Mitochondrial depolarization and repolarization in the early stages of acetaminophen hepatotoxicity in mice.

Mitochondrial injury and depolarization are primary events in acetaminophen hepatotoxicity. Previous studies have shown that restoration of mitochondrial function in surviving hepatocytes, which is critical to recovery, is at least partially accomplished via biogenesis of new mitochondria. However, other studies indicate that mitochondria also have the potential to spontaneously repolarize. Although repolarization was previously observed only at a sub-hepatotoxic dose of acetaminophen, we postulated that mitochondrial repolarization in hepatocytes outside the centrilobular regions of necrosis might contribute to recovery of mitochondrial function following acetaminophen-induced injury. Our studies utilized longitudinal intravital microscopy of millimeter-scale regions of the mouse liver to characterize the spatio-temporal relationship between mitochondrial polarization and necrosis early in acetaminophen-induced liver injury. Treatment of male C57BL/6J mice with a single intraperitoneal 250 mg/kg dose of acetaminophen resulted in hepatotoxicity that was apparent histologically within 2 h of treatment, leading to 20 and 60-fold increases in serum aspartate aminotransferase and alanine aminotransferase, respectively, within 6 h. Intravital microscopy of the livers of mice injected with rhodamine123, TexasRed-dextran, propidium iodide and Hoechst 33342 detected centrilobular foci of necrosis within extended regions of mitochondrial depolarization within 2 h of acetaminophen treatment. Although regions of necrosis were more apparent 6 h after acetaminophen treatment, the vast majority of hepatocytes with depolarized mitochondria did not progress to necrosis, but rather recovered mitochondrial polarization within 6 h. Recovery of mitochondrial function following acetaminophen hepatotoxicity thus involves not only biogenesis of new mitochondria, but also repolarization of existing mitochondria. These studies also revealed a spatial distribution of necrosis and mitochondrial depolarization whose single-cell granularity is inconsistent with the hypothesis that communication between neighboring cells plays an important role in the propagation of necrosis during the early stages of APAP hepatotoxicity. Small islands of healthy, intact cells were frequently found surrounded by necrotic cells, and small islands of necrotic cells were frequently found surrounded by healthy, intact cells. Time-series studies demonstrated that these "islands", consisting in some cases of single cells, are persistent; over a period of hours, injury does not spread from individual necrotic cells to their neighbors.

Endothelial STAT3 Modulates Protective Mechanisms in a Mouse Ischemia-Reperfusion Model of Acute Kidney Injury.

STAT3 is a transcriptional regulator that plays an important role in coordinating inflammation and immunity. In addition, there is a growing appreciation of the role STAT3 signaling plays in response to organ injury following diverse insults. Acute kidney injury (AKI) from ischemia-reperfusion injury is a common clinical entity with devastating consequences, and the recognition that endothelial alterations contribute to kidney dysfunction in this setting is of growing interest. Consequently, we used a mouse with a genetic deletion of Stat3 restricted to the endothelium to examine the role of STAT3 signaling in the pathophysiology of ischemic AKI. In a mouse model of ischemic AKI, the loss of endothelial STAT3 signaling significantly exacerbated kidney dysfunction, morphologic injury, and proximal tubular oxidative stress. The increased severity of ischemic AKI was associated with more robust endothelial-leukocyte adhesion and increased tissue accumulation of F4/80+ macrophages. Moreover, important proximal tubular adaptive mechanisms to injury were diminished in association with decreased tissue mRNA levels of the epithelial cell survival cytokine IL-22. In aggregate, these findings suggest that the endothelial STAT3 signaling plays an important role in limiting kidney dysfunction in ischemic AKI and that selective pharmacologic activation of endothelial STAT3 signaling could serve as a potential therapeutic target.

Inhibition of Toll-Like Receptor 4 Signaling Mitigates Microvascular Loss but Not Fibrosis in a Model of Ischemic Acute Kidney Injury.

