Carbon monoxide as a cellular protective agent in a swine model of cardiac arrest protocol.

Out-of-hospital cardiac arrest (OHCA) affects over 360,000 adults in the United States each year with a 50-80% mortality prior to reaching medical care. Despite aggressive supportive care and targeted temperature management (TTM), half of adults do not live to hospital discharge and nearly one-third of survivors have significant neurologic injury. The current treatment approach following cardiac arrest resuscitation consists primarily of supportive care and possible TTM. While these current treatments are commonly used, mortality remains high, and survivors often develop lasting neurologic and cardiac sequela well after resuscitation. Hence, there is a critical need for further therapeutic development of adjunctive therapies. While select therapeutics have been experimentally investigated, one promising agent that has shown benefit is CO. While CO has traditionally been thought of as a cellular poison, there is both experimental and clinical evidence that demonstrate benefit and safety in ischemia with lower doses related to improved cardiac/neurologic outcomes. While CO is well known for its poisonous effects, CO is a generated physiologically in cells through the breakdown of heme oxygenase (HO) enzymes and has potent antioxidant and anti-inflammatory activities. While CO has been studied in myocardial infarction itself, the role of CO in cardiac arrest and post-arrest care as a therapeutic is less defined. Currently, the standard of care for post-arrest patients consists primarily of supportive care and TTM. Despite current standard of care, the neurological prognosis following cardiac arrest and return of spontaneous circulation (ROSC) remains poor with patients often left with severe disability due to brain injury primarily affecting the cortex and hippocampus. Thus, investigations of novel therapies to mitigate post-arrest injury are clearly warranted. The primary objective of this proposed study is to combine our expertise in swine models of CO and cardiac arrest for future investigations on the cellular protective effects of low dose CO. We will combine our innovative multi-modal diagnostic platform to assess cerebral metabolism and changes in mitochondrial function in swine that undergo cardiac arrest with therapeutic application of CO.

The Rise in Tubular pH during Hypercalciuria Exacerbates Calcium Stone Formation.

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca2+ entry triggers a transepithelial Ca2+ flux to regulate proximal tubular (PT) luminal [Ca2+], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca2+ entry) inhibitors (Pyr10), Ca2+ entry by WTT cells was moderately inhibited, suggesting that the Ca2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Sex-specific Stone-forming Phenotype in Mice During Hypercalciuria/Urine Alkalinization.

Sex differences in kidney stone formation are well known. Females generally have slightly acidic blood and higher urine pH when compared with males, which makes them more vulnerable to calcium stone formation, yet the mechanism is still unclear. We aimed to examine the role of sex in stone formation during hypercalciuria and urine alkalinization through acetazolamide and calcium gluconate supplementation, respectively, for 4 weeks in wild-type (WT) and moderately hypercalciuric [TRPC3 knockout [KO](-/-)] male and female mice. Our goal was to develop calcium phosphate (CaP) and CaP+ calcium oxalate mixed stones in our animal model to understand the underlying sex-based mechanism of calcium nephrolithiasis. Our results from the analyses of mice urine, serum, and kidney tissues show that female mice (WT and KO) produce more urinary CaP crystals, higher [Ca2+], and pH in urine compared to their male counterparts. We identified a sex-based relationship of stone-forming phenotypes (types of stones) in our mice model following urine alkalization/calcium supplementation, and our findings suggest that female mice are more susceptible to CaP stones under those conditions. Calcification and fibrotic and inflammatory markers were elevated in treated female mice compared with their male counterparts, and more so in TRPC3 KO mice compared with their WT counterparts. Together these findings contribute to a mechanistic understanding of sex-influenced CaP and mixed stone formation that can be used as a basis for determining the factors in sex-related clinical studies.

Differential Interferon Signaling Regulation and Oxidative Stress Responses in the Cerebral Cortex and Cerebellum Could Account for the Spatiotemporal Pattern of Neurodegeneration in Niemann-Pick Disease Type C.

