Counter Regulation Research Articles

TRPC5 controls the adrenaline-mediated counter regulation of hypoglycemia

Hypoglycemia triggers autonomic and endocrine counter-regulatory responses to restore glucose homeostasis, a response that is impaired in patients with diabetes and its long-term complication hypoglycemia-associated autonomic failure (HAAF). We show that insulin-evoked hypoglycemia is severely aggravated in mice lacking the cation channel proteins TRPC1, TRPC4, TRPC5, and TRPC6, which cannot be explained by alterations in glucagon or glucocorticoid action. By using various TRPC compound knockout mouse lines, we pinpointed the failure in sympathetic counter-regulation to the lack of the TRPC5 channel subtype in adrenal chromaffin cells, which prevents proper adrenaline rise in blood plasma. Using electrophysiological analyses, we delineate a previously unknown signaling pathway in which stimulation of PAC1 or muscarinic receptors activates TRPC5 channels in a phospholipase-C-dependent manner to induce sustained adrenaline secretion as a crucial step in the sympathetic counter response to insulin-induced hypoglycemia. By comparing metabolites in the plasma, we identified reduced taurine levels after hypoglycemia induction as a commonality in TRPC5-deficient mice and HAAF patients.

A potential role for GPR107 in the development of Hypoglycemia Unawareness

Diabetes remains one of the largest health burdens in modern society and while an estimated 37.3 million people suffer from either type 1 or type 2 diabetes worldwide, a further 96 million people are currently estimated to be prediabetic. So far, most efforts for diabetic therapies have been focused on insulin-replacement therapies and secretagogues; however, a common risk associated with these therapies is hypoglycemia. Hypoglycemia is defined as low plasma glucose levels (< 70 mg/dl) and can lead to loss of consciousness and death. Recently, it has been established that repeated exposure to bouts of hypoglycemia in diabetic individuals likely impairs sympathoadrenal counter regulation, and shifts the glycemic threshold for symptoms to lower plasma glucose concentrations, close to levels which result in cognitive failure. This pathology has been titled hypoglycemia unawareness (HU) and constitutes a major risk factor for developing severe hypoglycemia. The development of severe hypoglycemia in diabetics accounts for a six-fold increase in mortality, compared to diabetic individuals who do not experience severe hypoglycemia. Thus, it is important to understand how to prevent and reverse its development. We have developed a novel rat model of HU in which rats are exposed to insulin-induced recurrent hypoglycemia for five consecutive days. In this model of HU, we found that GPR107 expression is dramatically increased in the hypothalamus of rats exhibiting loss of hypoglycemia-induced food intake. Our group previously identified GPR107 as a receptor for neuronostatin (NST), a 13 amino acid peptide derived from the somatostatin prepro-hormone, which can exert anorexigenic actions in the hypothalamus. Using a GPR107-flox rat model we are currently evaluating the effect of hypothalamic knock-down of GPR107 on the development of HU and basal feeding behavior. We anticipate that if knock-down of GPR107 reverses HU, the development of NST antagonists could potentially be useful therapeutically for diabetic patients who experience recurrent hypoglycemia. Our GPR107-flox rat model will enable us to identify upstream and downstream interacting partners of GPR107/NST. This could reveal hypothalamic pathways involved in the development of HU that also could serve as potential therapeutic targets for this devastation consequence of iatrogenic hypoglycemia. Internal Funds St. Louis University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In mouse chronic pancreatitis CD25+FOXP3+ regulatory T cells control pancreatic fibrosis by suppression of the type 2 immune response

Chronic pancreatitis (CP) is characterized by chronic inflammation and the progressive fibrotic replacement of exocrine and endocrine pancreatic tissue. We identify Treg cells as central regulators of the fibroinflammatory reaction by a selective depletion of FOXP3-positive cells in a transgenic mouse model (DEREG-mice) of experimental CP. In Treg-depleted DEREG-mice, the induction of CP results in a significantly increased stroma deposition, the development of exocrine insufficiency and significant weight loss starting from day 14 after disease onset. In CP, FOXP3+CD25+ Treg cells suppress the type-2 immune response by a repression of GATA3+ T helper cells (Th2), GATA3+ innate lymphoid cells type 2 (ILC2) and CD206+ M2-macrophages. A suspected pathomechanism behind the fibrotic tissue replacement may involve an observed dysbalance of Activin A expression in macrophages and of its counter regulator follistatin. Our study identified Treg cells as key regulators of the type-2 immune response and of organ remodeling during CP. The Treg/Th2 axis could be a therapeutic target to prevent fibrosis and preserve functional pancreatic tissue.

