Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and Genetically Encoded Tool Delivery in the Central and Peripheral Nervous Systems.

CRISPR/Cas9 gene editing represents an exciting avenue to study genes of unknown function and can be combined with genetically encoded tools such as fluorescent proteins, channelrhodopsins, DREADDs, and various biosensors to more deeply probe the function of these genes in different cell types. However, current strategies to also manipulate or visualize edited cells are challenging due to the large size of Cas9 proteins and the limited packaging capacity of adeno-associated viruses (AAVs). To overcome these constraints, we developed an alternative gene editing strategy using a single AAV vector and mouse lines that express Cre-dependent Cas9 to achieve efficient cell-type specific editing across the nervous system. Expressing Cre-dependent Cas9 from a genomic locus affords space to package guide RNAs for gene editing together with Cre-dependent, genetically encoded tools to manipulate, map, or monitor neurons using a single virus. We validated this strategy with three common tools in neuroscience: ChRonos, a channelrhodopsin, for studying synaptic transmission using optogenetics, GCaMP8f for recording Ca2+ transients using photometry, and mCherry for tracing axonal projections. We tested these tools in multiple brain regions and cell types, including GABAergic neurons in the nucleus accumbens, glutamatergic neurons projecting from the ventral pallidum to the lateral habenula, dopaminergic neurons in the ventral tegmental area, and proprioceptive neurons in the periphery. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection.

Transcriptomic landscape of mammalian ventral pallidum at single-cell resolution.

The ventral pallidum (VP) is critical for motivated behaviors. While contemporary work has begun to elucidate the functional diversity of VP neurons, the molecular heterogeneity underlying this functional diversity remains incompletely understood. We used snRNA-seq and in situ hybridization to define the transcriptional taxonomy of VP cell types in mice, macaques, and baboons. We found transcriptional conservation between all three species, within the broader neurochemical cell types. Unique dopaminoceptive and cholinergic subclusters were identified and conserved across both primate species but had no homolog in mice. This harmonized consensus VP cellular atlas will pave the way for understanding the structure and function of the VP and identified key neuropeptides, neurotransmitters, and neuro receptors that could be targeted within specific VP cell types for functional investigations.

Projection-TAGs enable multiplex projection tracing and multi-modal profiling of projection neurons.

Single-cell multiomic techniques have sparked immense interest in developing a comprehensive multi-modal map of diverse neuronal cell types and their brain wide projections. However, investigating the spatial organization, transcriptional and epigenetic landscapes of brain wide projection neurons is hampered by the lack of efficient and easily adoptable tools. Here we introduce Projection-TAGs, a retrograde AAV platform that allows multiplex tagging of projection neurons using RNA barcodes. By using Projection-TAGs, we performed multiplex projection tracing of the mouse cortex and high-throughput single-cell profiling of the transcriptional and epigenetic landscapes of the cortical projection neurons. Projection-TAGs can be leveraged to obtain a snapshot of activity-dependent recruitment of distinct projection neurons and their molecular features in the context of a specific stimulus. Given its flexibility, usability, and compatibility, we envision that Projection-TAGs can be readily applied to build a comprehensive multi-modal map of brain neuronal cell types and their projections.

Cell specific single viral vector CRISPR/Cas9 editing and genetically encoded tool delivery in the central and peripheral nervous systems.

