Small molecule selectin ligand inhibition improves outcome in ischemic acute renal failure.

BACKGROUND: The pathophysiologic and potential therapeutic role of selectins in renal ischemia-reperfusion injury (IRI) is not fully understood, due in part to redundancy in the roles of individual selectins. We hypothesized that blockade of ligands for all three selectins using a novel small molecule (TBC-1269) would improve the course of renal IRI by overcoming redundancy issues. This was investigated in a rat model of renal IRI. METHODS: Rats were treated with TBC-1269 either during or post-IRI. The effects of TBC-1269 were investigated in two models of renal IRI: moderate IRI (30 minutes bilateral renal artery clamping) and severe IRI (45 minutes clamping). The combination of anti-E- and anti-P-selectin antibodies also was investigated in rats subjected to moderate IRI. Renal function, histological injury and mortality were assessed. RESULTS: Rats treated with TBC-1269 during moderate IRI showed significantly reduced serum creatinine (SCr) and tubular necrosis post-ischemia compared to control animals. By contrast, delayed treatment (post-IRI) did not show a reduction in SCr. In rats with severe IRI, TBC-1269 treatment during IRI significantly reduced mortality at 48 hours post-ischemia. Rats with moderate IRI and treated with the combination of anti-E- and anti-P-selectin antibodies showed significantly reduced SCr compared to control rats at 24 hours post-ischemia. CONCLUSIONS: Small molecule selectin ligand inhibition provides a novel and effective approach to attenuate ischemic acute renal failure. Timing of treatment is crucial to success.

Identification of the CD4(+) T cell as a major pathogenic factor in ischemic acute renal failure.

Leukocytes have been implicated in the pathogenesis of ischemic acute renal failure (ARF), but the roles of the individual cell types involved are largely unknown. Recent indirect evidence suggests that T cells may play an important role in a murine model of ARF. In the current study, we found that mice deficient in T cells (nu/nu mice) are both functionally and structurally protected from postischemic renal injury. Reconstitution of nu/nu mice with wild-type T cells restored postischemic injury. We then analyzed the contribution of the individual T cell subsets to postischemic injury and found that mice deficient in CD4(+) T cells, but not mice deficient in CD8(+) T cells, were significantly protected from ARF. Direct evidence for a pathophysiologic role of the CD4(+) T cell was obtained when reconstitution of CD4-deficient mice with wild-type CD4(+) T cells restored postischemic injury. In addition, adoptive transfers of CD4(+) T cells lacking either the costimulatory molecule CD28 or the ability to produce IFN-gamma were inadequate to restore injury phenotype. These results demonstrate that the CD4(+) T cell is an important mediator of ischemic ARF, and targeting this cell may yield novel therapies.

IL-1 and TNF independent pathways mediate ICAM-1/VCAM-1 up-regulation in ischemia reperfusion injury.

In vitro studies have suggested that targeting interleukin (IL)-1 and tumor necrosis factor (TNF) can be used to regulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and potentially treat kidney inflammation. We therefore evaluated ICAM-1 and VCAM-1 regulation in knockout (KO) mice deficient in both IL-1 receptor 1 (R1) and TNF-R1 during renal ischemia reperfusion injury. ICAM-1 and VCAM-1 mRNA expression was measured with specific murine probes and Northern blotting (n =4/group). Protein expression was measured using immunohistochemistry. Serum creatinine (SCr), tubular histology, and neutrophil infiltration into postischemic kidneys were also quantified. ICAM-1 and VCAM-1 mRNA expression increased in both wild-type (WT) and KO mice at 2, 6, and 24 h. Protein expression of ICAM-1 and VCAM-1 was also increased at 24 h postischemia. SCr levels and tubular necrosis scores were comparable in WT and KO mice at 24 and 48 h. Neutrophil migration in KO mice was decreased at 24 h but comparable to WT at 48 h. These data demonstrate that IL-1 and TNF are not essential for postischemic increases in ICAM-1 and VCAM-1.