381-P: Recurrent Hypoglycemia Alters Endocrine Counterregulation during Insulin-Induced Hypoglycemia in Healthy and Diabetic Göttingen Minipigs

Hypoglycemia is a common adverse effect of insulin treatment in people with diabetes and recurrent episodes can alter counter regulation, ultimately increasing the risk of severe hypoglycemic episodes. The aim of this study was to investigate the effect of recurrent hypoglycemia on endocrine counter regulation during insulin-induced hypoglycemia in healthy and diabetic Göttingen Minipigs. Twelve adult, female, intact Göttingen Minipigs with (n=6) and without (n=6) streptozotocin-induced diabetes were included. Three pigs in each group were preconditioned with either hypoglycemia (RH) or normoglycemia (RN) on three consecutive days using a three-hour hyperinsulinemic clamp procedure (16 pmol/kg/min human insulin and variable glucose infusion) targeted a fasted minipig euglycemic level of 63 mg/dL plasma glucose in RN and 27 mg/dL in RH. On day four, all groups were subjected to a four-hour hypoglycemic clamp with 18 mg/dL target. Samples for plasma glucose, glucagon and epinephrine were collected at predefined time points. Influence of diabetes status and preconditioning on endocrine counter regulation during the final clamp were investigated using a repeated measures linear mixed model. Clamp glucose targets were reached in all groups. A blunted glucagon response was seen during the final hypoglycemic clamp in the RH groups (median 26.7 pM (IQR 21.6-33.2)) compared to RN groups (56.3 pM (46.3-63.5) , P=0.008) . Diabetes status did not influence the findings and no significant differences in plasma epinephrine were found between groups. Göttingen Minipigs with and without streptozotocin induced diabetes exhibit blunted glucagon response to hypoglycemia after recurrent hypoglycemia similar to humans, indicating impaired counter regulatory response. Epinephrine response was not significantly affected. Further studies are needed to investigate the effect of RH on epinephrine counter regulation in the Göttingen Minipig. Disclosure M.Lyhne: None. K.Hinz: Employee; Antag Therapeutics, Novo Nordisk. A.Vegge: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. J.Sturis: Employee; Novo Nordisk A/S. J.Kildegaard: Employee; Novo Nordisk A/S. U.Pedersen-bjergaard: Advisory Panel; Novo Nordisk A/S, Sanofi. L.Olsen: None.

1303-P: Decreased Hepatic HMGB1 in Type 2 Diabetes Increases Mitochondrial Glucose Uptake by Regulating Insulin/AKT Signaling

Introduction: High Mobility Group Box-1 (HMGB1) is strongly associated with Type 2 Diabetes (T2D) and hepatocyte function. HMGB1 can regulate mitochondrial function under high glucose conditions. Our group has shown that knockdown of HMGB1 reduces hyperglycemia with decrease insulin activity. We want to further understand the role of HMGB1 in hepatocyte mitochondrial function and the insulin/AKT signaling, a vital pathway in glucose hemostasis. We hypothesize that by decreasing HMGB1, hepatocytes increase glucose uptake by regulating mitochondrial function with more effective Insulin/AKT signaling. Methods: Primary hepatocytes and liver lysates were isolated from inducible HMGB1 KO (iHMGB1 KO) and HMGB1 Flox (wildtype) mice after Tamoxifen and Streptozotocin IP injections for HMGB1 knockout and T2D-10-weeks development. HMGB1 knockdown and insulin/AKT signaling were assessed by RT-PCR and immunoblot. Flow cytometry was performed on primary hepatocyte cell suspension for mitochondrial function/mass (TMRE, Mitotracker) and glucose uptake (NDBG) cellular markers. Results: Liver lysates showed significant decreased expression/activity of HMGB1, Insulin Receptor-B, phosphorylated AKT, PDK and SIRT3 in iHMGB1 KO T2D mice. A counter regulator of metabolic and glucose activity, phosphorylated PDH, was found increased in iHMGB1 KO mice. Flow cytometry of isolated hepatocytes showed that iHMGB1 KO T2D animals had significantly less mitochondrial mass and functioning mitochondria, however showed increased glucose uptake. Conclusion: We found a potential mechanism by which HMGB1 regulates glucose uptake by regulating mitochondrial function and mass potentially by regulating the effectiveness of the Insulin/Akt signaling in T2D. Future studies will be directed to test glucose transport/metabolism in HMGB1 KO hepatocytes guided by more effective Insulin activity in the mediation of mitochondria function at the different stages of T2D progression. Disclosure Z.Liu: None. C.Kaltenmeier: None. R.I.Mota alvidrez: None. Funding This work was funded by department of surgery support for RIMA