Gene manipulation strategies using germline knockout, conditional knockout, and more recently CRISPR/Cas9 are crucial tools for advancing our understanding of the nervous system. However, traditional gene knockout approaches can be costly and time consuming, may lack cell-type specificity, and can induce germline recombination. Viral gene editing presents and an exciting alternative to more rapidly study genes of unknown function; however, current strategies to also manipulate or visualize edited cells are challenging due to the large size of Cas9 proteins and the limited packaging capacity of adeno-associated viruses (AAVs). To overcome these constraints, we have developed an alternative gene editing strategy using a single AAV vector and mouse lines that express Cre-dependent Cas9 to achieve efficient cell-type specific editing across the nervous system. Expressing Cre-dependent Cas9 in specific cell types in transgenic mouse lines affords more space to package guide RNAs for gene editing together with Cre-dependent, genetically encoded tools to manipulate, map, or monitor neurons using a single virus. We validated this strategy with three commonly used tools in neuroscience: ChRonos, a channelrhodopsin, for manipulating synaptic transmission using optogenetics; GCaMP8f for recording Ca2+ transients using fiber photometry, and mCherry for anatomical tracing of axonal projections. We tested these tools in multiple brain regions and cell types, including GABAergic neurons in the nucleus accumbens (NAc), glutamatergic neurons projecting from the ventral pallidum (VP) to the lateral habenula (LHb), dopaminergic neurons in the ventral tegmental area (VTA), and parvalbumin (PV)-positive proprioceptive neurons in the periphery. This flexible approach should be useful to identify novel genes that affect synaptic transmission, circuit activity, or morphology with a single viral injection.

Muscle-Specific Ablation of Glucose Transporter 1 (GLUT1) Does Not Impair Basal or Overload-Stimulated Skeletal Muscle Glucose Uptake.

Glucose transporter 1 (GLUT1) is believed to solely mediate basal (insulin-independent) glucose uptake in skeletal muscle; yet recent work has demonstrated that mechanical overload, a model of resistance exercise training, increases muscle GLUT1 levels. The primary objective of this study was to determine if GLUT1 is necessary for basal or overload-stimulated muscle glucose uptake. Muscle-specific GLUT1 knockout (mGLUT1KO) mice were generated and examined for changes in body weight, body composition, metabolism, systemic glucose regulation, muscle glucose transporters, and muscle [3H]-2-deoxyglucose uptake ± the GLUT1 inhibitor BAY-876. [3H]-hexose uptake ± BAY-876 was also examined in HEK293 cells-expressing GLUT1-6 or GLUT10. mGLUT1KO mice exhibited no impairments in body weight, lean mass, whole body metabolism, glucose tolerance, basal or overload-stimulated muscle glucose uptake. There was no compensation by the insulin-responsive GLUT4. In mGLUT1KO mouse muscles, overload stimulated higher expression of mechanosensitive GLUT6, but not GLUT3 or GLUT10. In control and mGLUT1KO mouse muscles, 0.05 µM BAY-876 impaired overload-stimulated, but not basal glucose uptake. In the GLUT-HEK293 cells, BAY-876 inhibited glucose uptake via GLUT1, GLUT3, GLUT4, GLUT6, and GLUT10. Collectively, these findings demonstrate that GLUT1 does not mediate basal muscle glucose uptake and suggest that a novel glucose transport mechanism mediates overload-stimulated glucose uptake.

Lactotrehalose, an Analog of Trehalose, Increases Energy Metabolism Without Promoting Clostridioides difficile Infection in Mice.