Genetic susceptibility to renal ischemia reperfusion injury revealed in a murine model.

BACKGROUND: The development of genetically engineered mice has led to increased use of mouse models to study renal ischemic reperfusion injury (IRI). We hypothesized that susceptibility to IRI could result from strain differences due to genetic factors. METHODS: Our study compared recovery subsequent to renal IRI in NIH Swiss, C57BL/6, and BALB/c mice. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were evaluated postischemia. We also conducted reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of renal cytokines and adhesion molecules postischemia. RESULTS: At 48 hr postischemia, renal dysfunction in NIH Swiss mice was significantly reduced, compared with other groups (P<0.01). BUN measurements confirmed renal protection at 48 hr in the NIH Swiss group. RT-PCR analysis of mRNA postischemia demonstrated that, between strains, there was little difference in mRNA expression for renal cytokines and adhesion molecules. CONCLUSIONS: NIH Swiss mice appear to be resistant in susceptibility to renal IRI. Early expression of pro-inflammatory genes was not associated with resistance to IRI, thus genetic factors could be important in outcome after renal IRI.

Fractional clearance of high molecular weight proteins in conscious rats using a continuous infusion method.

BACKGROUND: The purported existence of "large pores" in the glomerular capillary wall has been derived primarily from studies using dextrans and Ficolls. Systematic studies using high molecular weight proteins have not been performed. One of the difficulties is that recent studies have demonstrated that albumin and other proteins undergo degradation during renal passage. Our study took into account this renal degradation in measuring the fractional clearance of various high molecular weight proteins (the hydrodynamic radii range was between 48 to 70 A). METHODS: Fractional clearances of tritium-labeled proteins were measured using ALZET osmotic pumps, which are designed to release a slow continuous infusion of tracer. Blood and urine collections were taken at 24-hour intervals over seven days and were counted for radioactivity, and glomerular filtration rate was measured by a creatinine assay. RESULTS: Steady-state levels of [3H]protein in plasma were obtained by day 6. The [3H]proteins in the plasma showed no degradation. The fractional clearances (mean +/- sd, N = 5) of the various proteins were albumin (radius = 36 A; 0.0023 +/- 0.0009), transferrin (48 A; 0.0046 +/- 0.0007), lactoperoxidase (58 A; 0. 0045 +/- 0.0005), immunoglobulin G (62 A; 0.0043 +/- 0.0009), lactate dehydrogenase (64 A; 0.0041 +/- 0.0009), and glucose oxidase (70 A; 0.0036 +/- 0.0011). CONCLUSIONS: These values suggest a weak dependence of fractional clearance on size-selective filtration, except for albumin, which undergoes a specific type of postglomerular processing. The fractional clearances were higher than expected from previous data on dextrans and Ficolls of equivalent hydrodynamic radius, and thus demonstrate that "large pores" may already exist in normal glomerular capillary walls.

Alterations in renal degradation of albumin in early experimental diabetes in the rat: a new factor in the mechanism of albuminuria.

1. Albumin is normally excreted as a mixture of intact protein and fragments that are produced during renal passage. The purpose of this study was to investigate the ratio of intact versus degraded forms of excreted albumin to ascertain whether changes in this ratio could account for the apparent increase in albumin excretion seen in diabetes, as measured by standard radioimmunoassay techniques. 2. Four-week male Sprague-Dawley rats with streptozotocin-induced diabetes and age-matched control rats were intravenously injected with [3H]albumin. Urine collected over 2 h was analysed by size exclusion chromatography and radioimmunoassay. A standard radioimmunoassay found a 7-fold increase in albumin excretion rate in diabetic rats, whereas there was only a 2-fold increase in albumin excretion (intact plus fragments). Urine analysed by size exclusion chromatography showed severe degradation for control rats (% monomer=4+/-2%); in diabetic rats there was a significant amount of monomer albumin excreted, along with moderately degraded and heavily degraded albumin (% monomer=17+/-5%). 3. This study has shown that the radioimmunoassay, which specifically detects intact albumin, considerably underestimates the amount of total urinary albumin which consists of intact and degraded material. The increase in albumin excretion rate observed in diabetes as measured by radioimmunoassay is mainly due to a change in the amount of intact albumin excreted and this is specifically due to the inhibition of albumin degradation at a post-glomerular site and not due to the onset of any type of glomerular 'shunt' pathway.