Regulation of Progressive Liver Disease by Mitochondrial Carboxylate Transport

Mitochondrial transporters link TCA cycle and cytosolic metabolism. Mitochondrial pyruvate import and citrate export are two especially important steps for metabolic regulation. Pyruvate is imported into mitochondria by the mitochondrial pyruvate carrier (MPC), whereas citrate is exported by the mitochondrial citrate carrier (CIC). During fatty liver disease and T2D, liver MPC expression increases and facilitates excessive gluconeogenesis driving chronic hyperglycemia. Liver MPC disruption attenuates hyperglycemia and decreases liver inflammation. Sustained MPC disruption provides long-term protection from hyperglycemia without apparent liver damage. Furthermore, MPC disruption markedly decreases oxidative stress-induced hepatocellular carcinoma, a highly lethal cancer for which fatty liver disease and fibrosis are a major risk factors. The mechanisms by which MPC disruption provides metabolic projection are not completely understood. One potential mechanism is that MPC disruption in-turn decreases mitochondrial citrate export. Cytosolic citrate stimulates increased gluconeogenesis and de novo lipogenesis, two features of fatty liver disease and T2D. Thus, CIC export of citrate derived from MPC-imported pyruvate could mediate these processes. To test the role of the CIC in fatty liver disease, we generated liver-specific CIC knockout mice. Liver CIC disruption markedly decreased de novo lipogenesis measured by deuterated water into lipids. To examine how altered transcriptional programming might contribute to decreased CIC LivKO lipogenesis, we analyzed WT and CIC LivKO livers by RNAseq and qPCR. Numerous genes involved in lipoprotein and fatty acid uptake were upregulated in CIC LivKO livers, including Acly and Fasn, suggesting transcriptional counter regulation for impaired lipogenesis. CIC disruption protected from glucose intolerance after Western diet feeding but did not change insulin tolerance. To understand this selective effect on glucose tolerance, we traced liver 13C-glucose metabolism in vivo. Mass spectrometry analysis of liver extracts showed that CIC disruption increased 13C enrichment into pyruvate and TCA cycle intermediates, consistent with disinhibited glycolysis and decreased mitochondrial citrate export. Together, these data provide evidence for an MPC-CIC axis as key mediator of fatty liver disease.

Antibiotic usage and resistance in Mexico: an update after a decade of change.

Ten years ago, a review on the status of resistance in Mexico was bleak: with antibiotics freely sold over the counter and poor regulation of generic drugs, among other conditions, resistance among relevant pathogens often ranked top, either among Latin American countries, or even worldwide. Since then, there have been some regulatory changes, along a decline in antibiotics usage: antibiotics are (supposedly) no longer sold without prescription, generic drugs (supposedly) have to demonstrate bioequivalence, and antibiotic usage has drop, from about 13 defined daily doses per 1,000 inhabitants per day, to 7. While these changes may sound encouraging, an analysis show that regulatory changes are incomplete at best, and usage decline may be the consequence of factors such as growing poverty. The assessment of resistance continues to be haphazard, without an organized network of laboratories providing a coherent picture. However, judging from a few nationwide reports, it appears that resistance among some nosocomial pathogens (MRSA, enterococci, Pseudomonas aeruginosa) is declining, as it is among pneumococci and enteropathogens; but it is rising among community-acquired, uropathogenic Escherichia coli. Resistance to colistin is slowly increasing; and worrisome resistance determinants, such as blaNDM-1 and mcr-1, appeared in Mexico shortly after their first report elsewhere. After four years from the United Nations General Assembly high-level meeting on antibiotic resistance, Mexico is yet to deploy the basic measures to assess and control antibiotic resistance. As such, and despite the regulatory changes, the 2010-2020 period looks like a "lost decade".