BACKGROUND & AIMS: Trehalose is a disaccharide that might be used in the treatment of cardiometabolic diseases. However, trehalose consumption promotes the expansion of Clostridioides difficile ribotypes that metabolize trehalose via trehalose-6-phosphate hydrolase. Furthermore, brush border and renal trehalases can reduce the efficacy of trehalose by cleaving it into monosaccharides. We investigated whether a trehalase-resistant analogue of trehalose (lactotrehalose) has the same metabolic effects of trehalose without expanding C difficile. METHODS: We performed studies with HEK293 and Caco2 cells, primary hepatocytes from mice, and human intestinal organoids. Glucose transporters were overexpressed in HEK293 cells, and glucose tra2nsport was quantified. Primary hepatocytes were cultured with or without trehalose or lactotrehalose, and gene expression patterns were analyzed. C57B6/J mice were given oral antibiotics and trehalose or lactotrehalose in drinking water, or only water (control), followed by gavage with the virulent C difficile ribotype 027 (CD027); fecal samples were analyzed for toxins A (ToxA) or B (ToxB) by enzyme-linked immunosorbent assay. Other mice were given trehalose or lactotrehalose in drinking water for 2 days before placement on a chow or 60% fructose diet for 10 days. Liver tissues were collected and analyzed by histologic, serum biochemical, RNA sequencing, autophagic flux, and thermogenesis analyses. We quantified portal trehalose and lactotrehalose bioavailability by gas chromatography mass spectrometry. Fecal microbiomes were analyzed by 16S ribosomal RNA sequencing and principal component analyses. RESULTS: Lactotrehalose and trehalose each blocked glucose transport in HEK293 cells and induced a gene expression pattern associated with fasting in primary hepatocytes. Compared with mice on the chow diet, mice on the high-fructose diet had increased circulating cholesterol, higher ratios of liver weight-to-body weight, hepatic lipid accumulation (steatosis), and liver gene expression patterns of carbohydrate-responsive de novo lipogenesis. Mice given lactotrehalose while on the high-fructose diet did not develop any of these features and had increased whole-body caloric expenditure compared with mice given trehalose or water and fed a high-fructose diet. Livers from mice given lactotrehalose had increased transcription of genes that regulate mitochondrial energy metabolism compared with liver from mice given trehalose or controls. Lactotrehalose was bioavailable in venous and portal circulation and fecal samples. Lactotrehalose reduced fecal markers of microbial branched-chain amino acid biosynthesis and increased expression of microbial genes that regulate insulin signaling. In mice given antibiotics followed by CD027, neither lactotrehalose nor trehalose increased levels of the bacteria or its toxin in stool-in fact, trehalose reduced the abundance of CD027 in stool. Lactotrehalose and trehalose reduced markers of inflammation in rectal tissue after CD027 infection. CONCLUSIONS: Lactotrehalose is a trehalase-resistant analogue that increases metabolic parameters, compared with trehalose, without increasing the abundance or virulence of C difficile strain CD027. Trehalase-resistant trehalose analogues might be developed as next-generation fasting-mimetics for the treatment of diabetes and nonalcoholic fatty liver disease.

Identification of druggable small molecule antagonists of the Plasmodium falciparum hexose transporter PfHT and assessment of ligand access to the glucose permeation pathway via FLAG-mediated protein engineering.

Although the Plasmodium falciparum hexose transporter PfHT has emerged as a promising target for anti-malarial therapy, previously identified small-molecule inhibitors have lacked promising drug-like structural features necessary for development as clinical therapeutics. Taking advantage of emerging insight into structure/function relationships in homologous facilitative hexose transporters and our novel high throughput screening platform, we investigated the ability of compounds satisfying Lipinksi rules for drug likeness to directly interact and inhibit PfHT. The Maybridge HitFinder chemical library was interrogated by searching for compounds that reduce intracellular glucose by >40% at 10 µM. Testing of initial hits via measurement of 2-deoxyglucose (2-DG) uptake in PfHT over-expressing cell lines identified 6 structurally unique glucose transport inhibitors. WU-1 (3-(2,6-dichlorophenyl)-5-methyl-N-[2-(4-methylbenzenesulfonyl)ethyl]-1,2-oxazole-4-carboxamide) blocked 2-DG uptake (IC50 = 5.8 ± 0.6 µM) with minimal effect on the human orthologue class I (GLUTs 1-4), class II (GLUT8) and class III (GLUT5) facilitative glucose transporters. WU-1 showed comparable potency in blocking 2-DG uptake in freed parasites and inhibiting parasite growth, with an IC50 of 6.1 ± 0.8 µM and EC50 of 5.5 ± 0.6 µM, respectively. WU-1 also directly competed for N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) binding and inhibited the transport of D-glucose with an IC50 of 5.9 ± 0.8 µM in liposomes containing purified PfHT. Kinetic analysis revealed that WU-1 acts as a non-competitive inhibitor of zero-trans D-fructose uptake. Decreased potency for WU-1 and the known endofacial ligand cytochalasin B was observed when PfHT was engineered to contain an N-terminal FLAG tag. This modification resulted in a concomitant increase in affinity for 4,6-O-ethylidene-α-D-glucose, an exofacially directed transport antagonist, but did not alter the Km for 2-DG. Taken together, these data are consistent with a model in which WU-1 binds preferentially to the transporter in an inward open conformation and support the feasibility of developing potent and selective PfHT antagonists as a novel class of anti-malarial drugs.