Niemann-Pick disease type C (NPC) is a fatal neurodegenerative condition caused by genetic mutations of the NPC1 or NPC2 genes that encode the NPC1 and NPC2 proteins, respectively, which are believed to be responsible for cholesterol efflux from late-endosomes/lysosomes. The pathogenic mechanisms that lead to neurodegeneration in NPC are not well understood. There are, however, well-defined spatiotemporal patterns of neurodegeneration that may provide insight into the pathogenic process. For example, the cerebellum is severely affected from early disease stages, compared with cerebral regions, which remain relatively spared until later stages. Using a genome-wide transcriptome analysis, we have recently identified an aberrant pattern of interferon activation in the cerebella of pre-symptomatic Npc1-/- mice. Here, we carried out a comparative transcriptomic analysis of cerebral cortices and cerebella of pre-symptomatic Npc1-/- mice and age-matched controls to identify differences that may help explain the pathological progression within the NPC brain. We report lower cerebral expression of genes within interferon signaling pathways, and significant differences in the regulation of oxidative stress, compared with the cerebellum. Our findings suggest that a delayed onset of interferon signaling, possibly linked to lower oxidative stress, may account for the slower onset of cerebral cortical pathology in the disease.

Investigation of Cerebral Mitochondrial Injury in a Porcine Survivor Model of Carbon Monoxide Poisoning.

INTRODUCTION: Carbon monoxide (CO) is a colorless and odorless gas that is a leading cause of environmental poisoning in the USA with substantial mortality and morbidity. The mechanism of CO poisoning is complex and includes hypoxia, inflammation, and leukocyte sequestration in brain microvessel segments leading to increased reactive oxygen species. Another important pathway is the effects of CO on the mitochondria, specifically at cytochrome c oxidase, also known as Complex IV (CIV). One of the glaring gaps is the lack of rigorous experimental models that may recapitulate survivors of acute CO poisoning in the early phase. The primary objective of this preliminary study is to use our advanced swine platform of acute CO poisoning to develop a clinically relevant survivor model to perform behavioral assessment and MRI imaging that will allow future development of biomarkers and therapeutics. METHODS: Four swine (10 kg) were divided into two groups: control (n = 2) and CO (n = 2). The CO group received CO at 2000 ppm for over 120 min followed by 30 min of re-oxygenation at room air for one swine and 150 min followed by 30 min of re-oxygenation for another swine. The two swine in the sham group received room air for 150 min. Cerebral microdialysis was performed to obtain semi real-time measurements of cerebral metabolic status. Following exposures, all surviving animals were observed for a 24-h period with neurobehavioral assessment and imaging. At the end of the 24-h period, fresh brain tissue (cortical and hippocampal) was immediately harvested to measure mitochondrial respiration. RESULTS: While a preliminary ongoing study, animals in the CO group showed alterations in cerebral metabolism and cellular function in the acute exposure phase with possible sustained mitochondrial changes 24 h after the CO exposure ended. CONCLUSIONS: This preliminary research further establishes a large animal swine model investigating survivors of CO poisoning to measure translational metrics relevant to clinical medicine that includes a basic neurobehavioral assessment and post exposure cellular measures.

Applications of near-infrared spectroscopy in neurocritical care.