The potential of various seeds as angiotensin-I converting enzyme inhibitory peptides derived from protein hydrolysate: a short review

Angiotensin-I converting enzyme (ACE) plays an imperative role in the blood pressure system. It generates intense vasoconstriction by converting angiotensin-I to angiotensin-II. Counter regulation of ACE may reduce blood pressure. One of the most frequently used medications for treating hypertension cases to inhibit ACE activity is a commercial synthetic drug. However, long-term consumption of those drugs could lead to suffering dangerous and unpredictable side effects. There have been many studies recently concerning the bioactive peptides as ACE inhibitors derived from various seeds. It has been reported that non-thermal extraction methods were used to obtain the protein from inside the cell. However, there was a lack of information focusing on ACE inhibitory peptides from plant biomass. Therefore, this review aimed to summarise and gather the point of view of the plant-derived ACE inhibitory peptides from raw material sources, processing, and peptide sequences studies. This paper contributed to explaining a comprehensive review of ACE inhibitory peptides from edible materials and proposes a fascinating discussion due to the sources being discovered derived from edible protein and safer grade. Various seeds in Indonesia may have future potential for ACE inhibitory peptides as natural therapeutic agents.

The COVID-19 Code – Cracked – WHO can Save The World (?)!

The first step in COVID-19 pathogenesis is the viral spike protein priming by Trans Membrane Peptide Receptor Serine S2 (TMPRSS2). TMPRSS2 promotes viral entry, cell to cell transmission, evasion of host immune response, and Angiotensin-Converting Enzyme 2 (ACE2) downregulation. Androgen through Androgen Receptor (AR) increases TMPRSS2 gene expression. Blocking AR may prevent viral entry and other TMPRSS2 mediated actions. ACE2 acts as an entry point for COVID-19 and as the counter regulator in Renin-Angiotensin-Aldosterone System (RAAS). RAAS maintains homeostasis of blood pressure, salt and water, inflammation, and immune response – through its two arms called “killer” and “protective pathways.” The balance between these two pathways determines life or death in disease states. ACE2 converts Angiotensin II to Angiotensin (1-7), which through Mas receptors mediates antiinflammatory, immune-modulatory, and anti-fibrotic actions. Angiotensin II also acts on Angiotensin type 2 Receptor (AT2R) to produce similar actions, called a "protective pathway." Further, Angiotensin II acts through its primary Angiotensin type 1 Receptor (AT1R), causing inflammatory, cytokine storm, and profibrotic response – called "Killer pathway." In COVID, down-regulated ACE2 leads to unabated Angiotensin II/AT1R – "Killer pathway" – actions producing a vicious cycle of "hyper-inflammatory state," resulting in ALI, ARDS, and death. AT1R activation further stimulates the secretion of aldosterone, which through Mineralocorticoid Receptor (MR), augments AT1R mediated 'killer pathway”. None of the COVID guideline drugs modulate this pathogenic mechanism. We examine the first time in history the scientific rationale for combined AR/AT1R/MR blockade for COVID-19 treatment and prevention.

Angiotensin-converting enzyme 2: a double-edged sword in COVID-19 patients with an increased risk of heart failure.