The development of chronic kidney disease (CKD) following an episode of acute kidney injury (AKI) is an increasingly recognized clinical problem. Inhibition of toll-like receptor 4 (TLR4) protects renal function in animal models of AKI and has become a viable therapeutic strategy in AKI. However, the impact of TLR4 inhibition on the chronic sequelae of AKI is unknown. Consequently, we examined the chronic effects of TLR4 inhibition in a model of ischemic AKI. Mice with a TLR4-deletion on a C57BL/6 background and wild-type (WT) background control mice (C57BL/6) were subjected to bilateral renal artery clamping for 19 min and reperfusion for up to 6 weeks. Despite the acute protective effect of TLR4 inhibition on renal function (serum creatinine 1.6 ± 0.4 mg/dL TLR4-deletion vs. 2.8 ± 0.3 mg/dL·WT) and rates of tubular apoptosis following ischemic AKI, we found no difference in neutrophil or macrophage infiltration. Furthermore, we observed significant protection from microvascular rarefaction at six weeks following injury with TLR4-deletion, but this did not alter development of fibrosis. In conclusion, we validate the acute protective effect of TLR4 signal inhibition in AKI but demonstrate that this protective effect does not mitigate the sequential fibrogenic response in this model of ischemic AKI.

Muc1 enhances the ß-catenin protective pathway during ischemia-reperfusion injury.

The hypoxia-inducible factor (HIF)-1 and ß-catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine mucin (Muc)1 protects kidney function and morphology in a mouse model of ischemia-reperfusion injury (IRI) by stabilizing HIF-1α, enhancing HIF-1 downstream signaling, and thereby preventing metabolic stress (Pastor-Soler et al. Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308: F1452-F1462, 2015). We asked if Muc1 regulates the ß-catenin protective pathway during IRI as 1) ß-catenin nuclear targeting is MUC1 dependent in cultured human cells, 2) ß-catenin is found in coimmunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, and 3) MUC1 prevents ß-catenin phosphorylation by glycogen synthase kinase (GSK)3ß and thereby ß-catenin degradation. Using the same mouse model of IRI, we found that levels of active GSK3ß were significantly lower in kidneys of control mice compared with Muc1 knockout (KO) mice. Consequently, ß-catenin was significantly upregulated at 24 and 72 h of recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both ß-catenin induction and nuclear targeting were absent in Muc1 KO mice. We also found downstream induction of ß-catenin prosurvival factors (activated Akt, survivin, transcription factor T cell factor 4 (TCF4), and its downstream target cyclin D1) and repression of proapoptotic factors (p53, active Bax, and cleaved caspase-3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during acute kidney injury proceeds by enhancing both the HIF-1 and ß-catenin protective pathways.

Muc1 is protective during kidney ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells.

The p53 inhibitor pifithrin-α can stimulate fibrosis in a rat model of ischemic acute kidney injury.

Inhibition of the tumor suppressor p53 diminishes tubular cell apoptosis and protects renal function in animal models of acute kidney injury (AKI). Therefore, targeting p53 has become an attractive therapeutic strategy in the approach to AKI. Although the acute protective effects of p53 inhibition in AKI have been examined, there is still relatively little known regarding the impact of acute p53 inhibition on the chronic sequelae of AKI. Consequently, we utilized the p53 inhibitor pifithrin-α to examine the long-term effects of p53 inhibition in a rodent model of ischemic AKI. Male Sprague-Dawley rats were subjected to bilateral renal artery clamping for 30 min followed by reperfusion for up to 8 wk. Pifithrin-α or vehicle control was administered at the time of surgery and then daily for 2 days [brief acute administration (BA)] or 7 days [prolonged acute administration (PA)]. Despite the acute protective effect of pifithrin-α in models of ischemic AKI, we found no protection in the microvascular rarefaction at 4 wk or development fibrosis at 8 wk with pifithrin-α administered on the BA schedule compared with vehicle control-treated animals. Furthermore, pifithrin-α administered on a PA schedule actually produced worse fibrosis compared with vehicle control animals after ischemic injury [21%/area (SD4.4) vs.16%/area (SD3.6)] as well as under sham conditions [2.6%/area (SD1.8) vs. 4.7%/area (SD1.3)]. The development of fibrosis with PA administration was independent of microvascular rarefaction. We identified enhanced extracellular matrix production, epithelial-to-mesenchymal transition, and amplified inflammatory responses as potential contributors to the augmented fibrosis observed with PA administration of pifithrin-α.

