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 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

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 influx of granulocytes in wild type and TLR4−/− mice.

<p>Influx of granulocytes in kidneys from wild type (white bars) and TLR4−/− (black bars) kidneys 1, 5 and 10 days after renal I/R injury or sham operation. One and ten days after I/R injury the number of granulocytes was significantly lower in kidneys of TLR4−/− mice than in kidneys of wild type mice as counted in 10 randomly selected high-power fields (HPFs) on outer medulla (magnification ×400). The amount of granulocytes from 8 mice per group were counted on renal tissue sections stained for Ly-6G and presented as mean±SEM. * p<0.05.</p

Renal function, injury and inflammatory influx in wild type, MyD88−/− and TRIF-mutant mice.

<p>Renal function of MyD88−/− and TRIF-mutant mice (black bars) did not differ compared with that of their wild type mice (white bars) one day after renal I/R as reflected by serum urea (A) and creatinine (B) levels and tubular injury (C). In addition, no differences were observed in the number of infiltrating granulocytes in kidneys of MyD88−/− and TRIF-mutant mice compared with their wild type mice (D). Data are mean±SEM of 6 (TRIF) or 8 (MyD88) mice per group (sham-operated animals: n = 2/group (TRIF) or n = 3/group (MyD88)). * p<0.05.</p

Scoring renal tubular damage of wild type and TLR4−/− mice.

<p>Score for histopathology after renal I/R injury (A) using PAS-D-stained renal tissue sections (B; representative for t = 1). Tubular damage was significantly lower in the outer medulla of kidneys of TLR4−/− mice (black bars) than in kidneys of wild type mice (white bars) one day after I/R injury. Data are mean±SEM of 8 mice per group (sham-operated animals: n = 3/group). * p<0.05.</p

Renal function parameters of wild type and TLR4−/− mice.

<p>Renal function of TLR4−/− mice (black bars) was improved compared with that of the wild type mice (white bars) one day after renal I/R as reflected by lower serum urea (A) and creatinine (B) levels. Data are mean±SEM of 8 mice per group (sham-operated animals: n = 3/group). * p<0.05.</p

Levels of proinflammatory cytokines and chemokines in wild type and TLR4−/− mice.

<p>Expression of proinflammatory chemokines KC and MCP-1 in kidney homogenates of wild type (white bars) and TLR4−/− (black bars) mice one day after I/R injury or sham-operation. KC levels were significantly lower in homogenate of TLR4−/− kidneys compared with wild type kidneys, whereas there were no differences observed in MCP-1 levels. Data are mean±SEM of 8 mice per group measured in duplicate (sham-operated animals: n = 3/group). * p<0.05.</p