Deficiency for the Chemokine Monocyte Chemoattractant Protein-1 Aggravates Tubular Damage after Renal Ischemia/Reperfusion Injury

<div><p>Temporal expression of chemokines is a crucial factor in the regulation of renal ischemia/reperfusion (I/R) injury and repair. Beside their role in the migration and activation of inflammatory cells to sites of injury, chemokines are also involved in other processes such as angiogenesis, development and migration of stem cells. In the present study we investigated the role of the chemokine MCP-1 (monocyte chemoattractant protein-1 or CCL2), the main chemoattractant for monocytes, during renal I/R injury. MCP-1 expression peaks several days after inducing renal I/R injury coinciding with macrophage accumulation. However, MCP-1 deficient mice had a significant decreased survival and increased renal damage within the first two days, i.e. the acute inflammatory response, after renal I/R injury with no evidence of altered macrophage accumulation. Kidneys and primary tubular epithelial cells from MCP-1 deficient mice showed increased apoptosis after ischemia. Taken together, MCP-1 protects the kidney during the acute inflammatory response following renal I/R injury.</p></div

Leukocyte influx in kidneys from MCP-1^+/+ (white bars) and MCP-1^-/- (black bars) mice 1 day after renal I/R or sham surgery.

<p>(a) The number of macrophages was determined in F4/80 stained paraffin kidney sections. Pictures (magnificantion 400x) of F4/80 stained kidney sections from MCP-1^+/+ (b) and MCP-1^-/- (c) after I/R injury. (d) Negative control of F4/80 staining. Renal mRNA expression of M1 macrophage markers iNOS (e) and CCR7 (f), and the M2 macrophage markers ARG1 (g) and YM1 (h) were determined in sham and I/R MCP-1^+/+ and MCP-1^-/- mice. (i) Renal MPO was significant increased in MCP-1^-/- compared with MCP-1^+/+ mice after I/R injury. However, Ly6-G stained kidneys sections (magnification 100x) revealed no difference between the total number of neutrophils in MCP-1^+/+ (j) and MCP-1^-/- (k) mice after I/R. (l) Negative control of Ly6-G staining. Data are presented as mean ± SEM, n = 4–5 (sham) and n = 9 (I/R). *<i>P</i><0.05 (Mann-Whitney U test).</p

Renal TGFβ1 and apoptosis and proliferation of TEC after simulated I/R in vitro.

<p>Total (a) and active (b) TGFβ1 was determined in kidneys of sham and ischemic operated MCP-1^+/+ (white bars) and MCP-1^-/- (black bars). (c-e)TEC were isolated from MCP-1^+/+ and MCP-1^-/- kidneys and grown to confluence. Subsequently TEC were subjected to simulated ischemia/reperfusion. Flowcytometry histograms of MCP-1^+/+ (c) and MCP-1^-/- (d) TEC; isolated nuclei were analyzed for propidium iodide staining of DNA. The percentage of apoptotic nuclei (broad hypodiploid peak) is given as a percentage of total TEC. The narrow peak (diploid) represents viable cells. (e) Graphic representation of the percentage of apoptotic and proliferating TEC of MCP-1^+/+ (white bars) and MCP-1^-/- (black bars) mice. Data are presented as mean ± SEM, n = 4–5 (sham) and n = 9 (I/R). or pooled from two independent experiments (in vitro) (n = 5–6). *<i>P</i><0.05 (Mann-Whitney U test (a-b) or unpaired Student’s <i>t</i> test).</p

Apoptotic and proliferating TEC in kidneys from MCP-1^+/+ (white bars) and MCP-1^-/- (black bars) mice 1 day after renal I/R injury or sham surgery.

<p>(a) The number of apoptotic TEC was quantified in caspase-3 stained paraffin kidney sections. Pictures (magnification 400x) of caspase-3 stained kidney sections from MCP-1^+/+ (b) and MCP-1^-/- (c) mice after renal I/R injury. (d) Negative control of caspase-3 staining. (e) The number of proliferating TEC was determined in Ki67 stained paraffin kidney sections. Pictures (magnification 400x) of Ki67 stained kidney sections from MCP-1^+/+ (f) and MCP-1^-/- (g) mice after renal I/R injury. (h) Negative control of Ki67 staining. Data are presented as mean ± SEM, n = 4–5 (sham) and n = 9 (I/R). *<i>P</i><0.05 (Mann-Whitney U test).</p

Renal MCP-1 expression 1, 7 and 14 days following I/R injury in MCP-1^+/+ mice (white bars) on mRNA (a) and protein (b) level.

