p<0.05 versus (vs) sham. (B) Representative micrographs (x10 magnification upper panel, x40 lower panel) of CD44 immunostaining in renal sections of WT sham and 24 hours LPS-treated mice. (C) Quantification of HABP immunostaining in kidneys of WT (white bars) and CD44 KO (black bars) mice. Data expressed as percent positive area of the total area analyzed. Mean + SEM, n=8, =p<0.05.Renal injury. (A) Renal function assessed by measurement of urea levels (mmol/L) in plasma of WT (white bars) and CD44 KO (black bars) mice. Mean + SEM, n=8, =p<0.05, WT vs CD44 KO =p<0.05, =P<0.01, vs sham. (B) Representative micrographs (x20) of Periodic acid-Schiff Diastase (PAS-D) stained renal sections of saline- and 24 hours LPS-treated WT and CD44 KO mice.Cytokines in the kidneys. Expression (pg/mg proteins) of (A) MCP-1, (B) IL-1, (C) TNF-, (D) IL-6 and (E) IL-10 in kidneys of WT (white bars) and CD44 KO (black bars) mice measured by ELISA. Data shown as mean + SEM, n=8, =p<0.05 augmented the renal levels of iNOS protein (Figure 6A), VCAM-1 (Figure 6B) and ICAM-1 mRNAs (Figure S1D). In CD44 KO kidneys, expression of iNOS was significantly lower at 2 and 4 hours in respect to WT kidneys (Figure 6A). Furthermore, at 2 hours CD44 KO kidneys revealed less VCAM-1 transcript expression (Figure 6B) and a trend (p=0.053) toward less ICAM-1 transcript expression (Figure S1D). Immunohistochemistry confirmed lower levels of VCAM-1 protein in CD44 KO kidneys at 2 hours (Figure 6C) instead, no significant differences were found in ICAM-1 protein levels 2 hours after LPS injection (Figure S1E).To obtain better insight into the role of CD44 in the primary cell response to LPS, we stimulated bone marrow-derived macrophages from WT and CD44 KO mice with 100ng/ml LPS for 4 and 24 hours. In line with the in vivo results, CD44 KO macrophages secreted significantly less TNF- and IL-6 after leukocyte influx in the kidneys. Quantifications of immunostainings for detection of (A) granulocytes, (B) macrophages, (C) T lymphocytes in paraffin renal sections of WT (white bars) and CD44 KO (black bars) mice. (A) Number of Ly6G-positive cells per HPF (x20). (B,C) Data expressed as percent positive area of the total areal analyzed. Mean + SEM, n=8, =p<0.05, WT vs CD44 KO =p<0.05, vs sham in (A) all groups vs sham.Renal endothelial activation. (A) iNOS expression detected by immunostaining in paraffin renal sections of WT (white bars) and CD44 KO (black bars) quantification shown as positive area percentage of total area. (B) Q-PCR analysis for renal expression of VCAM-1 data normalized for TBP expression levels. (C) Digital analysis of VCAM-1 protein expression 2 hours after LPS injection, assessed by immunohistochemistry on frozen renal sections. Results expressed as positive area percentage of total area. Mean + SEM, n=8, =p<0.05 =p<0.01, WT vs CD44 KO =p<0.05, =P<0.01, vs sham.Macrophage response to LPS in presence/absence of CD44. In vitro LPS (100ng/ml) stimulation of WT (white bars) and CD44 KO (black bars) bone marrow-derived macrophages (BMM) for 4 and 24 hours. Secretion levels (pg/ml) of (A) TNF- and (B) IL-6 detected by ELISA. (C) Gene expression of IL-1 and (D) IFN normalized for TBP transcript levels, assessed by Q-PCR. (E) ELISA measurement of IL-10 supernatant concentration (pg/ml). Mean + SEM, n=10, =p<0.05, =p<0.001. (F) WB assay of cell lysates from WT and CD44 KO BMM for detection of cytoplasmatic phosphorylated (p)-IB and nuclear p65. -actin or LaminA/C used as loading controls. Fold-increase in protein expression in respect to control is indicated hours stimulation, while after 24 hours the pro-inflammatory cytokine production was equal between the two groups (Figure 7A,B). In addition, the gene expression of IL-1 (p=0.014) and IFN (p=0.056) was lower in CD44 KO macrophages as compared to WT macrophages after 4 hours of stimulation (Figure 7C,D). On the contrary, the amount of secreted IL-10 was higher in the supernatant of CD44 KO cells at both 4 and 24 hours of stimulation (Figure 7E). TLR-4 activates the MyD88-dependent and Trif-dependent pathways. The first one leads to activation of NF-B and AP-1 inducing several proinflammatory cytokines, while the second pathway leads to IRF3 activation and hence induction of type I IFN, particularly IFN [28]. The lower expression of pro-inflammatory cytokines, including IFN, by CD44 KO macrophages suggests that both MyD88- and Trif-dependent pathways are less activated in these cells. To determine if this phenotype was due to lower expression levels of the molecules involved in sensing LPS and in mediating the intracellular pathways, we assessed in WT and CD44 KO macrophages mRNA levels of the LPS sensors TLR-4 and CD11b, and protein levels of MD-2, MyD88, Trif and TRAF6, another adaptor protein of the TLR-pathway [28]. None of these molecules showed differential expression in WT and