Zoya Plotkin

P53 Mediates the Apoptotic Response to GTP Depletion after Renal Ischemia-Reperfusion: Protective Role of a p53 Inhibitor

Ischemic injury to the kidney is characterized in part by nucleotide depletion and tubular cell death in the form of necrosis or apoptosis. GTP depletion was recently identified as an important inducer of apoptosis during chemical anoxia in vitro and ischemic injury in vivo. It has also been shown that GTP salvage with guanosine prevented apoptosis and protected function. This study investigates the role of p53 in mediating the apoptotic response to GTP depletion. Male Sprague-Dawley rats underwent bilateral renal artery clamp for 30 min followed by reperfusion. p53 protein levels increased significantly in the medulla over 24 h post-ischemia. The provision of guanosine inhibited the increase in p53. Pifithrin-alpha, a specific inhibitor of p53, mimicked the effects of guanosine. It had no effect on necrosis, yet it prevented apoptosis and protected renal function. Pifithrin-alpha was protective when given up to 14 h after the ischemic insult. The effects of pifithrin-alpha on p53 included inhibition of transcriptional activation of downstream p53 targets like p21 and Bax and inhibition of p53 translocation to the mitochondria. Similar results were obtained in cultured renal tubular cells. It is concluded that p53 is an important mediator of apoptosis during states of GTP depletion. Inhibitors of p53 should be considered in the treatment of ischemic renal injury.

Endotoxin Uptake by S1 Proximal Tubular Segment Causes Oxidative Stress in the Downstream S2 Segment

Gram-negative sepsis carries high morbidity and mortality, especially when complicated by acute kidney injury (AKI). The mechanisms of AKI in sepsis remain poorly understood. Here we used intravital two-photon fluorescence microscopy to investigate the possibility of direct interactions between filtered endotoxin and tubular cells as a possible mechanism of AKI in sepsis. Using wild-type (WT), TLR4-knockout, and bone marrow chimeric mice, we found that endotoxin is readily filtered and internalized by S1 proximal tubules through local TLR4 receptors and through fluid-phase endocytosis. Only receptor-mediated interactions between endotoxin and S1 caused oxidative stress in neighboring S2 tubules. Despite significant endotoxin uptake, S1 segments showed no oxidative stress, possibly as a result of the upregulation of cytoprotective heme oxygenase-1 and sirtuin-1 (SIRT1). Conversely, S2 segments did not upregulate SIRT1 and exhibited severe structural and functional peroxisomal damage. Taken together, these data suggest that the S1 segment acts as a sensor of filtered endotoxin, which it takes up. Although this may limit the amount of endotoxin in the systemic circulation and the kidney, it results in severe secondary damage to the neighboring S2 segments.

p53 Is Renoprotective after Ischemic Kidney Injury by Reducing Inflammation

In the rat, p53 promotes tubular apoptosis after ischemic AKI. Acute pharmacologic inhibition of p53 is protective in this setting, but chronic inhibition enhances fibrosis, demonstrating that the role of p53 in ischemic AKI is incompletely understood. Here, we investigated whether genetic absence of p53 is also protective in ischemic AKI. Surprisingly, p53-knockout mice (p53(-/-)) had worse kidney injury, compared with wild-type mice, and exhibited increased and prolonged infiltration of leukocytes after ischemia. Acute inhibition of p53 with pifithrin-α in wild-type mice mimicked the observations in p53(-/-) mice. Chimeric mice that lacked p53 in leukocytes sustained injury similar to p53(-/-) mice, suggesting an important role for leukocyte p53 in ischemic AKI. Compared with wild-type mice, a smaller proportion of macrophages in the kidneys of p53(-/-) and pifithrin-α-treated mice after ischemic injury were the anti-inflammatory M2 phenotype. Ischemic kidneys of p53(-/-) and pifithrin-α-treated mice also showed reduced expression of Kruppel-like factor-4. Finally, models of peritonitis in p53(-/-) and pifithrin-α-treated mice confirmed the anti-inflammatory role of p53 and its effect on the polarization of macrophage phenotype. In summary, in contrast to the rat, inflammation characterizes ischemic AKI in mice; leukocyte p53 is protective by reducing the extent and duration of this inflammation and by promoting the anti-inflammatory M2 macrophage phenotype.

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.

p53 regulates renal expression of HIF-1α 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.