Significance: Acute brain injuries are commonly encountered in the intensive care unit. Alterations in cerebrovascular physiology triggered by the initial insult can lead to neurological worsening, further brain injury, and poor outcomes. Robust methods for assessing cerebrovascular physiology continuously at the bedside are limited. Aim: In this review, we aim to assess the potential of near-infrared spectroscopy (NIRS) as a bedside tool to monitor cerebrovascular physiology in critically ill patients with acute brain injury as well as those who are at high risk for developing brain injury. Approach: We first review basic principles of cerebral blood flow regulation and how these are altered after brain injury. We then discuss the potential role for NIRS in different acute brain injuries. We pay specific attention to the potential for NIRS to (1) identify new brain injuries and clinical worsening, (2) non-invasively measure intracranial pressure (ICP) and cerebral autoregulation, and (3) identify optimal blood pressure (BP) targets that may improve patient outcomes. Results: A growing body of work supports the use of NIRS in the care of brain injured patients. NIRS is routinely used during cardiac surgeries to identify acute neurologic events, and there is some evidence that treatment algorithms using cerebral oximetry may result in improved outcomes. In acute brain injury, NIRS can be used to measure autoregulation to identify an "optimum" BP where autoregulation status is best preserved. Finally, NIRS has been utilized to identify oximetry thresholds that correlate with poor outcome as well as identify new focal intracranial hemorrhages. Conclusions: NIRS is emerging as a tool that can non-invasively measure brain function in critically ill patients. Future work will be aimed at technical refinements to improve diagnostic accuracy, as well as larger scale clinical trials that can establish a definitive impact on patient outcomes.

Exploring the Therapeutic Potential of Phosphorylated Cis-Tau Antibody in a Pig Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) results in the generation of tau. As hyperphosphorylated tau (p-tau) is one of the major consequences of TBI, targeting p-tau in TBI may lead to the development of new therapy. Twenty-five pigs underwent a controlled cortical impact. One hour after TBI, pigs were administered either vehicle (n = 13) or PNT001 (n = 12), a monoclonal antibody for the cis conformer of tau phosphorylated at threonine 231. Plasma biomarkers of neural injury were assessed for 14 days. Diffusion tensor imaging was performed at day 1 and 14 after injury, and these were compared to historical control animals (n = 4). The fractional anisotropy data showed significant white matter injury for groups at 1 day after injury in the corona radiata. At 14 days, the vehicle-treated pigs, but not the PNT001-treated animals, exhibited significant white matter injury compared to sham pigs in the ipsilateral corona radiata. The PNT001-treated pigs had significantly lower levels of plasma glial fibrillary acidic protein (GFAP) at day 2 and day 4. These findings demonstrate a subtle reduction in the areas of white matter injury and biomarkers of neurological injury after treatment with PNT001 following TBI. These findings support additional studies for PNT001 as well as the potential use of this agent in clinical trials in the near future.

Cell-Free DNA as a Biomarker in a Rodent Model of Chlorpyrifos Poisoning Causing Mitochondrial Dysfunction.

INTRODUCTION: Organophosphates (OPs) are a major public health problem worldwide due to ease of access and high toxicity lacking effective biomarkers and treatment. Cholinergic agents such as OPs and carbamates are responsible for many pesticide-related deaths. While the inhibition of AChE is thought to be the main mechanism of injury, there are other important pathways that contribute to the overall toxicity of OPs such as mitochondrial dysfunction. An existing gap in OP poisoning are biomarkers to gauge severity and prognosis. Cell-free DNA (cfDNA) are novel biomarkers that have gained increased attention as a sensitive biomarker of disease with novel use in acute poisoning. This study investigates alterations in cerebral mitochondrial function in a rodent model of chlorpyrifos poisoning with the use of cfDNA as a potential biomarker. METHODS: Twenty rodents were divided into two groups: Control (n = 10) and Chlorpyrifos (n = 10). Chlorpyrifos was administered through the venous femoral line with a Harvard Apparatus 11 Elite Syringe pump (Holliston, MA, USA) at 2 mg/kg. Animals were randomized to receive chlorpyrifos versus the vehicle (10% DMSO) for 60 min which would realistically present an acute exposure with continued absorption. At the end of the exposure (60 min), isolated mitochondria were measured for mitochondrial respiration along with measures of acetylcholinesterase activity, cfDNA, cytokines and western blot. RESULTS: The Chlorpyrifos group showed a significant decrease in heart rate but no change in the blood pressure. There was a significant increase in bulk cfDNA concentrations and overall decrease in mitochondrial respiration from brain tissue obtained from animals in the Chlorpyrifos group when compared to the Control group with no difference in acetylcholinesterase activity. In addition, there was a significant increase in both IL-2 and IL-12 in the Chlorpyrifos group. CONCLUSIONS: In our study, we found that the total cfDNA concentration may serve as a more accurate biomarker of OP exposure compared to acetylcholinesterase activity. In addition, there was an overall decrease in cerebral mitochondrial function in the Chlorpyrifos group when compared to the Control group.

