Spatiotemporal organisation of protein processing in the kidney.

The kidney regulates plasma protein levels by eliminating them from the circulation. Proteins filtered by glomeruli are endocytosed and degraded in the proximal tubule and defects in this process result in tubular proteinuria, an important clinical biomarker. However, the spatiotemporal organization of renal protein metabolism in vivo was previously unclear. Here, using functional probes and intravital microscopy, we track the fate of filtered proteins in real time in living mice, and map specialized processing to tubular structures with singular value decomposition analysis and three-dimensional electron microscopy. We reveal that degradation of proteins requires sequential, coordinated activity of distinct tubular sub-segments, each adapted to specific tasks. Moreover, we leverage this approach to pinpoint the nature of endo-lysosomal disorders in disease models, and show that compensatory uptake in later regions of the proximal tubule limits urinary protein loss. This means that measurement of proteinuria likely underestimates severity of endocytotic defects in patients.

Neurogenic and pericytic plasticity of conditionally immortalized cells derived from renal erythropoietin-producing cells.

In adult mammals, the kidney is the main source of circulating erythropoietin (Epo), the master regulator of erythropoiesis. In vivo data in mice demonstrated multiple subtypes of interstitial renal Epo-producing (REP) cells. To analyze the differentiation plasticity of fibroblastoid REP cells, we used a transgenic REP cell reporter mouse model to generate conditionally immortalized REP-derived (REPD) cell lines. Under nonpermissive conditions, REPD cells ceased from proliferation and acquired a stem cell-like state, with strongly enhanced hypoxia-inducible factor 2 (HIF-2α), stem cell antigen 1 (SCA-1), and CD133 expression, but also enhanced alpha-smooth muscle actin (αSMA) expression, indicating myofibroblastic signaling. These cells maintained the "on-off" nature of Epo expression observed in REP cells in vivo, whereas other HIF target genes showed a more permanent regulation. Like REP cells in vivo, REPD cells cultured in vitro generated long tunneling nanotubes (TNTs) that aligned with endothelial vascular structures, were densely packed with mitochondria and became more numerous under hypoxic conditions. Although inhibition of mitochondrial oxygen consumption blunted HIF signaling, removal of the TNTs did not affect or even enhance the expression of HIF target genes. Apart from pericytes, REPD cells readily differentiated into neuroglia but not adipogenic, chondrogenic, or osteogenic lineages, consistent with a neuronal origin of at least a subpopulation of REP cells. In summary, these results suggest an unprecedented combination of differentiation features of this unique cell type.

Intravital kidney microscopy: entering a new era.

The development of intravital imaging with multiphoton microscopy has had a major impact on kidney research. It provides the unique opportunity to visualize dynamic behavior of cells and organelles in their native environment and to relate this to the complex 3-dimensional structure of the organ. Moreover, changes in cell/organelle function can be followed in real time in response to physiological interventions or disease-causing insults. However, realizing the enormous potential of this exciting approach has necessitated overcoming several substantial practical hurdles. In this article, we outline the nature of these challenges and how a variety of technical advances have provided effective solutions. In particular, improvements in laser/microscope technology, fluorescent probes, transgenic animals, and abdominal windows are collectively making previously opaque processes visible. Meanwhile, the rise of machine learning-based image analysis is facilitating the rapid generation of large amounts of quantitative data, amenable to deeper statistical interrogation. Taken together, the increased capabilities of multiphoton imaging are opening up huge new possibilities to study structure-function relationships in the kidney in unprecedented detail. In addition, they are yielding important new insights into cellular mechanisms of tissue damage, repair, and adaptive remodeling during disease states. Thus, intravital microscopy is truly entering an exciting new era in translational kidney research.

The iron chelator Deferasirox causes severe mitochondrial swelling without depolarization due to a specific effect on inner membrane permeability.