The coronavirus disease (COVID-19) pandemic is a global health priority. Given that cardiovascular diseases (CVD) are the leading cause of morbidity around the world and that several trials have reported severe cardiovascular damage in patients infected with SARS-CoV-2, a substantial number of COVID-19 patients with underlying cardiovascular diseases need to continue their medications in order to improve myocardial contractility and to prevent the onset of major adverse cardiovascular events (MACEs), including heart failure. Some of the current life-saving medications may actually simultaneously expose patients to a higher risk of severe COVID-19. Angiotensin-converting enzyme 2 (ACE2), a key counter regulator of the renin-angiotensin system (RAS), is the main entry gate of SARS-CoV-2 into human host cells and an established drug target to prevent heart failure. In fact, ACE inhibitors, angiotensin II receptor blockers, and mineralocorticoid antagonists may augment ACE2 levels to protect organs from angiotensin II overload. Elevated ACE2 expression on the host cell surface might facilitate viral entrance, at the same time sudden nonadherence to these medications triggers MACEs. Hence, safety issues in the use of RAS inhibitors in COVID-19 patients with cardiac dysfunction remain an unsolved dilemma and need paramount attention. Although ACE2 generally plays an adaptive role in both healthy subjects and patients with systolic and/or diastolic dysfunction, we conducted a literature appraisal on its maladaptive role. Understanding the exact role of ACE2 in COVID-19 patients at risk of heart failure is needed to safely manage RAS inhibitors in frail and non-frail critically ill patients.

Glycyl-l-glutamine attenuates NPY-induced hyperphagia via the melanocortin system

This study aimed to determine whether glycyl-l-glutamine (Gly-Gln; β-endorphin (30–31)), a non-opioid peptide derived from β-endorphin processing, modulates neuropeptide Y (NPY)-induced feeding and hypothalamic mRNA expression of peptide hormones in male broiler chicks. Intracerebroventricular injection of NPY (235 pmol) generated a hyperphagic response in ad libitum chicks within 30 min. Co-administration of Gly-Gln (100 nmol) attenuated this response, inducing a 30 % decrease. This was not attributable to Gly-Gln hydrolysis because co-administration of glycine (Gly) and glutamine (Gln) had no effect on NPY-induced hyperphagia. Gly-Gln injected alone also showed no effect. The hypothalamic pro-opiomelanocortin mRNA expression in the co-injection group was significantly higher than that in the NPY alone group. These data indicate that endogenous Gly-Gln may contribute to regulate feeding behavior via the central melanocortin system in chicks and acts as a counter regulator of the neural activity in energy metabolism.

Hormonal Blood Pressure Regulationduring General Anesthesia Usinga Standardized Propofol Dosagein Children and Adolescents SeemsNot to Be Affected by Body Weight.

Obesity in pediatric surgical patients is a challenge for the anesthesiologist. Despite potentially beneficial properties, propofol might also induce hypotension. This study examined whether a dose adjustment in overweight children could avoid hypotension and if there would be differences regarding hormonal regulation in children under anesthesia. Fifty-nine children undergoing surgery under general anesthesia were enrolled in this prospective observational trial. Participants were allocated into two groups according to their BMI. The induction of anesthesia was conducted using propofol (“overweight”: 2 mg/kgBW, “regular”: 3.2 mg/kgBW). The maintenance of anesthesia was conducted as total intravenous anesthesia. Hormone levels of renin, angiotensin II, aldosterone, copeptin, norepinephrine and epinephrine were assessed at different timepoints. Blood pressure dropped after the administration of propofol in both groups, with a nadir 2 min after administration—but without a significant difference in the strength of reduction between the two groups. As a reaction, an increase in the plasma levels of renin, angiotensin and aldosterone was observed, while levels of epinephrine, norepinephrine and copeptin dropped. By adjusting the propofol dosage in overweight children, the rate of preincision hypotension could be reduced to the level of normal-weight patients with a non-modified propofol dose. The hormonal counter regulation was comparable in both groups. The release of catecholamines and copeptin as an indicator of arginine vasopressin seemed to be inhibited by propofol.