<p>(c) Kaplan-Meier survival curve revealed significant (<i>P</i> = 0.002) decreased survival in MCP-1^-/- compared with MCP-1^+/+ mice following renal I/R injury. One day after renal ischemia/reperfusion injury (I/R) plasma ureum (d), and creatinine (e) were determined in MCP-1^+/+ (white bars) and MCP-1^-/- (black bars). (f) Tubular damage was assessed in the outer cortex of MCP-1^-/- (white bars) and MCP-1^+/+ (black bars) following renal I/R injury. Representative pictures of MCP-1^+/+ and MCP-1^-/- PasD stained ischemic kidneys (10x magnification) are shown. Plasma levels of (g) LDH, (h) ASAT, and (i) ALAT were determined in sham and ischemic operated MCP-1^+/+ (white bars) and MCP-1^-/- (black bars) mice. Renal expression of tubular injury markers kidney injury molecule-1 (KIM-1, j) and neutrophil gelatinase-associated lipocalin (NGAL, k) one day following ischemic injury (I/R) in MCP-1^+/+ (white bars) and MCP-1^-/- (black bars) mice. Data are presented as mean ± SEM, n = 4–5 (sham), n = 9 (I/R), n = 22 (survival). *<i>P</i><0.05 (a,b,d-k: Mann-Whitney U test; c: Kaplan Meier) compared with sham (a,b) or MCP-1^+/+ (c-h).</p

SCF reduces hypoxia induced IM-PTEC apoptosis and induces phosphorylation of Bad.

<p>(A) SCF reduces caspase 3 activity following hypoxia. IM-PTEC cells were subjected to in vitro hypoxia and cultured with 0 ng or 100 ng SCF/ml medium for 24 hours. Cell lysates were collected and assayed for caspase 3 activity. To correct for equal input, values are expressed as activity per µg protein. SCF reduces caspase 3 activity in IM-PTEC cells following in vitro hypoxia (*<i>P</i> = 0.026). Data are expressed as mean±SEM. Results are obtained from 2 separate experiments with 6 measurements each. (B) IM-PTEC cells were serum starved and exposed to 0, 10 or 100 ng SCF/ml for 5, 10 and 15 minutes. Protein samples were analyzed using Western blot. SCF induced phosphorylation of Tyr719 of c-KIT; β actin was used as loading control. (C) SCF results in phosphorylation of Ser473 of Akt; total Akt expression was used as loading control (upper panel); SCF induces phosphorylation of Ser136 of Bad; β actin was used as loading control (lower panel). Normalized densitometric analyses of the immunoblots are presented as the ratio phosphorylated protein/loading control. Data shown here are representative results obtained from 3 separate experiments. (D) Renal tissue samples obtained from sham and animals subjected to ischemia were analyzed for phosphorylation of Akt (p-Ser473 Akt) and c-KIT (p-Tyr719 c-KIT). Expression of total Akt and c-KIT were used as loading control.</p

Effect of SCF ASON treatment on inflammation and TEC apoptosis.

<p>(A and B) The concentration of renal (A) KC and (B) IL-1β was measured by ELISA in whole kidney homogenates. An increase in the concentration KC was detected in samples obtained from ASON treated animals (▪) subjected to ischemia compared to controls (□) (*<i>P</i> = 0.03). No significant difference was detected in the concentration of IL-1β between NSON (□) and ASON (▪) treated animals. Data are expressed as mean±SEM. (C) Gr-1 positive cells were counted per hpf. Few Gr-1 positive passenger leukocytes were detected in sham operated animals. In contrast, the number of Gr-1 positive cells increased at day 1 after ischemia. No significant statistical difference was detected between the number of positive cells per hpf in NSON (□) or ASON (▪) treated animals. Data are expressed as mean±SEM. (D) Apoptosis of TEC was determined by immunostaining of tissue sections using an antibody to the active form of caspase 3. Positive TEC were counted per hpf, significantly more apoptotic TEC were detected in ASON treated animals (▪) compared to controls (□) at day 1 after ischemia (*<i>P</i> = 0.0005). Data are expressed as mean±SEM. (E) Proliferation of TEC was determined using an antibody to Ki.67 (right). Kidneys from ASON treated animals (▪) contained less proliferating TEC compared to NSON controls (□) at day 1 after ischemia (*<i>P</i> = 0.0009). Data are expressed as mean±SEM.</p

Tissue distribution of ODN.

<p>Distribution of FITC-labeled ODN was examined after two intraperitoneal administrations given with a 24 hour interval. Tissue was collected at 5 hours after the last administration. FITC was stained (brown) using a specific antibody showing (A) extensive uptake of FITC-labeled ODN by most tubule segments in the corticomedullary area and uptake by the parietal epithelium of the glomerulus (marked Gl) but (D) no significant uptake of ODN by the tubules located in the renal papilla. (C) CD10 expression (blue) by tubular epithelium co-stained for FITC (brown) demonstrates uptake of ODN by proximal tubules. (F) and (H) uptake of ODN by cells in liver and spleen and (G) no uptake of FITC-labeled ODN by lung tissue. Sections of (B) renal corticomedullary area, (E) renal papilla, (I) liver, (J) lung and (K) spleen labeled with secondary antibody only. Nuclei were counterstained with methyl green. Original magnification of all images: 40×.</p

Hypoxia of IM-PTEC results in SCF secretion.