Non-invasive diffuse optical monitoring of cerebral physiology in an adult swine-model of impact traumatic brain injury.

In this study, we used diffuse optics to address the need for non-invasive, continuous monitoring of cerebral physiology following traumatic brain injury (TBI). We combined frequency-domain and broadband diffuse optical spectroscopy with diffuse correlation spectroscopy to monitor cerebral oxygen metabolism, cerebral blood volume, and cerebral water content in an established adult swine-model of impact TBI. Cerebral physiology was monitored before and after TBI (up to 14 days post injury). Overall, our results suggest that non-invasive optical monitoring can assess cerebral physiologic impairments post-TBI, including an initial reduction in oxygen metabolism, development of cerebral hemorrhage/hematoma, and brain swelling.

Stabilization of uric acid mixed crystals by melamine.

Melamine stabilizes heterogeneous nucleation of calcium crystals by increasing the retention time and decreasing the rate of dissolution. Stabilization of such mixed crystals limit the efficacy of non-invasive treatment options for kidney stones. Crystalline forms of uric acid (UA) are also involved in urolithiasis or UA kidney stones; however, its interactions with contaminating melamine and the resulting effects on the retention of kidney stones remain unknown. Since melamine augments calcium crystal formation, it provides an avenue for us to understand the stability of UA-calcium phosphate (CaP) crystals. We show here that melamine facilitates UA+CaP crystal formation, resulting in greater aggregates. Moreover, melamine induced mixed crystal retention through a time-dependent manner in presence and/or absence of hydroxycitrate (crystal inhibitor), indicating its abridged effectiveness as conventional remedy. CaP was also shown to modify optical properties of UA+CaP mixed crystals. Differential staining of individual crystals revealed enhanced co-aggregation of UA and CaP. The dissolution rate of UA in presence of melamine was faster than its heterogeneous crystallization form with CaP, although the size was comparatively much smaller, suggesting disparity in regulation between UA and CaP crystallization. While melamine stabilized UA, CaP and mixed crystals in relatively physiological conditions (artificial urine), the retentions of those crystals were further augmented by melamine, even in presence of hydroxycitrate, thus reducing treatment efficacy.

Ablation of TRPC3 disrupts Ca2+ signaling in salivary ductal cells and promotes sialolithiasis.

Clinical studies and structural analyses of salivary stones strongly suggest a linkage between higher saliva calcium (Ca2+) and salivary stone formation, sialolithiasis; however, the process and the mechanism leading to Ca2+ overload during sialolithiasis is not well understood. Here, we show that TRPC3 null (-/-) mice presented with a reduction in Ca2+ entry and current in ductal cells with higher saliva [Ca2+] suggesting diminished transepithelial Ca2+ flux across the salivary ductal cells, leaving more Ca2+ in ductal fluid. Significantly, we found that TRPC3 was expressed in mice and human salivary ductal cells, while intraductal stones were detected in both mice (TRPC3-/-) and patient (sialolithiasis) salivary glands. To identify the mechanism, we found that TRPC3 was crucial in preventing the expression of calcification genes (BMP2/6, Runx2) in ductal cells which may be due to higher extracellular Ca2+ in SMG tissues. Similarly, inflammatory (IL6, NLRP3), fibrotic (FN1, TGFß1) and apoptotic (Bax1/Bcl2) markers were also elevated, suggesting that the loss of TRPC3 induces genetic changes that leads to salivary gland cell death and induction of inflammatory response. Overall, ablation of TRPC3-/- leads to higher saliva [Ca2+], along with elevated detrimental gene expressions, altogether contributing to salivary gland stone formation.

Reduction of TRPC1/TRPC3 mediated Ca2+-signaling protects oxidative stress-induced COPD.