Metabolic and Cardiac Adaptation to Chronic Pharmacologic Blockade of Facilitative Glucose Transport in Murine Dilated Cardiomyopathy and Myocardial Ischemia.

GLUT transgenic and knockout mice have provided valuable insight into the role of facilitative glucose transporters (GLUTs) in cardiovascular and metabolic disease, but compensatory physiological changes can hinder interpretation of these models. To determine whether adaptations occur in response to GLUT inhibition in the failing adult heart, we chronically treated TG9 mice, a transgenic model of dilated cardiomyopathy and heart failure, with the GLUT inhibitor ritonavir. Glucose tolerance was significantly improved with chronic treatment and correlated with decreased adipose tissue retinol binding protein 4 (RBP4) and resistin. A modest improvement in lifespan was associated with decreased cardiomyocyte brain natriuretic peptide (BNP) expression, a marker of heart failure severity. GLUT1 and -12 protein expression was significantly increased in left ventricular (LV) myocardium in ritonavir-treated animals. Supporting a switch from fatty acid to glucose utilization in these tissues, fatty acid transporter CD36 and fatty acid transcriptional regulator peroxisome proliferator-activated receptor α (PPARα) mRNA were also decreased in LV and soleus muscle. Chronic ritonavir also increased cardiac output and dV/dt-d in C57Bl/6 mice following ischemia-reperfusion injury. Taken together, these data demonstrate compensatory metabolic adaptation in response to chronic GLUT blockade as a means to evade deleterious changes in the failing heart.

Evaluating the Efficacy of GLUT Inhibitors Using a Seahorse Extracellular Flux Analyzer.

Glucose is metabolized through anaerobic glycolysis and aerobic oxidative phosphorylation (OXPHOS). Perturbing glucose uptake and its subsequent metabolism can alter both glycolytic and OXPHOS pathways and consequently lactate and/or oxygen consumption. Production and secretion of lactate, as a consequence of glycolysis, leads to acidification of the extracellular medium. Molecular oxygen is the final electron acceptor in the electron transport chain, facilitating oxidative phosphorylation of ADP to ATP. The alterations in extracellular acidification and/or oxygen consumption can thus be used as indirect readouts of glucose metabolism and assessing the impact of inhibiting glucose transport through specific glucose transporters (GLUTs). The Seahorse bioenergetics analyzer can measure both the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The proposed methodology affords a robust, high-throughput method to screen for GLUT inhibition in cells engineered to express specific GLUTs, providing live cell read-outs upon GLUT inhibition.

Development of GLUT4-selective antagonists for multiple myeloma therapy.

Cancer cells consume more glucose to fuel metabolic programs fundamental to sustaining their survival, growth and proliferation. Among the fourteen SLC2A family members, GLUTs 1 and 4 are high-affinity glucose transporters. GLUT4 (SLC2A4) is highly expressed in muscle and adipose tissue. Basally retained within the cell, GLUT4 traffics to the plasma membrane (PM) in response to insulin and exercise-stimulation. The plasma cell malignancy multiple myeloma (MM) exhibits increased constitutive expression of GLUT4 on the PM, co-opting use of GLUT4 for survival and proliferation. GLUT4 inhibition by knockdown or treatment with the FDA-approved HIV protease inhibitor ritonavir leads to cytostatic and/or cytotoxic and chemosensitizing effects in tumor cells both in vitro and in vivo. We recently reported our generation of GLUT4 homology models and virtual high-throughput screening (vHTS) to identify multiple series of novel GLUT4 antagonists. In this report, we describe our initial hit-to-lead optimization to synthesize new analogs with improved potency and selectivity for GLUT4, and the biological characterization of these compounds in a variety of assays. We show that our lead compound (compound 20) decreases glucose uptake and cell proliferation as well as inhibits the expression of pro-survival MCL-1 in MM similar to the effect observed via knockdown of GLUT4 expression. Compound 20 is also effective at chemosensitizing multiple myeloma cell lines and patient samples to venetoclax, dexamethasone and melphalan. In sum, we report development of selective GLUT4 inhibitors lacking inhibitory activity against GLUT1 and GLUT8. We show that selective pharmacological inhibition of GLUT4 is feasible and this may represent a novel strategy for the treatment and chemosensitization of multiple myeloma to standard therapeutics.