Counter Regulation of Spic by NF-κB and STAT Signaling Controls Inflammation and Iron Metabolism in Macrophages

SUMMARYActivated macrophages must carefully calibrate their inflammatory responses to balance efficient pathogen control with inflammation-mediated tissue damage, but the molecular underpinnings of this “balancing act” remain unclear. Using genetically engineered mouse models and primary macrophage cultures, we show that Toll-like receptor (TLR) signaling induces the expression of the transcription factor Spic selectively in patrolling monocytes and tissue macrophages by a nuclear factor κB (NF-κB)-dependent mechanism. Functionally, Spic downregulates pro-inflammatory cytokines and promotes iron efflux by regulating ferroportin expression in activated macrophages. Notably, interferon-gamma blocks Spic expression in a STAT1-dependent manner. High levels of interferon-gamma are indicative of ongoing infection, and in its absence, activated macrophages appear to engage a “default” Spic-dependent anti-inflammatory pathway. We also provide evidence for the engagement of this pathway in sterile inflammation. Taken together, our findings uncover a pathway wherein counter-regulation of Spic by NF-κB and STATs attune inflammatory responses and iron metabolism in macrophages.

Serum progranulin/tumor necrosis factor-\u03b1 ratio as independent predictor of systolic blood pressure in overweight hypertensive patients: a cross-sectional study

BackgroundVascular inflammation plays a key role in the progression of hypertension. Progranulin (PGRN), an anti-inflammatory growth factor, mediated inhibition of tumor necrosis factor-α (TNF-α), a pleiotropic cytokine, activity has been well-established. Despite the role of chronic low-grade inflammation in hypertension, serum levels of PGRN and PGRN/TNF-α ratio and, their association with systolic and diastolic blood pressure has not been determined in hypertensive patients till now. This study aims to find and correlate the serum levels of pro-inflammatory cytokine (TNF-α), anti-inflammatory growth factor (PGRN), and PGRN/TNF-α ratio with the blood pressure in systolic-diastolic hypertension (SDH) and isolated systolic hypertension (ISH) patients.ResultsA cross-sectional study was conducted on SDH patients (mean age, 52.95 ± 12.6 years; male/female (M/F) number = 15/10) and ISH patients (mean age, 55.80 ± 9.40 years; M/F number = 12/13) (n = 25 each). Twenty-five age and body mass index (BMI)-matched healthy subjects (mean age, 56.00 ± 8.55 years; male/female number = 11/14) were considered as control. All patients and healthy subjects were overweight (BMI, 25–30 kg/m2). Overnight fasting blood samples of subjects were taken and levels of PGRN and TNF-α were measured using ELISA diagnostic kits. PGRN and TNF-α levels were found significantly high, whereas PGRN/TNF ratio was found very low, in SDH and ISH patients as compared to healthy subjects. Reduced PGRN/TNF-α ratio and pulse pressure were found as independent predictors of SBP both in SDH and ISH patients.ConclusionsFindings of elevated PGRN levels in response to raised TNF-α levels depict the counter regulation by PGRN to neutralize TNF-α. Findings of reduced PGRN/TNF ratio, and it being an independent predictor of SBP, ascertain the key role of imbalance in pro- and anti-inflammatory environment in hypertension. Thus, it strengthens the cross-link between the concept of immunity–adiposity–inflammation–blood pressure¸ a vicious network. Further, this cross-link of SBP and progranulin must be explored in longitudinal studies. New researches should be focused not only on impact of pro-inflammatory environment rather to find on a balance between pro- and anti-inflammatory status, so that new target sites could be explored for therapeutic management of hypertension.

Hypoglycemia awareness and burden among hospitalized patients at high risk for hypoglycemia