Oxidative stress is a predisposing factor in Chronic Obstructive Pulmonary Disease (COPD). Specifically, pulmonary epithelial (PE) cells reduce antioxidant capacity during COPD because of the continuous production of reactive oxygen species (ROS). However, the molecular pathogenesis that governs such ROS activity is unclear. Here we show that the dysregulation of intracellular calcium concentration ([Ca2+]i) in PE cells from COPD patients, compared to the healthy PE cells, is associated with the robust functional expressions of Transient Receptor Potential Canonical (TRPC)1 and TRPC3 channels, and Ca2+ entry (SOCE) components, Stromal Interaction Molecule 1 (STIM1) and ORAI1 channels. Additionally, the elevated expression levels of fibrotic, inflammatory, oxidative, and apoptotic markers in cells from COPD patients suggest detrimental pathway activation, thereby reducing the ability of lung remodeling. To further delineate the mechanism, we used human lung epithelial cell line, A549, since the behavior of SOCE and the expression patterns of TRPC1/C3, STIM1, and ORAI1 were much like PE cells. Notably, the knockdown of TRPC1/C3 in A549 cells substantially reduced the SOCE-induced [Ca2+]i rise, and reversed the ROS-mediated oxidative, fibrotic, inflammatory, and apoptotic responses, thus confirming the role of TRPC1/C3 in SOCE driven COPD-like condition. Higher TRPC1/C3, STIM1, and ORAI1 expressions, along with a greater Ca2+ entry, via SOCE in ROS-induced A549 cells, led to the rise in oxidative, fibrotic, inflammatory, and apoptotic gene expression, specifically through the extracellular signal-regulated kinase (ERK) pathway. Abatement of TRPC1 and/or TRPC3 reduced the mobilization of [Ca2+]i and reversed apoptotic gene expression and ERK activation, signifying the involvement of TRPC1/C3. Together these data suggest that TRPC1/C3 and SOCE facilitate the COPD condition through ROS-mediated cell death, thus implicating their likely roles as potential therapeutic targets for COPD. SUMMARY: Alterations in Ca2+ signaling modalities in normal pulmonary epithelial cells exhibit COPD through oxidative stress and cellular injury, compromising repair, which was alleviated through inhibition of store-operated calcium entry. SUBJECT AREA: Calcium, ROS, Cellular signaling, lung disease.

Advanced Neuromonitoring Modalities on the Horizon: Detection and Management of Acute Brain Injury in Children.

Timely detection and monitoring of acute brain injury in children is essential to mitigate causes of injury and prevent secondary insults. Increasing survival in critically ill children has emphasized the importance of neuroprotective management strategies for long-term quality of life. In emergent and critical care settings, traditional neuroimaging modalities, such as computed tomography and magnetic resonance imaging (MRI), remain frontline diagnostic techniques to detect acute brain injury. Although detection of structural and anatomical abnormalities remains crucial, advanced MRI sequences assessing functional alterations in cerebral physiology provide unique diagnostic utility. Head ultrasound has emerged as a portable neuroimaging modality for point-of-care diagnosis via assessments of anatomical and perfusion abnormalities. Application of electroencephalography and near-infrared spectroscopy provides the opportunity for real-time detection and goal-directed management of neurological abnormalities at the bedside. In this review, we describe recent technological advancements in these neurodiagnostic modalities and elaborate on their current and potential utility in the detection and management of acute brain injury.

Brain Targeted Xenon Protects Cerebral Vasculature After Traumatic Brain Injury.