SLC2A8 (GLUT8) is a mammalian trehalose transporter required for trehalose-induced autophagy.

Trehalose is a disaccharide demonstrated to mitigate disease burden in multiple murine neurodegenerative models. We recently revealed that trehalose rapidly induces hepatic autophagy and abrogates hepatic steatosis by inhibiting hexose transport via the SLC2A family of facilitative transporters. Prior studies, however, postulate that intracellular trehalose is sufficient to induce cellular autophagy. The objective of the current study was to identify the means by which trehalose accesses the hepatocyte cytoplasm, and define the distal signaling mechanisms by which trehalose induces autophagy. We provide gas chromatographic/mass spectrometric, fluorescence microscopic and radiolabeled uptake evidence that trehalose traverses the plasma membrane via SLC2A8 (GLUT8), a homolog of the trehalose transporter-1 (Tret1). Moreover, GLUT8-deficient hepatocytes and GLUT8-deficient mice exposed to trehalose resisted trehalose-induced AMP-activated protein kinase (AMPK) phosphorylation and autophagic induction in vitro and in vivo. Although trehalose profoundly attenuated mTORC1 signaling, trehalose-induced mTORC1 suppression was insufficient to activate autophagy in the absence of AMPK or GLUT8. Strikingly, transient, heterologous Tret1 overexpression reconstituted autophagic flux and AMPK signaling defects in GLUT8-deficient hepatocyte cultures. Together, these data suggest that cytoplasmic trehalose access is carrier-mediated, and that GLUT8 is a mammalian trehalose transporter required for hepatocyte trehalose-induced autophagy and signal transduction.

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters.

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z' factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC50) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening.

Trehalose inhibits solute carrier 2A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis.

Trehalose is a naturally occurring disaccharide that has gained attention for its ability to induce cellular autophagy and mitigate diseases related to pathological protein aggregation. Despite decades of ubiquitous use as a nutraceutical, preservative, and humectant, its mechanism of action remains elusive. We showed that trehalose inhibited members of the SLC2A (also known as GLUT) family of glucose transporters. Trehalose-mediated inhibition of glucose transport induced AMPK (adenosine 5'-monophosphate-activated protein kinase)-dependent autophagy and regression of hepatic steatosis in vivo and a reduction in the accumulation of lipid droplets in primary murine hepatocyte cultures. Our data indicated that trehalose triggers beneficial cellular autophagy by inhibiting glucose transport.

A mouse model of Townes-Brocks syndrome expressing a truncated mutant Sall1 protein is protected from acute kidney injury.