The iron chelator Deferasirox (DFX) causes severe toxicity in patients for reasons that were previously unexplained. Here, using the kidney as a clinically relevant in vivo model for toxicity together with a broad range of experimental techniques, including live cell imaging and in vitro biophysical models, we show that DFX causes partial uncoupling and dramatic swelling of mitochondria, but without depolarization or opening of the mitochondrial permeability transition pore. This effect is explained by an increase in inner mitochondrial membrane (IMM) permeability to protons, but not small molecules. The movement of water into mitochondria is prevented by altering intracellular osmotic gradients. Other clinically used iron chelators do not produce mitochondrial swelling. Thus, DFX causes organ toxicity due to an off-target effect on the IMM, which has major adverse consequences for mitochondrial volume regulation.

Overcoming Endocytosis Deficiency by Cubosome Nanocarriers.

The use of lipid-based nanoparticles for the delivery of biomacromolecules has attracted considerable attention due to the current interest in protein-based therapeutics. Cubosomes protect the incorporated therapeutics, which are susceptible to degradation by enzymes, thereby improving their bioavailability, and concomitantly enhance cellular uptake. The cubosome nanoparticles presented herein were loaded with bovine serum albumin (BSA) and characterized by small-angle X-ray scattering and dynamic light scattering techniques, while the BSA encapsulation and its release were evaluated in vitro. The ability of this formulation to increase the cellular uptake of albumin by 2-fold was tested on various types of renal tubular cells and confirmed by in vivo renal uptake experiments in mice. The obtained results show that cubosomes are able to deliver BSA inside the cell through distinct uptake and intracellular routing. These data were substantiated, with evidence of a high cubosome-mediated uptake of BSA in Clcn5 knockout mice characterized by defective receptor-mediated endocytosis. The use of cubosomes as a delivery system thus represents a promising approach to overcome the low endocytic uptake in diseased epithelial cells and to treat dysfunctions of the kidney proximal tubule.

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis.

BACKGROUND: The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown. METHODS: To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach. RESULTS: Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology. CONCLUSIONS: Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

The prostaglandin E2 receptor EP3 controls CC-chemokine ligand 2-mediated neuropathic pain induced by mechanical nerve damage.

Prostaglandin (PG) E2 is an important lipid mediator that is involved in several pathophysiological processes contributing to fever, inflammation, and pain. Previous studies have shown that early and continuous application of nonsteroidal anti-inflammatory drugs significantly reduces pain behavior in the spared nerve injury (SNI) model for trauma-induced neuropathic pain. However, the role of PGE2 and its receptors in the development and maintenance of neuropathic pain is incompletely understood but may help inform strategies for pain management. Here, we sought to define the nociceptive roles of the individual PGE2 receptors (EP1-4) in the SNI model using EP knockout mice. We found that PGE2 levels at the site of injury were increased and that the expression of the terminal synthase for PGE2, cytosolic PGE synthase was up-regulated in resident positive macrophages located within the damaged nerve. Only genetic deletion of the EP3 receptor affected nociceptive behavior and reduced the development of late-stage mechanical allodynia as well as recruitment of immune cells to the injured nerve. Importantly, EP3 activation induced the release of CC-chemokine ligand 2 (CCL2), and antagonists against the CCL2 receptor reduced mechanical allodynia in WT but not in EP3 knockout mice. We conclude that selective inhibition of EP3 might present a potential approach for reducing chronic neuropathic pain.

The targeted anti-oxidant MitoQ causes mitochondrial swelling and depolarization in kidney tissue.

Kidney proximal tubules (PTs) contain a high density of mitochondria, which are required to generate ATP to power solute transport. Mitochondrial dysfunction is implicated in the pathogenesis of numerous kidney diseases. Damaged mitochondria are thought to produce excess reactive oxygen species (ROS), which can lead to oxidative stress and activation of cell death pathways. MitoQ is a mitochondrial targeted anti-oxidant that has shown promise in preclinical models of renal diseases. However, recent studies in nonkidney cells have suggested that MitoQ might also have adverse effects. Here, using a live imaging approach, and both in vitro and ex vivo models, we show that MitoQ induces rapid swelling and depolarization of mitochondria in PT cells, but these effects were not observed with SS-31, another targeted anti-oxidant. MitoQ consists of a lipophilic cation (Tetraphenylphosphonium [TPP]) joined to an anti-oxidant component (quinone) by a 10-carbon alkyl chain, which is thought to insert into the inner mitochondrial membrane (IMM). We found that mitochondrial swelling and depolarization was also induced by dodecyltriphenylphosphomium (DTPP), which consists of TPP and the alkyl chain, but not by TPP alone. Surprisingly, MitoQ-induced mitochondrial swelling occurred in the absence of a decrease in oxygen consumption rate. We also found that DTPP directly increased the permeability of artificial liposomes with a cardiolipin content similar to that of the IMM. In summary, MitoQ causes mitochondrial swelling and depolarization in PT cells by a mechanism unrelated to anti-oxidant activity, most likely because of increased IMM permeability due to insertion of the alkyl chain.