CD44 KO cells (data not shown). To prove the activation of NFB, the phosphorylation of the inhibitor of NF-B (IB) and the nuclear translocation of the NF-B p65-subunit were evaluated (Figure 7F). Upon LPS-stimulation both phosphorylated IB and p65 were increased, respectively, in the cytoplasmatic and nuclear fractions. Relatively to control, the fold-increase, and 24 hours, in phosphorylated IB was equal to 2.8 and 4.2 in WT BMM, and to 1.6 and 3.2 in CD44 KO cells. WT macrophages displayed a 3.4 (4 hours) and a 1.4 (24 hours) fold-increase in nuclear p65, whereas in CD44 KO BMM p65 was augmented only at 4 hours (3.1 fold-increase). We next wondered whether CD44-activation by ligation would affect the cell response to LPS in terms of cytokine production. In WT macrophages CD44 ligation induced a further increase in TNF- secretion in response to LPS (Figure 8A) this effect was absent in CD44 KO cells. In WT BMM ligation of CD44 on itself did not increase cytokine secretion (data not shown). CD44 mediates activation of several pathways, including mitogen-activated protein kinases (MAPK) and phosphoinositide 3-OH kinase (PI3K)/Akt pathways [29], which have been shown to mediate LPS-elicited NF-B activation [28,30-33]. Blocking p38 MAPK with SB203580 and PI3K with 3-MA, prior to LPS-exposure, significantly diminished TNF- secretion in WT macrophages and not in CD44 KO BMM (Figure 8B). After treatment with the inhibitors, WT BMM cytokine-secretion was reduced to the levels of CD44 KO BMM stimulated with LPS alone. Inhibition of TLR4 with Cli095, instead, blocked completely cytokine-secretion in both cell groups. 19276360These findings suggest that CD44 is not completely necessary for TLR-4-activation, whereas is required for the LPS-induced p38 MAPK and PI3K signaling. Finally, we evaluated renal expression of phosphorylated Akt and p38 MAPK by immunohistochemistry (Figure S1F,G). No statistically significant differences were found, presumably LPS-challenged macrophages after CD44-ligation and inhibitors pre-treatment. In vitro LPS (100ng/ml) stimulation of WT (white bars) and CD44 KO (black bars) BMM for 4 hours after CD44-ligation. (A) ELISA for detection of TNF- in supernatants data shown as fold-increase compared to LPS-stimulated BMM. (B) TNF- levels (pg/ml) in presence or not of 10 SB203580, 10mM 3-MA, or 0.5ug/ml Cli095 (ELISA). Mean + SEM, n=6, =p<0.05, WT vs CD44 KO =p<0.05, =p<0.01, vs WT +LPS (no inhibitors) ^^=p<0.01, vs CD44 KO+LPS (no inhibitors)because of the high expression of both proteins in their phosphorylated state in tubular cells. Given that WT TEC lack CD44, they behave similarly to CD44 KO TEC, as previously shown by morphological observation, analysis of tubular injury markers and TEC apoptosis rate.Since less inflammatory cell influx was found in CD44 KO kidneys in vivo and CD44 is known to play a role in cell migration, the migratory ability of LPS-treated blood cells from WT and CD44 KO mice was investigated by Transwell migration assay. In absence of CD44, a lower number of CD11b- and Ly6G-positive cells migrated into the lower chamber containing medium supplemented with MIP-2 and MCP-1 after 24 hours exposure (Figure 9).Sepsis is a major public health problem it is the leading cause of death in non-coronary intensive care units and is the 11th cause of death in the United States. Therefore, better understanding of the pathophysiology of sepsis and identification of new targets is very needed to improve septic shock treatment [34]. Our study demonstrates that upon LPS exposure, lack of CD44 impairs the early pro-inflammatory cytokine response, inflammatory cell migration/chemotaxis, endothelial activation, and therefore delays the onset of endotoxin shock-induced renal dysfunction. Since sepsis is defined as a systemic inflammatory response syndrome, we evaluated the plasma pro-inflammatory cytokine levels, which are known to be upregulated in septic patients and correlate with mortality [2,35], and the cytokine expression in kidneys. In both compartments, and particularly in kidneys, the levels of pro-inflammatory cytokines were lower in CD44deficient mice. The fact that cytokine expression in kidneys presents greater differences between the mice strains compared to plasma cytokine levels can be attributed to the secondary amplification of inflammation. Through extrarenal TLR-4, LPS causes an early rise in cytokines, which in turn activate a second level of inflammatory cascade including further cytokine production, NO synthesis, upregulation of cell adhesion molecules that results in inflammatory cells migration into tissues, endothelial injury and organ damage [2]. Hence, a relative small difference in the cytokine plasma levels may produce a greater effect in organs. This could result from the CD44 interaction with molecules present in circulation or ECM, such as HA.