<p>(A) To validate the effect of paraffin oil immersion on IM-PTEC, KC and MIP-2 levels were measured in culture medium 24 hrs after hypoxia. In accordance with previous reports, our <i>in vitro</i> hypoxia model led to increased KC and MIP-2 secretion by IM-PTEC. Data are expressed as mean±SEM, results are combined of data from two independent experiments (*<i>P</i> = 0.03). (B) Normoxic cells (left) displayed normal epithelial morphology with the appearance of cell-cell contacts. Cell subjected to 60 minutes of hypoxia (right) displayed cellular retraction and loss of cell-cell contacts, indicative of damage by hypoxic stress. Original magnification: 20×. (C) Transcription of c-KIT and SCF was determined in normoxic control cells (nO₂) and following hypoxia (hyp) and SCF stimulation following hypoxia (100 ng/ml, hyp SCF). Samples were obtained at 24 hours following hypoxia. Dual bands for SCF show the two splice variants designated as full-length Kit Ligand (KL) -1 and KL-2, which lacks exon 6. TATA box binding protein (TBP) was used as a loading control. Data shown here are representative results obtained from 3 separate experiments. (D) Levels of SCF were measured in medium samples from cells subjected to <i>in vitro</i> hypoxia using a murine SCF specific ELISA. The concentration SCF obtained from control samples was low and values were just above the detection limit of the ELISA. Hypoxia induced a significant increase in medium SCF. (E) Western blot analysis of conditioned culture medium samples of control cells (nO2), hypoxic cells (hyp), hypoxic cells cultured with 10 ng SCF/ml medium (hyp + SCF 10 ng/ml) or hypoxic cells cultured with 100 ng SCF/ml medium (hyp + SCF 100 ng/ml). Upper panel: 45 kD bands representing full length membrane SCF (KL-1) appear after short exposure. Lower panel: longer exposure reveals a smaller SCF variant approximately 31–32 kD in size and recombinant SCF (approx. 18 kD). Hypoxia leads to increased levels of KL-1 in medium samples whereas the addition of SCF induces increased levels of the smaller SCF forms in the medium. All samples were collected 24 hours following hypoxia. Data shown here are representative results obtained from 3 separate experiments (*<i>P</i> = 0.04). (F) Western blot analysis of cell lysates obtained from cells 24 hours after being subjected to hypoxia. Phosphorylation of tyrosine 719 of c-KIT (pTyr719) was present in samples from normoxic control cells. Hypoxia increases the relative rate of c-KIT phosphorylation. Upper panel: phospho Tyr719 c-KIT. Lower panel: total c-KIT. Densitometric analysis of the relative increase of Tyr719 c-KIT phosphorylation versus total c-KIT following hypoxia. Phosphorylation of Tyr719 phosphorylation in control cells was set as 1. Western blot data shown here are representative results obtained from 3 separate experiments; data from all experiments were used for the densitometric analysis as shown here (*<i>P</i> = 0.05).</p

SCF ASON treatment increases renal injury.

<p>(A) Whole kidney sample analysis of SCF expression by ELISA did not show any significant difference in SCF levels between NSON (□) and ASON (▪) treated animals (<i>P</i> = 0.09, non significant). Data are expressed as mean±SEM. (B) Digital image analysis of SCF immunostainings per hpf of the corticomedullary region demonstrated significantly less staining on tissue sections from ASON treated animals (▪) compared to controls (□) at day 1 after ischemia (*<i>P</i> = 0.003). Data are expressed as mean±SEM. (C) and (D) Representative SCF immunostainings of ischemic kidney sections used for image analysis of NSON (c) and ASON (d) treated animals; original magnification: 40×. (E) Tubular damage was scored in a semi-quantitative fashion. The degree of injury one day after ischemia was significantly increased in ASON treated animals (▪) compared to NSON controls (□) (*<i>P</i> = 0.005). Data are expressed as mean±SEM. (F) and (G) Renal function was determined by measurement of (f) plasma urea and (g) creatinine values. ASON treatment (▪) resulted in significantly elevated plasma urea (*<i>P</i> = 0.0047) and creatinine (*<i>P</i> = 0.001) compared to NSON controls (□) at day one after ischemia. Data are expressed as mean±SEM. (H) Representative PAS-D stainings illustrating tubular injury as scored in (E). Kidneys from sham operated animals after NSON and ASON treatment show no histologic signs of tubular injury. In contrast, at day 1 after ischemia most tubules in the corticomedullary area appear dilated with loss of the brush border or display denudation of the tubular basement membrane. In NSON-treated animals, more undamaged tubules were present compared to ASON-treated animals.</p