Glutamine donor pretreatment in rat kidney transplants with severe preservation reperfusion injury.

BACKGROUND: Glutamine (GLN) has been shown to confer cytoprotection by enhancing endogenous heat shock protein (HSP) expression. We hypothesized that GLN donor pretreatment protects rat renal grafts against severe preservation reperfusion injury (PRI). MATERIALS AND METHODS: GLN (0.75 g/kg) or saline was administered i.p. to male donor rats 24 h and 6 h before donor nephrectomy. Kidneys (n = 6/group) were cold-stored in UW solution for 40 h and transplanted into bilaterally nephrectomized syngeneic recipients. Grafts were removed after 24 h. Renal HSP 70 expression was determined by Western blotting. Graft function was assessed by serum creatinine. Renal cross sections were microscopically examined for acute tubular necrosis, apoptosis, tubular proliferation, and macrophage infiltration. RESULTS: GLN donor pretreatment significantly increased intragraft HSP 70 expression. Serum creatinine was not different between groups: 2.6 +/- 0.2 mg/dL (saline) versus 2.7 +/- 0.5 mg/dL (GLN). Both treatment groups showed severe tubular damage with significantly less papillary necrosis in the GLN group (P < 0.05). GLN significantly reduced the number of apoptotic tubular cells in the cortex, medulla, and papilla (P < 0.001 versus saline). Postinjury tubular proliferation, measured by PCNA antigen expression, and intragraft macrophage infiltration was not influenced by GLN. CONCLUSIONS: In rat renal grafts suffering severe PRI pharmacological preconditioning with GLN attenuates early structural damage, especially tubular cell apoptosis. Stimulation of renal HSP 70 expression could be an important mechanism of GLN-induced cytoprotection. Our findings may have implications for the treatment of delayed graft function in recipients of marginal donor kidneys.

Chemokine receptor CCR1 but not CCR5 mediates leukocyte recruitment and subsequent renal fibrosis after unilateral ureteral obstruction.

As chemokine receptor CCR1 and CCR5 expression on circulating leukocytes is thought to contribute to leukocyte recruitment during renal fibrosis, the authors examined the effects of unilateral ureteral obstruction (UUO) in mice deficient for CCR1 or CCR5. Analysis of UUO kidneys from CCR1-deficient mice revealed a reduction of interstitial macrophages and lymphocytes (35% and 55%, respectively) compared with wild-type controls. CCR1-deficient mice had reduced CCR5 mRNA levels in UUO kidneys, which correlated with a reduction of CCR5+ T cell infiltrate as determined by flow cytometry. Interstitial fibroblasts, renal TGF-beta1 mRNA expression, interstitial volume, and collagen I deposits were all significantly reduced in CCR1-deficient mice. In contrast, renal leukocytes and fibrosis were unaffected in CCR5-deficient mice with UUO. However, if treated with the CCR1 antagonist BX471, CCR5-deficient mice showed a similar reduction of renal leukocytes and fibrosis as CCR1-deficient mice. To determine the underlying mechanism labeled macrophages and T cells isolated from either wild-type, CCR1-deficient, or CCR5-deficient mice were injected into wild-type mice with UUO. Three hours later, renal cell recruitment was reduced for CCR1-deficient cells or cells pretreated with BX471 compared with CCR5-deficient or wild-type cells. Thus, CCR1 but not CCR5 is required for leukocyte recruitment and fibrosis after UUO in mice. Therefore, CCR1 is a promising target for therapeutic intervention in leukocyte-mediated fibrotic tissue injury, e.g. progressive renal fibrosis.

Activation of toll-like receptor-9 induces progression of renal disease in MRL-Fas(lpr) mice.

How bacterial or viral infections trigger flares of autoimmunity is poorly understood. As toll-like receptor (TLR)-9 activation by exogenous or endogenous CpG-DNA may contribute to disease activity of systemic lupus erythematosus, we examined the effects of CpG-oligodeoxynucleotides (ODN) or DNA derived from Escherichia coli (E. coli) on the course of nephritis in MRL(lpr/lpr) mice. In kidneys of these mice, TLR9 localized to glomerular, tubulointerstitial, and perivascular infiltrates. After intraperitoneal injection labeled CpG-ODN localized to glomerular and interstitial macrophages and dendritic cells in nephritic kidneys of MRL(lpr/lpr) mice but not in healthy MRL controls. Furthermore, murine J774 macrophages and splenocytes from MRL(lpr/lpr) mice, but not tubular epithelial cells, renal fibroblasts, or mesangial cells, expressed TLR9 and up-regulated CCL5/RANTES mRNA upon stimulation with CpG-ODN in vitro. In vivo both E. coli DNA and CpG-ODN increased serum DNA autoantibodies of the IgG2a isotype in MRL(lpr/lpr) mice. This was associated with progression of mild to crescentic glomerulonephritis, interstitial fibrosis, and heavy proteinuria. CpG-ODN increased renal CCL2/MCP-1 and CCL5/RANTES expression associated with increased glomerular and interstitial leukocyte recruitment. In contrast control GpC-ODN had no effect. We conclude that TLR9 activation triggers disease activity of systemic autoimmunity, for example, lupus nephritis, and that adaptive and innate immune mechanisms contribute to the CpG-DNA-induced progression of lupus nephritis.

