Takashi Hato

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.

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.

Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures

In the live animal, tissue autofluorescence arises from a number of biologically important metabolites, such as the reduced form of nicotinamide adenine dinucleotide. Because autofluorescence changes with metabolic state, it can be harnessed as a label-free imaging tool with which to study metabolism in vivo Here, we used the combination of intravital two-photon microscopy and frequency-domain fluorescence lifetime imaging microscopy (FLIM) to map cell-specific metabolic signatures in the kidneys of live animals. The FLIM images are analyzed using the phasor approach, which requires no prior knowledge of metabolite species and can provide unbiased metabolic fingerprints for each pixel of the lifetime image. Intravital FLIM revealed the metabolic signatures of S1 and S2 proximal tubules to be distinct and resolvable at the subcellular level. Notably, S1 and distal tubules exhibited similar metabolic profiles despite apparent differences in morphology and autofluorescence emission with traditional two-photon microscopy. Time-lapse imaging revealed dynamic changes in the metabolic profiles of the interstitium, urinary lumen, and glomerulus-areas that are not resolved by traditional intensity-based two-photon microscopy. Finally, using a model of endotoxemia, we present examples of the way in which intravital FLIM can be applied to study kidney diseases and metabolism. In conclusion, intravital FLIM of intrinsic metabolites is a bias-free approach with which to characterize and monitor metabolism in vivo, and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.

Tamm-Horsfall Protein Regulates Granulopoiesis and Systemic Neutrophil Homeostasis

Tamm-Horsfall protein (THP) is a glycoprotein uniquely expressed in the kidney. We recently showed an important role for THP in mediating tubular cross-talk in the outer medulla and in suppressing neutrophil infiltration after kidney injury. However, it remains unclear whether THP has a broader role in neutrophil homeostasis. In this study, we show that THP deficiency in mice increases the number of neutrophils, not only in the kidney but also in the circulation and in the liver, through enhanced granulopoiesis in the bone marrow. Using multiplex ELISA, we identified IL-17 as a key granulopoietic cytokine specifically upregulated in the kidneys but not in the liver of THP(-/-) mice. Indeed, neutralization of IL-17 in THP(-/-) mice completely reversed the systemic neutrophilia. Furthermore, IL-23 was also elevated in THP(-/-) kidneys. We performed real-time PCR on laser microdissected tubular segments and FACS-sorted renal immune cells and identified the S3 proximal segments, but not renal macrophages, as a major source of increased IL-23 synthesis. In conclusion, we show that THP deficiency stimulates proximal epithelial activation of the IL-23/IL-17 axis and systemic neutrophilia. Our findings provide evidence that the kidney epithelium in the outer medulla can regulate granulopoiesis. When this novel function is added to its known role in erythropoiesis, the kidney emerges as an important regulator of the hematopoietic system.

A Practical Method for Monitoring FRET-based Biosensors in Living Animals Using Two-photon Microscopy

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

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.

Tamm-Horsfall Protein Regulates Mononuclear Phagocytes in the Kidney

Tamm-Horsfall protein (THP), also known as uromodulin, is a kidney-specific protein produced by cells of the thick ascending limb of the loop of Henle. Although predominantly secreted apically into the urine, where it becomes highly polymerized, THP is also released basolaterally, toward the interstitium and circulation, to inhibit tubular inflammatory signaling. Whether, through this latter route, THP can also regulate the function of renal interstitial mononuclear phagocytes (MPCs) remains unclear, however. Here, we show that THP is primarily in a monomeric form in human serum. Compared with wild-type mice, THP-/- mice had markedly fewer MPCs in the kidney. A nonpolymerizing, truncated form of THP stimulated the proliferation of human macrophage cells in culture and partially restored the number of kidney MPCs when administered to THP-/- mice. Furthermore, resident renal MPCs had impaired phagocytic activity in the absence of THP. After ischemia-reperfusion injury, THP-/- mice, compared with wild-type mice, exhibited aggravated injury and an impaired transition of renal macrophages toward an M2 healing phenotype. However, treatment of THP-/- mice with truncated THP after ischemia-reperfusion injury mitigated the worsening of AKI. Taken together, our data suggest that interstitial THP positively regulates mononuclear phagocyte number, plasticity, and phagocytic activity. In addition to the effect of THP on the epithelium and granulopoiesis, this new immunomodulatory role could explain the protection conferred by THP during AKI.