Activation of the ERK pathway precedes tubular proliferation in the obstructed rat kidney.

BACKGROUND: In vitro studies suggest that activation of the extracellular signal-regulated kinase (ERK) pathway plays a critical role in the proliferation of tubular epithelial and myofibroblast-like cells. However, little is known of ERK activation in individual cell types in normal or diseased kidney. The aims of this study were to (1) localize ERK activation within the kidney, and (2) examine the relationship between ERK activation and cell proliferation in the injured kidney. METHODS: Unilateral ureteric obstruction (UUO) was induced in groups of six Wistar rats, which were killed at 30 minutes, 6 hours, and 1, 4, or 7 days after obstruction. Activation of ERK was identified using antibodies specific for the phosphorylated form of ERK (pERK) in Western blots and immunostaining. Proliferating cells were detected using bromodeoxyuridine (BrdU). RESULTS: Western blotting showed abundant expression of the two ERK isoforms, ERK-1 and ERK-2, in normal rat kidney. Low levels of activated ERK (pERK-2> pERK-1) were detected in normal rat kidney by Western blotting. Immunostaining showed that ERK activation in normal kidney was largely restricted to collecting ducts in the outer medulla. Within 30 minutes of ureter obstruction, Western blotting showed a sixfold increase in ERK activation followed by a second peak (14-fold increase) on days 4 and 7. The initial peak of ERK activation was localized to medullary collecting ducts and the thick ascending limb of Henle (TALH), whereas the second peak corresponded to a progressive increase in ERK activation in dilated collecting ducts and in interstitial cells in the cortex. Proliferation of tubular epithelial cells closely followed the pattern of ERK activation, being evident first in medullary collecting ducts and TALH on day 1, and then in cortical collecting ducts from day 4. CONCLUSION: This study has identified a discrete pattern of ERK activation in normal rat kidney and an increase in ERK activation following obstruction. The temporal and spatial relationship in which ERK activation preceded tubular cell proliferation suggest that ERK signaling plays a key role in tubular epithelial cell proliferation in the injured kidney.

Role of interleukin-10 in rat mesangioproliferative glomerulonephritis.

Interleukin-10 (IL-10) has been recognized as a growth factor for rat mesangial cells in vitro; however, its role in mesangioproliferative glomerulonephritis is unknown. We studied the expression of IL-10 mRNA in the rat anti-Thy-1 model of mesangioproliferative glomerulonephritis (experiment 1) and, subsequently, the effects of blocking IL-10 during anti-Thy-1 nephritis using the IL-10 inhibitor, AS101 (experiment 2). In experiment 1, PCR analysis failed to detect IL-10 mRNA in normal rat kidney, however, a clear signal for IL-10 mRNA was evident on day 6 of anti-Thy-1 nephritis. In situ hybridization showed IL-10 mRNA expression in focal glomerular areas in anti-Thy-1 nephritis. Combined in situ hybridization and immunohistochemistry showed that glomerular IL-10 mRNA was expressed by both macrophages and mesangial cells. In experiment 2, treatment with AS101 significantly downregulated renal IL-10 gene expression, as demonstrated by semiquantitative PCR. However, the induction of glomerular hypercellularity, mesangial proliferation (PCNA+ cells), mesangial cell activation (alpha-SMA expression) and macrophage accumulation (ED1+ cells) seen in saline-treated anti-Thy-1 nephritis was unaffected by AS101 treatment. In conclusion, renal IL-10 gene expression is upregulated during pathological mesangial cell proliferation in rats with anti-Thy-1 nephritis. However, the inability of IL-10 suppression with AS101 to prevent anti-Thy-1 disease suggests that IL-10 is not essential for pathological mesangial cell proliferation.

Macrophage accumulation at a site of renal inflammation is dependent on the M-CSF/c-fms pathway.

Production of macrophage-colony stimulating factor (M-CSF), the major macrophage growth factor, is increased in tissues during inflammation. Therefore, we determined whether M-CSF, acting through its receptor c-fms, contributes to macrophage accumulation at a site of tissue injury. Daily treatment with anti-c-fms or control antibody was given to mice with renal inflammation resulting from unilateral ureteric obstruction (UUO). Following UUO, kidney M-CSF mRNA increased in association with macrophage accumulation (days 1, 5, and 10) and local macrophage proliferation (days 5 and 10). Anti-c-fms treatment caused a minor inhibition of monocyte recruitment at day 1, reduced macrophage accumulation by 75% at day 10, but did not affect blood monocyte counts or the CD4 and CD8 lymphocytic infiltrate. Prevention of macrophage accumulation by anti-c-fms treatment was associated with a 90% reduction in local macrophage proliferation at days 5 and 10 without evidence of increased macrophage apoptosis. Therefore, M-CSF/c-fms signaling plays a key role in macrophage accumulation during tissue injury.