CC chemokine ligand 5/RANTES chemokine antagonists aggravate glomerulonephritis despite reduction of glomerular leukocyte infiltration.

The chemokine CC chemokine ligand (CCL)5/RANTES as well as its respective receptor CCR5 mediate leukocyte infiltration during inflammation and are up-regulated early during the course of glomerulonephritis (GN). We tested the effects of the two CCL5/RANTES blocking analogs, Met-RANTES and amino-oxypentane-RANTES, on the course of horse apoferritin (HAF)-induced GN. HAF-injected control mice had proliferative GN with mesangial immune complex deposits of IgG and HAF. Daily i.p. injections of Met-RANTES or amino-oxypentane-RANTES markedly reduced glomerular cell proliferation and glomerular macrophage infiltration, which is usually associated with less glomerular injury and proteinuria in HAF-GN. Surprisingly, however, HAF-GN mice treated with both analogs showed worse disease with mesangiolysis, capillary obstruction, and nephrotic range albuminuria. These findings were associated with an enhancing effect of the CCL5/RANTES analogs on the macrophage activation state, characterized by a distinct morphology and increased inducible NO synthetase expression in vitro and in vivo, but a reduced uptake of apoptotic cells in vivo. The humoral response and the Th1/Th2 balance in HAF-GN and mesangial cell proliferation in vitro were not affected by the CCL5/RANTES analogs. We conclude that, despite blocking local leukocyte recruitment, chemokine analogs can aggravate some specific disease models, most likely due to interactions with systemic immune reactions, including the removal of apoptotic cells and inducible NO synthetase expression.

Bacterial CpG-DNA aggravates immune complex glomerulonephritis: role of TLR9-mediated expression of chemokines and chemokine receptors.

Immune complex glomerulonephritis (GN) often deteriorates during infection with viruses and bacteria that, in contrast to mammals, have DNA that contains many unmethylated CpG motifs. Balb/c mice with horse apoferritin-induced GN (HAF-GN) were treated with either saline, CpG-oligodeoxynucleotides (ODN), or control GpC-ODN. Only CpG-ODN exacerbated HAF-GN with an increase of glomerular macrophages, which was associated with massive albuminuria and increased renal MCP-1/CCL2, RANTES/CCL5, CCR1, CCR2, and CCR5 mRNA expression. CpG-ODN induced a Th1 response as indicated by serum anti-HAF IgG(2a) titers, mesangial IgG(2a) deposits, and splenocyte IFN-gamma secretion. Messenger RNA for the CpG-DNA receptor Toll-like reeptor 9 (TLR9) was present in kidneys with HAF-GN but not in normal kidneys. The source of TLR9 mRNA in HAF-GN could be infiltrating macrophages or intrinsic renal cells, e.g., mesangial cells; but, in vitro, only murine J774 macrophages expressed TLR9. In J774 cells, CpG-ODN induced the chemokines MCP-1/CCL2 and RANTES/CCL5 and the chemokine receptors CCR1 and CCR5. It is concluded that CpG-DNA can aggravate preexisting GN via a shift toward a Th1 response but also by a novel pathway involving TLR9-mediated chemokine and chemokine receptor expression by macrophages, which may contribute to the enhanced glomerular macrophage recruitment and activation. This mechanism may be relevant during infection-triggered exacerbation of human immune-complex GN and other immune-mediated diseases in general.

A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation.

The expression of chemokines and their receptors is thought to contribute to leukocyte infiltration and progressive renal fibrosis after unilateral ureter obstruction (UUO). We hypothesized that blocking the chemokine receptor CCR1 using the nonpeptide antagonist BX471 could reduce leukocyte infiltration and renal fibrosis after UUO. UUO kidneys from BX471-treated mice (day 0-10 and day 6-10) revealed a 40-60% reduction of interstitial macrophage and lymphocyte infiltrate compared with controls. Treated mice also showed a marked reduction of CCR1 and CCR5 mRNA levels, and FACS analysis showed a comparable reduction of CD8+/CCR5+ T cells. Markers of renal fibrosis, such as interstitial fibroblasts, interstitial volume, mRNA and protein expression for collagen I, were all significantly reduced by BX471-treatment compared with vehicle controls. By contrast treatment was ineffective when the drug was supplied only from days 0 to 5. In summary, blockade of CCR1 substantially reduces cell accumulation and renal fibrosis after UUO. Most interestingly, late onset of treatment is also effective. We therefore conclude that CCR1 blockade may represent a new therapeutic strategy for reducing cellular infiltration and renal fibrosis as major factors in the progression to end-stage renal failure.