New frontiers in intravital microscopy of the kidney.

PURPOSE OF REVIEW: Intravital imaging with multiphoton microscopy enables the detailed study of dynamic cellular processes within functioning organs in living animals, in ways that would not otherwise be possible. It therefore represents a powerful tool in translational kidney research. In this article, we will discuss several new technical developments that have significantly increased the capabilities of kidney imaging. RECENT FINDINGS: Important contemporary advances in biomedical imaging technology include longer wavelength excitation lasers, far-red emitting fluorescent reporters, highly sensitive detectors, fluorescence lifetime measurements, adaptive optics, microendoscopes, high-throughput automated analysis algorithms and tissue clearing techniques. Several recent studies have utilized intravital microscopy to gain valuable new insights into important physiological and pathophysiological processes in the kidney, such as renal handling of albumin and the cellular pathogenesis of acute kidney injury in sepsis. SUMMARY: Major technological advances are rapidly expanding the frontiers of intravital microscopy, which is likely to play an increasingly important role in preclinical kidney research in the coming years.

Mitochondria as therapeutic targets in acute kidney injury.

PURPOSE OF REVIEW: Mitochondria are complex intracellular organelles with a variety of important functions. The kidney tubule is densely packed with mitochondria, and mitochondrial dysfunction is thought to be central to the pathogenesis of acute kidney injury (AKI). Mitochondria therefore represent potential targets for novel therapeutic interventions in AKI. RECENT FINDINGS: Several mitochondrial targeted approaches have shown promise in recent preclinical studies of AKI, including measures to: reduce oxidative stress within mitochondria; prevent mitochondrial fission and activation of cell death pathways; enhance recycling of damaged mitochondria via autophagy and mitophagy; and accelerate mitochondrial biogenesis postinsult. SUMMARY: Recent studies show that it is now eminently feasible to pharmacologically manipulate various key aspects of mitochondrial biology in the kidney, and this has much potential for the future treatment of AKI. However, significant hurdles will have to be overcome in the translational pathway for these strategies to successfully migrate to the clinic.

Design and performance of an ultra-flexible two-photon microscope for in vivo research.

We present a cost-effective in vivo two-photon microscope with a highly flexible frontend for in vivo research. Our design ensures fast and reproducible access to the area of interest, including rotation of imaging plane, and maximizes space for auxiliary experimental equipment in the vicinity of the animal. Mechanical flexibility is achieved with large motorized linear stages that move the objective in the X, Y, and Z directions up to 130 mm. 360° rotation of the frontend (rotational freedom for one axis) is achieved with the combination of a motorized high precision bearing and gearing. Additionally, the modular design of the frontend, based on commercially available optomechanical parts, allows straightforward updates to future scanning technologies. The design exceeds the mobility of previous movable microscope designs while maintaining high optical performance.

Prostacyclin regulates spinal nociceptive processing through cyclic adenosine monophosphate-induced translocation of glutamate receptors.

