MMP-9 gene deletion mitigates microvascular loss in a model of ischemic acute kidney injury.

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.

N-cadherin is depleted from proximal tubules in experimental and human acute kidney injury.

Ischemia remains the most common cause of acute kidney injury (AKI). Decreased intercellular adhesion and alterations in adhesion molecules may contribute to the loss of renal function observed in AKI. In the present study, we evaluated the distribution of adhesion molecules in the human kidney and analyzed their expression in human and experimental AKI. Specimens of human kidneys obtained from patients with and without AKI were stained for the cell adhesion molecules E-cadherin, N-cadherin and beta-catenin. Experimental AKI in rats was induced by renal artery clamping. Immunostaining and immunoblotting were carried out for E-cadherin, N-cadherin and beta-catenin. Proximal tubule cells from opossum kidneys (OKs) were used to analyze the effect of chemical hypoxia (ATP depletion) in vitro. In the adult human kidney, N-cadherin was expressed in proximal tubules, while E-cadherin was expressed in other nephron segments. beta-Catenin was expressed in both proximal and distal tubules. In human AKI and in ischemic rat kidneys, N-cadherin immunostaining was depleted from proximal tubules. There was no change in E-cadherin or beta-catenin. In vitro, OK cells expressed N-cadherin only in the presence of collagen, and ATP depletion led to a depletion of N-cadherin. Collagen IV staining was reduced in ischemic rat kidneys compared to controls. The results of the study suggest that N-cadherin may play a significant role in human and experimental AKI.

Evidence for involvement of nonesterified fatty acid-induced protonophoric uncoupling during mitochondrial dysfunction caused by hypoxia and reoxygenation.

BACKGROUND: Proximal tubules subjected to hypoxia in vitro under conditions relevant to ischaemia in vivo develop an energetic deficit that is not corrected even after full reoxygenation. We have provided evidence that accumulation of nonesterified fatty acids (NEFA) is the primary reason for this energetic deficit. In this study, we have further investigated the mechanism for the NEFA-induced energetic deficit. METHODS: Mitochondrial membrane potential (Deltapsi) was measured in digitonin-permeabilized, freshly isolated proximal tubules by safranin O uptake. Addition of the potassium/proton exchanger nigericin enables the determination of the mitochondrial proton motive force (Deltap) and the proton gradient (DeltapH). ATP was measured luminometrically and NEFA colorimetrically. RESULTS: Tubule ATP content was depleted after hypoxia and recovered incompletely, even after full reoxygenation. Mitochondrial safranin O uptake was decreased in proximal tubules after hypoxia and reoxygenation (H/R). This decrease was attenuated by delipidated bovine serum albumin (dBSA) or citrate. Addition of nigericin increased safranin O uptake of mitochondria in normoxic proximal tubules, but not in proximal tubules after H/R. Addition of dBSA restored the effect of nigericin to increase mitochondrial safranin O uptake. Addition of the NEFA oleate had the same impact on mitochondrial safranin O uptake as subjecting proximal tubules to H/R. CONCLUSION: The mechanism of the NEFA-induced energetic deficit in freshly isolated rat proximal tubules induced by H/R is characterized by impaired ATP production after full reoxygenation, impaired recovery of Deltapsi and Deltap, abrogation of DeltapH and sensitivity to citrate, consistent with involvement of the tricarboxylate carrier. The data support the concept that protonophoric uncoupling by NEFA movement on anion carriers plays a critical role in proximal tubule mitochochondrial dysfunction after H/R.

Acute and chronic microvascular alterations in a mouse model of ischemic acute kidney injury.

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.

Imaging vascular pathology.

Examining the mechanisms of renal microvascular alterations in disease and the ramifications they have for overall organ function has remained a challenge due to the complexity of the renal microvascular system and the inability to examine it with sufficient resolution in vivo. However, advances in intravital microscopy have provided opportunities to meet these challenges. In this review we will examine specific areas where intravital imaging has advanced our knowledge of renal disease processes and present areas where these techniques, especially intravital multiphoton microscopy, have even further potential to integrate our knowledge of renal vascular pathology.

Digital fluorescence imaging of organic cation transport in freshly isolated rat proximal tubules.

The secretion of cationic drugs and endogenous metabolites is a major function of the kidney. This is accomplished by organic cation transport systems, mainly located in the proximal tubules. Here, we describe a model for continuous measurement of organic cation (OC) transport. In this model, organic cation transport in individual freshly isolated rat proximal tubules is investigated by use of digital fluorescence imaging. To directly measure organic cation transport across the basolateral membrane, the fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP+) is used with a customized perfusion chamber. ASP+ uptake in this model displayed the characteristics of organic cation transport. Over the tested range of 1 to 50 microM, it showed a concentration-dependent uptake across the basolateral membrane. In the presence of competitive inhibitors of OC transport such as N1-methylnicotinamide+, tetraethylammonium+, and choline+, a concentration-dependent and reversible inhibition of ASP+ uptake could be documented. In conclusion, continuous measurement of organic cation transport in freshly isolated rat proximal tubules by digital fluorescence imaging using ASP+ is a useful tool for investigation of drug transport and interactions and, furthermore, may be helpful for investigation of organic cation transport under pathophysiological conditions.

ATP protects, by way of receptor-mediated mechanisms, against hypoxia-induced injury in renal proximal tubules.

We examined the effect of ATP on hypoxia-induced injury in freshly isolated rat renal proximal tubules and compared it with the effects of stable ATP analogues and ATP degradation products. Extracellular ATP significantly reduced hypoxia-induced structural cell damage (lactate dehydrogenase release). P(2)-receptor agonistic ATP analogues, including 2'-methylthio-ATP (2-Me-S-ATP), were also protective. In contrast, the P(1)-agonistic degradation products AMP and adenosine were not protective. Hypoxia-induced functional cell damage (loss of cellular potassium) was not changed by ATP or 2-Me-S-ATP. We therefore conclude that the protective property of ATP is not based on an effect of the degradation products or on a direct effect on cellular energy metabolism. The data indicate that the protective effect of ATP is mediated by P(2) receptors.