BackgroundHypoglycemia, a common complication of insulin therapy in patients with diabetes, is associated with increased hospital morbidity and mortality. Hypoglycemia may be underrecognized in hospitalized patients due to impaired counter regulation or reduced recognition. Beta blocker (BB) use may also affect hypoglycemia recognition. AimTo characterize hypoglycemia unawareness and patient perceived hypoglycemia burden in hospitalized patients. Materials and methodsOver a 2-month period, we prospectively identified non-critically ill cognitively intact hospitalized insulin-requiring patients who were undergoing bedside glucose monitoring. Participants were included if they reported any episode of hypoglycemia in the 30 days prior to admission and either had no BB use or were on stable BB for 90 days. Hypoglycemia unawareness was assessed using the Clarke score and burden of hypoglycemia was assessed with the (TRIM-HYPO) score. ResultsOf the 46 participants, 20 were not taking a BB and 26 were taking a BB. Predictors of hypoglycemia during admission included glucose coefficient of variation and hospital length of stay. Nine participants (20%) had hypoglycemia unawareness (Clarke score ≥4). Participants with a history of coronary artery disease were less likely to have impaired awareness. Burden of hypoglycemia was associated with more hypoglycemia and longer hospital length of stay. There were no differences in measures of hypoglycemia unawareness or burden according to BB use. ConclusionsBB use was not related to hypoglycemia unawareness, or burden in hospitalized high risk patients. Hypoglycemia burden may identify patients at higher risk of hypoglycemia during admission.

IFN Regulatory Factor 4 Controls Post-ischemic Inflammation and Prevents Chronic Kidney Disease.

Ischemia reperfusion injury (IRI) of the kidney results in interferon regulatory factor 4 (IRF4)–mediated counter-regulation of the acute inflammatory response. Beyond that, IRF4 exerts important functions in controlling the cytokine milieu, T-cell differentiation, and macrophage polarization. The latter has been implicated in tissue remodeling. It therefore remains elusive what the role of IRF4 is in terms of long-term outcome following IRI. We hypothesized that an inability to resolve chronic inflammation in Irf4−/− mice would promote chronic kidney disease (CKD) progression. To evaluate the effects of IRF4 in chronic upon acute injury in vivo, a mouse model of chronic injury following acute IRI was employed. The expression of Irf4 increased within 10 days after IRI in renal tissue. Both mRNA and protein levels remained high up to 5 weeks upon IRI, suggesting a regulatory function in the chronic phase. Mice deficient in IRF4 display increased tubular cell loss and defective clearance of infiltrating macrophages. These phenomena were associated with increased expression of pro-inflammatory macrophage markers together with reduced expression of alternatively activated macrophage markers. In addition, IRF4-deficient mice showed defective development of alternatively activated macrophages. Hints of a residual M1 macrophage signature were further observed in human biopsy specimens of patients with hypertensive nephropathy vs. living donor specimens. Thus, IRF4 restricts CKD progression and kidney fibrosis following IRI, potentially by enabling M2 macrophage polarization and restricting a Th1 cytokine response. Deteriorated alternative macrophage subpopulations in Irf4−/− mice provoke chronic intrarenal inflammation, tubular epithelial cell loss, and renal fibrosis in the long course after IRI in mice. The clinical significance of these finding for human CKD remains uncertain at present and warrants further studies.

Abstract P2011: Chronic Systemic Administration of Pro-Inflammatory Cytokines Causes Cardiovascular Dysfunction and Downregulates Brain Expression of Regulator of G-Protein Signaling-10 in Rats

Systemic pro-inflammatory cytokines (PICs) promote central oxidative stress and neuroinflammation that contribute to sympathetic excitation and cardiovascular dysfunction in hypertension and heart failure. However, the key molecular regulators of neuroinflammatory responses in the brain remain unclear. Regulator of G-protein Signaling-10 (RGS10), a GTPase-accelerating protein that is expressed predominantly in the brain, has recently emerged as an important counter regulator of PICs-induced downstream signaling in the central nervous system. RGS10 negatively regulates NF-kB-mediated inflammatory responses in microglia and antagonizes neurotoxic effects of peripheral inflammation. The present study examined whether peripherally administered PICs alter RGS10 expression in cardiovascular/autonomic brain regions like subfornical organ (SFO) and hypothalamic paraventricular nucleus (PVN). Male SD rats received a 2-week subcutaneous infusion of vehicle, interleukin (IL)-1β (400 ng/day) and tumor necrosis factor (TNF)-α (600 ng/day). Rats treated with IL-1β (n=6) or TNF-α (n=6) had significantly (*P<0.05) reduced mRNA level (fold change) of RGS10 in SFO (0.60±0.15* or 0.69±0.16* vs 1.03±0.28; IL-1β or TNF-α, vs vehicle treatment) and PVN (0.72±0.15* or 0.71±0.19* vs 1.02±0.24), and lessoned mRNA level of NF-kB inhibitor IkB-α (SFO: 0.60±0.21* or 0.55±0.16* vs 1.07±0.46; PVN: 0.70±0.31* or 0.82±0.16* vs 1.00±0.07). Treatments with IL-1β or TNF-α increased mRNA level of NADPH oxidase 2 (SFO: 1.96±0.91* or 1.62±0.47* vs 1.03±0.28; PVN: 2.00±0.82* or 1.74±0.70* vs 1.00±0.15) and cyclooxygenase-2 (SFO: 1.78±0.76* or 1.98±1.04* vs 1.00±0.13; PVN: 1.68±0.34* or 1.62±0.57* vs 1.02±0.24). Rats treated with IL-1β or TNF-α had elevated mean blood pressure (98±11* or 100±9* vs 84±5 mmHg) and increased sympathetic activity as indicated by pressure response to ganglionic blockade (38±7 * or 36±6* vs 28±4 mmHg). Additionally, IL-1β or TNF-α-treated rats had increased cardiac fibrotic markers and impaired cardiac diastolic function. These results suggest that circulating PICs reduce RGS10 expression in the brain, which may contribute to sympathetic excitation and cardiovascular dysfunction in inflammatory conditions.

