Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure 1 . AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury 1 . This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized 1,2 . Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules 1,2 , the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types 3 . Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely 1,2 . However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals 4,5 . The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish 6 . This provides a unique opportunity to image cell and molecular changes in realtime, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish 7,8 . However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell ...

Various techniques have been utilized historically to generate acute pulmonary inflammation in the murine system. Crystalline silica exposure results in acute inflammation followed by pulmonary fibrosis. Methods of exposure are varied in their techniques, as well as types of anesthesia. Therefore, the current study sought to compare the effects of two major anesthesia (isoflurane and ketamine) and three routes of instillation, intranasal (IN), intratracheal (IT), and trans-oral (TO), on markers of inflammation. Mice were anesthetized with isoflurane or ketamine and instilled IN with silica or phosphate buffered saline (PBS). Mice were sacrificed and lavaged after three days. To assess inflammation, alveolar cells were assessed by cytospin and lavage fluid was analyzed for inflammatory cytokines and total protein. While all parameters were increased in silica-exposed groups, regardless of anesthesia type, there was significant increase in neutrophils and total protein in mice anesthetized with ketamine, compared to isoflurane. In comparing instillation techniques, mice were anesthetized with isoflurane and instilled IN, IT, or TO with silica. Increases were observed in all parameters, except tumor necrosis factor-α, following IT silica instillation as compared to the IN and TO instillation groups. In addition, fluorescent microsphere uptake by alveolar macrophages supported the notion that all methods of instillation were uniform, but IT had significantly greater dispersion. Taken together, these data show that each method of exposure tested, generated significant inflammation among the silica groups, and any differences in parameters or techniques should be taken into consideration when developing an animal model to study pulmonary diseases.

Dietary changes associated with industrialization substantially increase the prevalence of chronic diseases, such as obesity, type II diabetes, and cardiovascular disease, major contributors to the public health burden. The high prevalence of these chronic diseases is often attributed to an 'evolutionary mismatch' between human physiology and modern nutritional environments. Western diets enriched with foods that were scarce throughout human evolutionary history (e.g., simple sugars and saturated fats) promote inflammation and disease relative to diets more akin to ancestral human hunter-gatherer diets, such as a Mediterranean diet. Peripheral blood monocytes, precursors to macrophages and important mediators of innate immunity and inflammation, are sensitive to the environment and may represent a critical intermediate in the pathway linking diet to disease. We evaluated the effects of 15 months of whole diet manipulations mimicking human Western or Mediterranean diet patterns on monocyte polarization using a well-established model of human health, the cynomolgus macaque (Macaca fascicularis). Monocyte transcriptional profiles differed markedly between the two diets, with 40% of transcripts showing differential expression (FDR < 0.05). Monocytes from Western diet consumers were polarized toward a more proinflammatory phenotype. Compared to the Mediterranean diet, the Western diet shifted the co-expression of 445 gene pairs, including small RNAs and transcription factors associated with metabolism and adiposity in humans, and dramatically altered behavior. For example, Western-fed individuals were more anxious and less socially integrated compared to the Mediterranean-fed subjects. These behavioral changes were also associated with some of the effects of diet on gene expression, suggesting an interaction between diet, central nervous system activity, and monocyte gene expression. The results of this study provide new insights into evolutionary mismatch at the molecular level and uncover new pathways through which Western diets alter monocyte polarization toward a proinflammatory phenotype.

Dietary composition is associated with the differential prevalence of psychiatric disorders; the Western diet confers increased risk, while the Mediterranean diet appears to reduce risk. In nonhuman primates, anxiety-like behaviors and social isolation have been linked to both Western diet consumption and increased inflammatory disease risk, and recent evidence suggests that diet composition may affect immune system function in part through its effects on behavior. This is particularly important in the context of the global COVID-19 pandemic in which social isolation has been associated with disease. Here, we examined the effects of Western- and Mediterranean-like diets on social behavior in a randomized, 34-month preclinical trial in middle-aged female cynomolgus macaques (Macaca fascicularis). Diet induced rapid and persistent changes in a suite of behaviors. After just three months of experimental diet consumption, a composite measure of diet-altered behavior (DAB) significantly differed between the two diets (p = 0.014) and remained different throughout the 24-month experimental observation period (p = 2.2 × 10−8). Monkeys fed the Western diet spent more time alone (FDR = 4.4 × 10−5) and displayed more anxiety behavior (FDR = 0.048), whereas monkeys fed the Mediterranean diet spent more time resting (FDR = 0.0013), attentive (FDR = 0.017), and in body contact with groupmates (FDR = 4.1 × 10−8). These differences were largely due to changes in behavior of animals fed the Mediterranean diet, while Western-diet-fed-animals exhibited similar behaviors compared to the eight-month baseline period, during which all monkeys consumed a common laboratory diet. These observations provide experimental support in a nonhuman primate model, demonstrating a potential therapeutic benefit of the Mediterranean diet consumption to reduce social isolation and anxiety and thus mitigate social isolation-associated disorders that often accompany illness and disability.