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.

Single cell sequencing studies have characterized the transcriptomic signature of cell types within the kidney. However, the spatial distribution of acute kidney injury (AKI) is regional and affects cells heterogeneously. We first optimized coordination of spatial transcriptomics and single nuclear sequencing datasets, mapping 30 dominant cell types to a human nephrectomy. The predicted cell type spots corresponded with the underlying histopathology. To study the implications of AKI on transcript expression, we then characterized the spatial transcriptomic signature of two murine AKI models: ischemia reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were associated with injury pathways. Using single cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. Neutrophils infiltrated the renal medulla in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubules. In the CLP model, infiltrating macrophages dominated the outer cortical signature and Mdk was identified as a corresponding chemotactic factor. The regional distribution of these immune cells was validated with multiplexed CO-Detection by inDEXing (CODEX) immunofluorescence. Spatial transcriptomic sequencing complements single cell sequencing by uncovering mechanisms driving immune cell infiltration and detection of relevant cell subpopulations.

The innate immune system is the first line of defense in response to nonself and danger signals from microbial invasion or tissue injury. It is increasingly recognized that each organ uses unique sets of cells and molecules that orchestrate regional innate immunity. The cells that execute the task of innate immunity are many and consist of not only "professional" immune cells but also nonimmune cells, such as renal epithelial cells. Despite a high level of sophistication, deregulated innate immunity is common and contributes to a wide range of renal diseases, such as sepsis-induced kidney injury, GN, and allograft dysfunction. This review discusses how the innate immune system recognizes and responds to nonself and danger signals. In particular, the roles of renal epithelial cells that make them an integral part of the innate immune apparatus of the kidney are highlighted.

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.

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 2/2 ) 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-a in wild-type mice mimicked the observations in p53 2/2 mice. Chimeric mice that lacked p53 in leukocytes sustained injury similar to p53 2/2 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 2/2 and pifithrin-a-treated mice after ischemic injury were the anti-inflammatory M2 phenotype. Ischemic kidneys of p53 2/2 and pifithrin-a-treated mice also showed reduced expression of Kruppel-like factor-4. Finally, models of peritonitis in p53 2/2 and pifithrin-a-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. Human AKI is a grave clinical condition frequently seen in the setting of nephrotoxic insults, sepsis, and hemodynamic compromise. Despite decades of clinical and laboratory studies, there are no established therapeutic modalities that are known to alter the course of AKI. Consequently, treatment for AKI to date is supportive and focused on the maintenance of body homeostasis through the control of fluids and electrolytes with modalities as aggressive as dialysis. 1,2 In addition, human AKI unfortunately carries significant morbidity and mortality and can frequently result in long-term loss of function, especially when superimposed on preexisting kidney disease. [3][4][5] Renal artery or pedicle clamp models of ischemiareperfusion injury (IRI) in mice and rats have primarily been utilized to study the pathophysiologic pathways involved in ischemic AKI. The emerging picture of this pathophysiology is that of a complex and often interrelated array of signaling events triggered primarily by hypoxia, reactive oxygen and nitrogen species, and nucleotide depletion. These triggering events in turn can activate or suppress a myriad of secondary effectors such as transcription factors, chemokines, and cytokines. 1 The ultimate result is tubular cell death in the form of apoptosis and necrosis, endothelial alterations, and infiltration of inflammatory cells. The final phenotype of AKI is likely dependent on the particular injury model used and possibly the animal species under investigation.

Sepsis is a dynamic state that progresses at variable rates and has life-threatening consequences. Staging patients along the sepsis timeline requires a thorough knowledge of the evolution of cellular and molecular events at the tissue level. Here, we investigated the kidney, an organ central to the pathophysiology of sepsis. Single-cell RNA-sequencing in a murine endotoxemia model revealed the involvement of various cell populations to be temporally organized and highly orchestrated. Endothelial and stromal cells were the first responders. At later time points, epithelial cells upregulated immune-related pathways while concomitantly downregulating physiological functions such as solute homeostasis. Sixteen hours after endotoxin, there was global cell–cell communication failure and organ shutdown. Despite this apparent organ paralysis, upstream regulatory analysis showed significant activity in pathways involved in healing and recovery. This rigorous spatial and temporal definition of murine endotoxemia will uncover precise biomarkers and targets that can help stage and treat human sepsis.

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 ironhandling 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. 26: 134726: -136226: , 201526: . doi: 10.1681 Gram-negative sepsis is a formidable and challenging clinical condition that carries very high morbidity and mortality. Indeed, it is estimated that mortality from sepsis can exceed 70%, especially when complicated by organ failure. 1 AKI is frequently seen in patients with sepsis and dramatically increases morbidity and mortality. To date, treatment of sepsis and sepsis-induced AKI remains supportive and relies primarily on antibiotic therapy, fluid and electrolyte management, and hemodynamic support. 2 One hallmark of sepsis is widespread tissue oxidative stress. Uncontrolled reactive oxygen species rapidly react with all biologic macromolecules and result in deleterious tissue damage, including organ failure. We and others have shown that the endotoxin (LPS) receptor Tolllike receptor 4 (TLR4) and coreceptor CD14 are expressed in renal tubules and upregulated in sepsis and ischemia. [3][4][5][6][7][8] We also showed that systemic endotoxin is freely filtered and interacts with S1 J Am Soc Nephrol

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 2/2 mice. Indeed, neutralization of IL-17 in THP 2/2 mice completely reversed the systemic neutrophilia. Furthermore, IL-23 was also elevated in THP 2/2 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.