It has been postulated that developmental pathways are reutilized during repair and regeneration after injury, but functional analysis of many genes required for kidney formation has not been performed in the adult organ. Mutations in SALL1 cause Townes-Brocks syndrome (TBS) and nonsyndromic congenital anomalies of the kidney and urinary tract, both of which lead to childhood kidney failure. Sall1 is a transcriptional regulator that is expressed in renal progenitor cells and developing nephrons in the embryo. However, its role in the adult kidney has not been investigated. Using a mouse model of TBS (Sall1TBS), we investigated the role of Sall1 in response to acute kidney injury. Our studies revealed that Sall1 is expressed in terminally differentiated renal epithelia, including the S3 segment of the proximal tubule, in the mature kidney. Sall1TBS mice exhibited significant protection from ischemia-reperfusion injury and aristolochic acid-induced nephrotoxicity. This protection from acute injury is seen despite the presence of slowly progressive chronic kidney disease in Sall1TBS mice. Mice containing null alleles of Sall1 are not protected from acute kidney injury, indicating that expression of a truncated mutant protein from the Sall1TBS allele, while causative of congenital anomalies, protects the adult kidney from injury. Our studies further revealed that basal levels of the preconditioning factor heme oxygenase-1 are elevated in Sall1TBS kidneys, suggesting a mechanism for the relative resistance to injury in this model. Together, these studies establish a functional role for Sall1 in the response of the adult kidney to acute injury.

The Glucose Transporter PfHT1 Is an Antimalarial Target of the HIV Protease Inhibitor Lopinavir.

Malaria and HIV infection are coendemic in a large portion of the world and remain a major cause of morbidity and mortality. Growing resistance of Plasmodium species to existing therapies has increased the need for new therapeutic approaches. The Plasmodium glucose transporter PfHT is known to be essential for parasite growth and survival. We have previously shown that HIV protease inhibitors (PIs) act as antagonists of mammalian glucose transporters. While the PI lopinavir is known to have antimalarial activity, the mechanism of action is unknown. We report here that lopinavir blocks glucose uptake into isolated malaria parasites at therapeutically relevant drug levels. Malaria parasites depend on a constant supply of glucose as their primary source of energy, and decreasing the available concentration of glucose leads to parasite death. We identified the malarial glucose transporter PfHT as a target for inhibition by lopinavir that leads to parasite death. This discovery provides a mechanistic basis for the antimalarial effect of lopinavir and provides a direct target for novel drug design with utility beyond the HIV-infected population.

In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy.

Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.

The role of tumor necrosis factor alpha in regulating the expression of Tamm-Horsfall Protein (uromodulin) in thick ascending limbs during kidney injury.

BACKGROUND: Tamm-Horsfall Protein (THP) is a glycoprotein expressed exclusively by cells of the thick ascending loop (TAL) of Henle. THP has a protective role in acute kidney injury (AKI), and its expression is downregulated in the early stages of injury. Tumor necrosis factor alpha (TNFα) is a cytokine endogenously expressed by the TAL and is also induced by AKI. Therefore, we hypothesized that TNFα is a key regulator of THP expression. METHODS: We used a mouse model of AKI (ischemia-reperfusion injury, IRI) and a cell culture system of a TAL cell line (MKTAL). RESULTS: We show that TNFα is upregulated by TAL cells early after AKI in vivo. The expression of THP and its transcription factor Hepatocyte nuclear factor 1ß (HNF1ß) were concomitantly decreased at the peak of injury. Furthermore, recombinant TNFα inhibits significantly, and in a dose-dependent manner, the expression of THP, but not HNF1ß in MKTAL cells. Interestingly, neither TNFα neutralization nor genetic deletion of TNFα increased THP or HNF levels after injury in vivo. CONCLUSION: Our data suggest that TNFα can inhibit the expression of THP in TAL cells via an HNF1ß-independent mechanism, but the downregulation of THP expression in the early AKI does not depend on TNFα. We propose that TNFα regulates THP expression in a homeostatic setting, but the impact of TNFα on THP during kidney injury is superseded by other factors that could inhibit HNF1ß-mediated expression of THP.

Tamm-Horsfall protein translocates to the basolateral domain of thick ascending limbs, interstitium, and circulation during recovery from acute kidney injury.