Abstract Cerebrovascular dysfunction following traumatic brain injury (TBI) is a well-characterized phenomenon. Given the therapeutic potential of xenon, we aimed to study its effects after localized delivery to the brain using microbubbles. We designed xenon-containing microbubbles stabilized by dibehenoylphosphatidylcholine (DBPC) and polyethylene glycol (PEG) attached to saturated phospholipid (DPSE-PEG5000). Using a pig model of TBI, these microbubbles were intravenously injected, and ultrasound was used to release xenon at the level of the carotid artery. The control group received perfluorobutane containing microbubbles. Diffusion tensor imaging (DTI) showed areas of higher fractional anisotropy for pigs receiving xenon microbubbles compared to the control group at 1 day after injury. Radial diffusivity analysis showed that this effect was mainly the result of acute edema. Pigs were euthanized at 5 days, and the brain tissues of xenon-treated animals showed reduction of perivascular inflammation and blood-brain barrier disruption. Endothelial cell culture experiments showed that glutamate reduces tight junction protein zona occludens-1 (ZO-1), but treatment with xenon microbubbles attenuates this effect. Xenon treatment protects cerebrovasculature and reduces astroglial reactivity after TBI. Further, these data support the future use of localized delivery of various therapeutic agents for brain injury using microbubbles in order to limit systemic side effects and reduce costs.

Alteration in Cerebral Metabolism in a Rodent Model of Acute Sub-lethal Cyanide Poisoning.

INTRODUCTION: Cyanide exposure can occur in various settings such as industry and metallurgy. The primary mechanism of injury is cellular hypoxia from Complex IV (CIV) inhibition. This leads to decreased ATP production and increased reactive oxygen species production. The brain and the heart are the organs most affected due to their high metabolic demand. While the cardiac effects of cyanide are well known, the cerebral effects on cellular function are less well described. We investigated cerebral metabolism with a combination of brain respirometry, microdialysis, and western blotting using a rodent model of sub-lethal cyanide poisoning. METHODS: Twenty rodents were divided into two groups: control (n = 10) and sub-lethal cyanide (n = 10). Cerebral microdialysis was performed during a 2 mg/kg/h cyanide exposure to obtain real-time measurements of cerebral metabolic status. At the end of the exposure (90 min), brain-isolated mitochondria were measured for mitochondrial respiration. Brain tissue ATP concentrations, acyl-Coenzyme A thioesters, and mitochondrial content were also measured. RESULTS: The cyanide group showed significantly increased lactate and decreased hypotension with decreased cerebral CIV-linked mitochondrial respiration. There was also a significant decrease in cerebral ATP concentration in the cyanide group and a significantly higher cerebral lactate-to-pyruvate ratio (LPR). In addition, we also found decreased expression of Complex III and IV protein expression in brain tissue from the cyanide group. Finally, there was no change in acyl-coenzyme A thioesters between the two groups. CONCLUSIONS: The key finding demonstrates mitochondrial dysfunction in brain tissue that corresponds with a decrease in mitochondrial function, ATP concentrations, and an elevated LPR indicating brain dysfunction at a sub-lethal dose of cyanide.

Anti-inflammatory role of extracellular l-arginine through calcium sensing receptor in human renal proximal tubular epithelial (HK-2) cells.

Renal tubular epithelial cells are capable of synthesizing interleukins (IL) in response to a variety of proinflammatory cytokines. Moreover, elevated urinary levels of IL have been shown in patients with various forms of nephritic diseases. However, the underlying intracellular signaling mechanism is unclear. Here we show the immunological signaling role of l-Arginine (l-Arg) through Ca2+-sensing receptor (CaSR) in human kidney 2 (HK-2) renal proximal tubular epithelial cells, using Ca2+ imaging and patch clamp techniques and its mechanistic link to the downstream cellular function. Both pharmacological and siRNA inhibitors support the activation CaSR by extracellular l-Arg to induced Ca2+ entry via a Transient receptor potential canonical (TRPC) channel in HK-2 cells mainly through the receptor operated Ca2+ entry (ROCE). Activation of CaSR by l-Arg led to the rise in p-p38/p38 expression suggesting [Ca2+]i as a regulator for p38-signaling pathways. Notably, l-Arg activated CaSR-induced Ca2+ signaling reduced the expressions of key fibrotic, inflammatory, and apoptotic genes, suggesting its nephroprotective role via Ca2+ signaling through CaSR in HK-2 cells. Since we found that the IL-6 expressions were inversely proportional to the increasing concentrations of l-Arg in HK-2 cells, we measured the release of IL-6, which steadily decreased as the concentrations of l-Arg were elevated. Taken together, extracellular l-Arg is a negative regulator for IL-6-induced inflammatory process, through the activation of CaSR and TRPC channel by ROCE pathway and can have a potential to alleviate inflammatory renal diseases.