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.

p53 regulates renal expression of HIF-1{alpha} and pVHL under physiological conditions and after ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) and is characterized by widespread tubular and microvascular damage. The tumor suppressor p53 is upregulated after IRI and contributes to renal injury in part by promoting apoptosis. Acute, short-term inhibition of p53 with pifithrin-alpha conveys significant protection after IRI. The hypoxia-inducible factor-1 (HIF-1) pathway is also activated after IRI and has opposing effects to those promoted by p53. The balance between the HIF-1 and p53 responses can determine the outcome of IRI. In this manuscript, we investigate whether p53 regulates the HIF-1 pathway in a rodent model of IRI. HIF-1alpha is principally expressed in the collecting tubules (CT) and thick ascending limbs (TAL) under physiological conditions. However, inhibition of p53 with pifithrin-alpha increases the faint expression of HIF-1alpha in proximal tubules (PT) under physiological conditions. Twenty-four hours after IRI, HIF-1alpha expression is decreased in both CT and TAL. HIF-1alpha expression in the PT is not significantly altered after IRI. Acute inhibition of p53 significantly increases HIF-1alpha expression in the PT after IRI. Additionally, pifithrin-alpha prevents the IRI-induced decrease in HIF-1alpha in the CT and TAL. Parallel changes are observed in the HIF-1alpha transcriptive target, carbonic anhydrase-9. Finally, inhibition of p53 prevents the dramatic changes in Von Hippel-Lindau protein morphology and expression after IRI. We conclude that activation of p53 after IRI mitigates the concomitant activation of the protective HIF-1 pathway. Modulating the interactions between the p53 and HIF-1 pathway can provide novel options in the treatment of AKI.

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.

Minocycline reduces renal microvascular leakage in a rat model of ischemic renal injury.

Tetracyclines exhibit significant anti-inflammatory properties, inhibit matrix metalloproteinases (MMPs), and are protective in models of ischemia-reperfusion injury (IRI). Both inflammatory cascades and MMP activation have been demonstrated to modulate microvascular permeability. Because increased microvascular permeability occurs during IRI in a variety of organ systems including the kidney, we hypothesized that minocycline, a semisynthetic tetracycline, would diminish microvascular leakage during renal IRI. To test this hypothesis, we used intravital 2-photon microscopy to examine leakage of fluorescent dextrans from the vasculature in a rodent model of IRI. Minocycline significantly reduced the extent of dextran (500 kDa) leakage from the renal microvasculature 24 h after ischemia. Although minocycline diminished leukocyte accumulation in the kidney following ischemia, areas of leukocyte accumulation did not correlate with areas of microvascular permeability in either the saline- or minocycline-pretreated animals. Minocycline diminished the perivascular increase in MMP-2 and MMP-9, as well as the increase in MMP-2 activity 24 h after ischemia. ABT-518, a specific inhibitor of MMP-2 and MMP-9, also significantly reduced the extent of dextran (500 kDa) leakage from the renal microvasculature 24 h after ischemia. Our results indicate that minocycline mitigates the renal microvascular permeability defect following IRI. This effect is spatially distinct from the effect of minocycline on leukocyte accumulation and may be related to diminished activity of MMPs on the integrity of the perivascular matrix.

Injury of the renal microvascular endothelium alters barrier function after ischemia.

The role of renal microvascular endothelial cell injury in the pathophysiology of ischemic acute renal failure (ARF) remains largely unknown. No consistent morphological alterations have been ascribed to the endothelium of the renal microvasculature as a result of ischemia-reperfusion injury. Therefore, the purpose of this study was to examine biochemical markers of endothelial injury and morphological changes in the renal microvascular endothelium in a rodent model of ischemic ARF. Circulating von Willebrand factor (vWF) was measured as a marker of endothelial injury. Twenty-four hours after ischemia, circulating vWF peaked at 124% over baseline values (P = 0.001). The FVB-TIE2/GFP mouse was utilized to localize morphological changes in the renal microvascular endothelium. Immediately after ischemia, there was a marked increase in F-actin aggregates in the basal and basolateral aspect of renal microvascular endothelial cells in the corticomedullary junction. After 24 h of reperfusion, the pattern of F-actin staining was more similar to that observed under physiological conditions. In addition, alterations in the integrity of the adherens junctions of the renal microvasculature, as demonstrated by loss of localization in vascular endothelial cadherin immunostaining, were observed after 24 h of reperfusion. This observation temporally correlated with the greatest extent of permeability defect in the renal microvasculature as identified using fluorescent dextrans and two-photon intravital imaging. Taken together, these findings indicate that renal vascular endothelial injury occurs in ischemic ARF and may play an important role in the pathophysiology of ischemic ARF.