Tamm-Horsfall protein (THP) is a glycoprotein normally targeted to the apical membrane domain of the kidney's thick ascending limbs (TAL). We previously showed that THP of TAL confers protection to proximal tubules against acute kidney injury (AKI) via a possible cross talk between the two functionally distinct tubular segments. However, the extent, timing, specificity, and functional effects of basolateral translocation of THP during AKI remain unclear. Using an ischemia-reperfusion (IRI) model of murine AKI, we show here that, while THP expression in TAL is downregulated at the peak of injury, it is significantly upregulated 48 h after IRI. Confocal immunofluorescence and immunoelectron microscopy reveal a major redirection of THP during recovery from the apical membrane domain of TAL towards the basolateral domain, interstitium, and basal compartment of S3 segments. This corresponds with increased THP in the serum but not in the urine. The overall epithelial polarity of TAL cells does not change, as evidenced by correct apical targeting of Na(+)-K(+)-2Cl cotransporter (NKCC2) and basolateral targeting of Na(+)-K(+)-ATPase. Compared with the wild-type, THP(-/-) mice show a significantly delayed renal recovery after IRI, due possibly to reduced suppression by THP of proinflammatory cytokines and chemokines such as monocyte chemoattractant protein-1 during recovery. Taken together, our data suggest that THP redistribution in the TAL after AKI is a protein-specific event and its increased interstitial presence negatively regulates the evolving inflammatory signaling in neighboring proximal tubules, thereby enhancing kidney recovery. The increase of serum THP may be used as a prognostic biomarker for recovery from AKI.

A formulation-enabled preclinical efficacy assessment of a farnesoid X receptor agonist, GW4064, in hamsters and cynomolgus monkeys.

The farnesoid X receptor (FXR) belongs to one of the human nuclear receptor superfamilies that regulate gene transcription. FXR is widely expressed in liver, gall bladder, intestine, kidney, and adrenal glands. It serves as a key controller of bile acid homeostasis through its regulation of bile acid synthesis, conjugation, secretion, and absorption. FXR is also known to play a role in lipid regulation, triglyceride synthesis, and lipoprotein metabolism and clearance. We used a commercially available FXR agonist, GW4064, as a model compound to assess preclinical efficacy in two species (hamster and cynomolgus monkey). The crystalline GW4064, however, was found to have limited solubility, which resulted in poor oral bioavailability. This made it difficult to assess in vivo efficacy at the exposure levels desired. The physiochemical properties of GW4064 were assessed and both salt and self-emulsifying drug delivery system (SEDDS) formulation were developed and tested. The SEDDS formulation was found to greatly improve the oral bioavailability of GW4064, and permitted the evaluation of FXR agonist target efficacy.

Tamm-Horsfall protein-deficient thick ascending limbs promote injury to neighboring S3 segments in an MIP-2-dependent mechanism.

Tamm-Horsfall protein (THP) is a glycoprotein expressed exclusively in thick ascending limbs (TAL) of the kidney. We recently described a novel protective role of THP against acute kidney injury (AKI) via downregulation of inflammation in the outer medulla. Our current study investigates the mechanistic relationships among the status of THP, inflammation, and tubular injury. Using an ischemia-reperfusion model in wild-type and THP-/- mice, we demonstrate that it is the S3 proximal segments but not the THP-deficient TAL that are the main targets of tubular injury during AKI. The injured S3 segments that are surrounded by neutrophils in THP-/- mice have marked overexpression of neutrophil chemoattractant MIP-2 compared with wild-type counterparts. Neutralizing macrophage inflammatory protein-2 (MIP-2) antibody rescues S3 segments from injury, decreases neutrophil infiltration, and improves kidney function in THP-/- mice. Furthermore, using immunofluorescence volumetric imaging of wild-type mouse kidneys, we show that ischemia alters the intracellular translocation of THP in the TAL cells by partially shifting it from its default apical surface domain to the basolateral domain, the latter being contiguous to the basolateral surface of S3 segments. Concomitant with this is the upregulation, in the basolateral surface of S3 segments, of the scavenger receptor SRB-1, a putative receptor for THP. We conclude that TAL affects the susceptibility of S3 segments to injury at least in part by regulating MIP-2 expression in a THP-dependent manner. Our findings raise the interesting possibility of a direct role of basolaterally released THP on regulating inflammation in S3 segments.