Ablation of TRPC3 compromises bicarbonate and phosphate transporter activity in mice proximal tubular cells.

Proximal tubular (PT) cells reabsorb most calcium (Ca2+ ), phosphate (PO4 3- ), bicarbonate (HCO3 - ), and oxalate (C2 O4 2- ) ions. We have shown that mice lacking Transient Receptor Potential Canonical 3 (TRPC3-/- ) channel are moderately hypercalciuric with presentation of luminal calcium phosphate (CaP) crystals at the loop of Henle (LOH). However, other predisposing factors for such crystal deposition are unknown. Thus, we examined the distinctions in functional status of HCO3 - , PO4 3- , and C2 O4 2- transporters in PT cells of wild type (WT) and TRPC3-/- mice by whole-cell patch clamp techniques to assess their contribution in the development of LOH CaP crystals. Here we show the development of concentration dependent HCO3 - -induced currents in all PT cells, which was confirmed by using specific HCO3 - channel inhibitor, S0859. Interestingly, such activities were diminished in PT cells from TRPC3-/- mice, suggesting reduced HCO3 - transport in absence of TRPC3. While PO4 3- -induced currents were also concentration dependent in all PT cells (confirmed by PO4 3- channel inhibitor, PF-06869206), those activities were reduced in absence of TRPC3, suggesting lower PO4 3- reabsorption that can leave excess luminal PO4 3- . Next, we applied thiosulfate (O3 S2 2 - ) as a competitive inhibitor of the SLC26a6 transporter upon C2 O4 2- current activation and observed a reduced C2 O4 2- -induced conductance which was greater in TRPC3-/- PT cells. Together, these results suggest that the reduced activities of HCO3 - , PO4 3- , and C2 O4 2- transporters in moderately hypercalciuric (TRPC3-/- ) PT cells can create a predisposing condition for CaP and CaP tubular crystallization, enabling CaP crystal formation in LOH of TRPC3-/- mice.

Imaging of White Matter Injury Correlates with Plasma and Tissue Biomarkers in Pediatric Porcine Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) causes significant white matter injury, which has been characterized by various rodent and human clinical studies. The exact time course of imaging changes in a pediatric brain after TBI and its relation to biomarkers of injury and cellular function, however, is unknown. To study the changes in major white matter structures using a valid model of TBI that is comparable to a human pediatric brain in terms of size and anatomical features, we utilized a four-week-old pediatric porcine model of injury with controlled cortical impact (CCI). Using diffusion tensor imaging differential tractography, we show progressive anisotropy changes at major white matter tracts such as the corona radiata and inferior fronto-occipital fasciculus between day 1 and day 30 after injury. Moreover, correlational tractography shows a large part of bilateral corona radiata having positive correlation with the markers of cellular respiration. In contrast, bilateral corona radiata has a negative correlation with the plasma biomarkers of injury such as neurofilament light or glial fibrillary acidic protein. These are expected correlational findings given that higher integrity of white matter would be expected to correlate with lower injury biomarkers. We then studied the magnetic resonance spectroscopy findings and report decrease in a N-acetylaspartate/creatinine (NAA/Cr) ratio at the pericontusional cortex, subcortical white matter, corona radiata, thalamus, genu, and splenium of corpus callosum at 30 days indicating injury. There was also an increase in choline/creatinine ratio in these regions indicating rapid membrane turnover. Given the need for a pediatric TBI model that is comparable to human pediatric TBI, these data support the use of a pediatric pig model with CCI in future investigations of therapeutic agents. This model will allow future TBI researchers to rapidly translate our pre-clinical study findings into clinical trials for pediatric TBI.