No abstract

Functional and structural abnormalities in the renal microvasculature are important processes contributing to the pathophysiology of ischemic acute kidney injury (AKI). In this study, we examine the contribution of endothelial cell loss via apoptosis on microvascular permeability and rarefaction in a mouse model of ischemic AKI. Three-dimensional reconstructions of microvascular networks obtained 24 h following acute ischemic injury demonstrate an intact endothelial monolayer in areas of increased microvascular permeability. A 45% decrease in microvascular density was observed 4 wk after acute ischemic injury. Examination of microvascular endothelial cells following acute ischemic injury did not reveal evidence of positive terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining at 1, 2, 8, and 16 days following ischemia; however, activation of caspase-3 was evident in endothelial cells following acute ischemic injury. Examination of angiopoietin (Ang) protein expression in the kidney 24 h after ischemic injury revealed an eightfold increase in Ang-1 but no significant change in Ang-2. No significant difference in the expression of vascular endothelial growth factor or Ang-2 was observed 4 wk after ischemic injury, although an almost twofold elevation in Ang-1 was observed. An increase in angiostatic breakdown products of collagen IV was observed at both 24 h and 4 wk after ischemic injury. Taken together, these findings indicate that the loss of endothelial cells following ischemic injury is not a major contributor to altered microvascular permeability, although renal microvascular endothelial cells are vulnerable to the initiation of apoptotic mechanisms following ischemic injury that can ultimately impact microvascular density.

Microvascular rarefaction following an episode of acute kidney injury (AKI) is associated with renal hypoxia and progression toward chronic kidney disease. The mechanisms contributing to microvascular rarefaction are not well-understood, although disruption in local angioregulatory substances is thought to contribute. Matrix metalloproteinase (MMP)-9 is an endopeptidase important in modifying the extracellular matrix (ECM) and remodeling the vasculature. We examined the role of MMP-9 gene deletion on microvascular rarefaction in a rodent model of ischemic AKI. MMP-9-null mice and background control (FVB/NJ) mice were subjected to bilateral renal artery clamping for 20 min followed by reperfusion for 14, 28, or 56 days. Serum creatinine level in MMP-9-null mice 24 h after injury [1.4 (SD 0.8) mg/dl] was not significantly different from FVB/NJ mice [1.5 (SD 0.6) mg/dl]. Four weeks after ischemic injury, FVB/NJ mice demonstrated a 30-40% loss of microvascular density compared with sham-operated (SO) mice. In contrast, microvascular density was not significantly different in the MMP-9-null mice at this time following injury compared with SO mice. FVB/NJ mice had a 50% decrease in tissue vascular endothelial growth factor (VEGF) 2 wk after ischemic insult compared with SO mice. A significant difference in VEGF was not observed in MMP-9-null mice compared with SO mice. There was no significant difference in the liberation of angioinhibitory fragments from the ECM between MMP-9-null mice and FVB/NJ mice following ischemic injury. In conclusion, MMP-9 deletion stabilizes microvascular density following ischemic AKI in part by preserving tissue VEGF levels.

Secretion of cationic drugs and endogenous metabolites is a major function of the kidney accomplished by tubular organic cation transport systems. A cationic styryl dye (ASP(+)) was developed as a fluorescent substrate for renal organic cation transporters. The dye was injected intravenously and continuously monitored in externalized rat kidneys by time-resolved two-photon laser scanning microscopy. To investigate changes in transport activity, cimetidine, a competitive inhibitor of organic cation transport was co-injected with ASP(+). Shortly after injection, fluorescence increased in peritubular capillaries. Simultaneously, fluorescence was transiently found at the basolateral membrane of the proximal and distal tubules at a higher intensity and shorter wavelength indicating membrane association of ASP(+). Subsequently, intracellular fluorescence increased steeply within 10 s. In the proximal tubules, intracellular fluorescence decreased by 50% within 5 min, while in the distal tubules the fluorescence decreased by only 5% within the same time frame. Intracellular uptake of ASP(+) into proximal tubules was significantly reduced by cimetidine. Our studies show that organic cation transport of the kidney can be visualized in vivo by two-photon laser scanning microscopy.