BACKGROUND: Prostacyclin (PGI2) is known to be an important mediator of peripheral pain sensation (nociception) whereas little is known about its role in central sensitization. METHODS: The levels of the stable PGI2-metabolite 6-keto-prostaglandin F1α (6-keto-PGF1α) and of prostaglandin E2 (PGE2) were measured in the dorsal horn with the use of mass spectrometry after peripheral inflammation. Expression of the prostanoid receptors was determined by immunohistology. Effects of prostacyclin receptor (IP) activation on spinal neurons were investigated with biochemical assays (cyclic adenosine monophosphate-, glutamate release-measurement, Western blot analysis) in embryonic cultures and adult spinal cord. The specific IP antagonist Cay10441 was applied intrathecally after zymosan-induced mechanical hyperalgesia in vivo. RESULTS: Peripheral inflammation caused a significant increase of the stable PGI2 metabolite 6-keto-PGF1α in the dorsal horn of wild-type mice (n = 5). IP was located on spinal neurons and did not colocalize with the prostaglandin E2 receptors EP2 or EP4. The selective IP-agonist cicaprost increased cyclic adenosine monophosphate synthesis in spinal cultures from wild-type but not from IP-deficient mice (n = 5-10). The combination of fluorescence-resonance-energy transfer-based cyclic adenosine monophosphate imaging and calcium imaging showed a cicaprost-induced cyclic adenosine monophosphate synthesis in spinal cord neurons (n = 5-6). Fittingly, IP activation increased glutamate release from acute spinal cord sections of adult mice (n = 13-58). Cicaprost, but not agonists for EP2 and EP4, induced protein kinase A-dependent phosphorylation of the GluR1 subunit and its translocation to the membrane. Accordingly, intrathecal administration of the IP receptor antagonist Cay10441 had an antinociceptive effect (n = 8-11). CONCLUSION: Spinal prostacyclin synthesis during early inflammation causes the recruitment of GluR1 receptors to membrane fractions, thereby augmenting the onset of central sensitization.

BKCa channels expressed in sensory neurons modulate inflammatory pain in mice.

Large conductance calcium-activated potassium (BKCa) channels are important regulators of neuronal excitability. Although there is electrophysiological evidence for BKCa channel expression in sensory neurons, their in vivo functions in pain processing have not been fully defined. Using a specific antibody, we demonstrate here that BKCa channels are expressed in subpopulations of peptidergic and nonpeptidergic nociceptors. To test a functional association of BKCa channel activity in sensory neurons with particular pain modalities, we generated mice in which BKCa channels are ablated specifically from sensory neurons and analyzed their behavior in various models of pain. Mutant mice showed increased nociceptive behavior in models of persistent inflammatory pain. However, their behavior in models of neuropathic or acute nociceptive pain was normal. Moreover, systemic administration of the BKCa channel opener, NS1619, inhibited persistent inflammatory pain. Our investigations provide in vivo evidence that BKCa channels expressed in sensory neurons exert inhibitory control on sensory input in inflammatory pain states.

Prostacyclin mediates neuropathic pain through interleukin 1ß-expressing resident macrophages.

Prostacyclin is an important mediator of peripheral pain sensation. Here, we investigated its potential participation in mediating neuropathic pain and found that prostacyclin receptor (IP) knockout mice exhibited markedly decreased pain behavior. Application of an IP antagonist to the injury site or selective IP deficiency in myeloid cells mimicked the antinociceptive effect observed in IP knockout mice. At the site of nerve injury, IP was expressed in interleukin (IL) 1ß-containing resident macrophages, which were less common in IP knockout mice. Local administration of the IP agonist cicaprost inhibited macrophage migration in vitro and promoted accumulation of IP- and IL1ß-expressing cells as well as an increase of IL1ß concentrations at the application site in vivo. Fittingly, the IL1-receptor antagonist anakinra (IL-1ra) decreased neuropathic pain behavior in wild-type mice but not in IP knockout mice. Finally, continuous, but not single administration, of the cyclooxygenase inhibitor meloxicam early after nerve injury decreased pain behavior and the number of resident macrophages. Thus, early synthesis of prostacyclin at the site of injury causes accumulation of IL1ß-expressing macrophages as a key step in neuropathic pain after traumatic injury.

Synthesis of lipid mediators during UVB-induced inflammatory hyperalgesia in rats and mice.

Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.

Overdose of the histamine H3 inverse agonist pitolisant increases thermal pain thresholds.