Anomalous decrease in dextran sulfate clearance in the diabetic rat kidney.

The anomalous increase in charge selectivity as previously observed with reduced dextran sulfate clearances in diabetic rats (L. D. Michels, M. Davidman, and W. F. Keane. Kidney Int. 21: 699-705, 1982) was confirmed in 4-wk streptozotocin (STZ) diabetic Sprague-Dawley rats using the isolated perfused kidney technique. The apparent charge selectivity in both control and diabetic rats could be abolished by increasing the dextran sulfate concentration to 200 micrograms/ml in the perfusate. This was demonstrated by a high rate of processing of dextran sulfate (approximately 1,700 ng.min-1.kidney-1) by glomeruli in both control and diabetic kidneys and by the fact that charge interaction could not explain the concentration dependence. The amount of urinary desulfation of dextran sulfate was also found to be significantly less in the diabetic kidney as was glomerular sulfatase activity compared with controls. Dextran sulfate glomerular processing is therefore altered in the STZ diabetic rat kidney but could be rationalized in terms of previous models of endothelial cell receptor-mediated uptake of dextran sulfate. The results are consistent with recent work demonstrating that there is little or no electrostatic charge interaction operating on dextran sulfate or other negatively charged molecules at the glomerular capillary wall.

Competition between polyanions in glomerular binding and renal clearance.

This study tests the hypothesis that exogenous polyanions may accumulate in the glomerular capillary wall and thereby provide fixed negative charges that will be effective, through electrostatic interactions, in governing the transcapillary transport of charged molecules. We have tested the effects of high concentrations of dextran sulfate, heparin, and orosomucoid on the fractional clearance of albumin and dextran sulfate using the isolated perfused kidney technique. We demonstrate that all these exogenous polyanions are without effect on the fractional clearance of albumin. Augmentation of glomerular charge through the binding of dextran sulfate or orosomucoid is very small compared to the charge augmentation by albumin. These results agree with previous studies which demonstrate that electrostatic interactions governing glomerular transcapillary wall transport are negligible. This study also demonstrates that there is no plausible correlation between the fixed negative charge contributed by endogenous metabolically labeled glomerular heparan sulfate and the manifestation of apparent charge selectivity as determined with dextran sulfate. The results are also consistent with previous studies that the renal processing of both dextran sulfate and albumin is associated with specific cellular uptake.

Glomerular processing of dextran sulfate during transcapillary transport.

The fractional clearance of dextran sulfate across the glomerular capillary wall in the isolated perfused kidney is shown to be dependent on the concentration of dextran sulfate in the perfusate and the degree of sulfate substitution on dextran sulfate. While normal charge selectivity is apparent at low dextran sulfate perfusate concentrations, the nature of the concentration dependence of the fractional clearance clearly eliminates charge-electrostatic interactions as being responsible. A strong correlation has been experimentally established between fractional clearance and the degree of desulfation of dextran sulfate found in the urine. The correlation was further established through the demonstration that the lysosomotropic agent NH4Cl partially inhibits charge selectivity as well as desulfation. The glomerular cellular processing of the dextran sulfate was further studied through dextran sulfate-mediated release of metabolically labeled glomerular heparan sulfate. A model of glomerular capillary wall transport, invoking endothelial cell processing of dextran sulfate, is proposed to explain the differences between the fractional clearance of dextran and dextran sulfate.