The Macrophage Mediates the Renoprotective Effects of Endotoxin Preconditioning

Preconditioning is a preventative approach, whereby minimized insults generate protection against subsequent larger exposures to the same or even different insults. In immune cells, endotoxin preconditioning downregulates the inflammatory response and yet, preserves the ability to contain infections. However, the protective mechanisms of preconditioning at the tissue level in organs such as the kidney remain poorly understood. Here, we show that endotoxin preconditioning confers renal epithelial protection in various models of sepsis in vivo. We also tested the hypothesis that this protection results from direct interactions between the preconditioning dose of endotoxin and the renal tubules. This hypothesis is on the basis of our previous findings that endotoxin toxicity to nonpreconditioned renal tubules was direct and independent of immune cells. Notably, we found that tubular protection after preconditioning has an absolute requirement for CD14-expressing myeloid cells and particularly, macrophages. Additionally, an intact macrophage CD14-TRIF signaling pathway was essential for tubular protection. The preconditioned state was characterized by increased macrophage number and trafficking within the kidney as well as clustering of macrophages around S1 proximal tubules. These macrophages exhibited increased M2 polarization and upregulation of redox and iron-handling molecules. In renal tubules, preconditioning prevented peroxisomal damage and abolished oxidative stress and injury to S2 and S3 tubules. In summary, these data suggest that macrophages are essential mediators of endotoxin preconditioning and required for renal tissue protection. Preconditioning is, therefore, an attractive model to investigate novel protective pathways for the prevention and treatment of sepsis.

Novel application of complementary imaging techniques to examine in vivo glucose metabolism in the kidney

The metabolic status of the kidney is a determinant of injury susceptibility and a measure of progression for many disease processes; however, noninvasive modalities to assess kidney metabolism are lacking. In this study, we employed positron emission tomography (PET) and intravital multiphoton microscopy (MPM) to assess cortical and proximal tubule glucose tracer uptake, respectively, following experimental perturbations of kidney metabolism. Applying dynamic image acquisition PET with 2-18fluoro-2-deoxyglucose (18F-FDG) and tracer kinetic modeling, we found that an intracellular compartment in the cortex of the kidney could be distinguished from the blood and urine compartments in animals. Given emerging literature that the tumor suppressor protein p53 is an important regulator of cellular metabolism, we demonstrated that PET imaging was able to discern a threefold increase in cortical 18F-FDG uptake following the pharmacological inhibition of p53 in animals. Intravital MPM with the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) provided increased resolution and corroborated these findings at the level of the proximal tubule. Extending our observation of p53 inhibition on proximal tubule glucose tracer uptake, we demonstrated by intravital MPM that pharmacological inhibition of p53 diminishes mitochondrial potential difference. We provide additional evidence that inhibition of p53 alters key metabolic enzymes regulating glycolysis and increases intermediates of glycolysis. In summary, we provide evidence that PET is a valuable tool for examining kidney metabolism in preclinical and clinical studies, intravital MPM is a powerful adjunct to PET in preclinical studies of metabolism, and p53 inhibition alters basal kidney metabolism.

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.

Endotoxin preconditioning reprograms S1 tubules and macrophages to protect the kidney

Preconditioning with a low dose of endotoxin confers unparalleled protection against otherwise lethal models of sepsis. The mechanisms of preconditioning have been investigated extensively in isolated immune cells such as macrophages. However, the role of tissue in mediating the protective response generated by preconditioning remains unknown. Here, using the kidney as a model organ, we investigated cell type-specific responses to preconditioning. Compared with preadministration of vehicle, endotoxin preconditioning in the cecal ligation and puncture mouse model of sepsis led to significantly enhanced survival and reduced bacterial load in several organs. Furthermore, endotoxin preconditioning reduced serum levels of proinflammatory cytokines, upregulated molecular pathways involved in phagocytosis, and prevented the renal function decline and injury induced in mice by a toxic dose of endotoxin. The protective phenotype involved the clustering of macrophages around S1 segments of proximal tubules, and full renal protection required both macrophages and renal tubular cells. Using unbiased S1 transcriptomic and tissue metabolomic approaches, we identified multiple protective molecules that were operative in preconditioned animals, including molecules involved in antibacterial defense, redox balance, and tissue healing. We conclude that preconditioning reprograms macrophages and tubules to generate a protective environment, in which tissue health is preserved and immunity is controlled yet effective. Endotoxin preconditioning can thus be used as a discovery platform, and understanding the role and participation of both tissue and macrophages will help refine targeted therapies for sepsis.