OBJECTIVE AND DESIGN: Pitolisant (BF2.649) is a selective inverse agonist for the histamine H(3) receptor and was developed for the treatment of excessive daytime sleepiness in Parkinson disease, narcolepsy, and schizophrenia. Since H(3)-ligands can decrease inflammatory pain, we tested Pitolisant in inflammatory and neuropathic pain models. MATERIALS AND TREATMENTS: Behavioral effects of pitolisant and the structural different H(3) receptor inverse agonists ciproxifan and ST-889 were tested in zymosan-induced inflammation and the spared nerve injury model for neuropathic pain. METHODS: Responses to mechanical and thermal stimuli were determined. Calcium imaging was performed with primary neuronal cultures of dorsal root ganglions. RESULTS: Clinically relevant doses of pitolisant (10 mg/kg) had no relevant effect on mechanical or thermal pain thresholds in all animal models. Higher doses (50 mg/kg) dramatically increased thermal but not mechanical pain thresholds. Neither ciproxifan nor ST-889 altered thermal pain thresholds. In peripheral sensory neurons high concentrations of pitolisant (30-500 µM), but not ciproxifan, partially inhibited calcium increases induced by capsaicin, a selective activator of transient receptor potential vanilloid receptor 1 (TRPV1). High doses of pitolisant induced a strong hypothermia. CONCLUSION: The data show a dramatic effect of high dosages of pitolisant on the thermosensory system, which appears to be H(3) receptor-independent.

Anti-inflammatory role of microsomal prostaglandin E synthase-1 in a model of neuroinflammation.

A major immunological response during neuroinflammation is the activation of microglia, which subsequently release proinflammatory mediators such as prostaglandin E(2) (PGE(2)). Besides its proinflammatory properties, cyclooxygenase-2 (COX-2)-derived PGE(2) has been shown to exhibit anti-inflammatory effects on innate immune responses. Here, we investigated the role of microsomal PGE(2) synthase-1 (mPGES-1), which is functionally coupled to COX-2, in immune responses using a model of lipopolysaccharide (LPS)-induced spinal neuroinflammation. Interestingly, we found that activation of E-prostanoid (EP)2 and EP4 receptors, but not EP1, EP3, PGI(2) receptor (IP), thromboxane A(2) receptor (TP), PGD(2) receptor (DP), and PGF(2) receptor (FP), efficiently blocked LPS-induced tumor necrosis factor α (TNFα) synthesis and COX-2 and mPGES-1 induction as well as prostaglandin synthesis in spinal cultures. In vivo, spinal EP2 receptors were up-regulated in microglia in response to intrathecally injected LPS. Accordingly, LPS priming reduced spinal synthesis of TNFα, interleukin 1ß (IL-1ß), and prostaglandins in response to a second intrathecal LPS injection. Importantly, this reduction was only seen in wild-type but not in mPGES-1-deficient mice. Furthermore, intrathecal application of EP2 and EP4 agonists as well as genetic deletion of EP2 significantly reduced spinal TNFα and IL-1ß synthesis in mPGES-1 knock-out mice after LPS priming. These data suggest that initial inflammation prepares the spinal cord for a negative feedback regulation by mPGES-1-derived PGE(2) followed by EP2 activation, which limits the synthesis of inflammatory mediators during chronic inflammation. Thus, our data suggest a role of mPGES-1-derived PGE(2) in resolution of neuroinflammation.

Evaluation of a pre-treatment assessment to select mand topographies for functional communication training.

Recent research has suggested that variables related to specific mand topographies targeted during functional communication training (FCT) can affect treatment outcomes. These include effort, novelty of mands, previous relationships with problem behavior, and preference. However, there is little extant research on procedures for identifying which mand topographies to incorporate into FCT. In the current study, a mand topography assessment was conducted following functional analyses to identify the proficiency with which individuals used several different mand topographies. Two mand topographies (high and low proficiency) were then compared during FCT-based treatments. FCT was more effective when the mand topography identified as high proficiency was incorporated into FCT as compared to FCT that included the lower proficiency response. The results are discussed in terms of the need for individualized assessment procedures for selecting mand topographies that are targeted during FCT.