Functional Topography in the Rat Rostral Ventrolateral Medulla (RVLM): Distribution of C1 Neurons that Respond to Cardiovascular versus Metabolic Stimuli

Neurons controlling cardiovascular responses and glucose counter regulation are co‐located within the RVLM, with activated neurons lying almost exclusively caudal to the caudal pole of the facial nucleus (FN). However, it is unclear whether the same neurons are involved in triggering both cardiovascular and metabolic responses. We compared the distributions of RVLM C1 neurons activated by cardiovascular or metabolic stimuli using Fos to identify activated neurons. To conscious male or female rats, we administered insulin (10 U/kg ip) or 2‐deoxyglucose (2DG; 400 mg/kg sc) to induce hypoglycaemia or nitroprusside (NP; 1 mg/ml iv), hydralazine (HDZ; 10 mg/kg iv) or diazoxide (DZX; 50 mg/kg iv) to cause hypotension. Rats were transcardially perfused with formaldehyde 90 or 120 minutes later. Coronal 30‐micrometer thick sections of medulla (1:4 series) from the spinomedullary junction to mid‐FN were immunoperoxidase‐stained to show Fos‐immunoreactivity plus phenylethanolamine N‐methyl transferase (PNMT)‐immunoreactivity to identify C1 neurons. The 6 sections containing the 600 micrometers caudal to the caudal pole of FN (sections FN0 to FN‐5) were each divided into 3 equal segments (medial, middle, lateral) and the total numbers of PNMT neurons with Fos nuclei (Fos+PNMT neurons) were counted. The numbers of Fos+PNMT neurons were greater in the medial and middle thirds than in the lateral third of the C1 cell column in insulin‐ and 2DG‐treated rats. In NP‐, DZX‐or HDZ‐treated rats, the numbers of Fos+PNMT neurons were greater in the middle and lateral two thirds of the C1 cell column. Two‐way ANOVA showed a very strong interaction between stimulus and laterality (p<0.0001), both for number of Fos+PNMT neurons and for percentage of PNMT neurons with Fos nuclei. These results reveal that the extent of Fos expression in PNMT neurons in the medial versus lateral zones depends on the type of stimulus. Post‐hoc analysis confirmed that, in the medial zone, the metabolic stimuli activated more PNMT neurons than cardiovascular stimuli, while it was the opposite in the lateral zone (p<0.0001 in both cases). These results show that hypoglycaemia‐responsive RVLM C1 neurons are concentrated in the medial and middle thirds of the C1 cell column whereas hypotension‐responsive RVLM C1 neurons are concentrated in the middle and lateral two thirds of the column. Hence, within the rat RVLM, C1 neurons are distributed topographically according to their function.Support or Funding InformationNHMRC 1025031; NIH HL096787 & HL130577This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract B206: Evaluation of tumor biomarkers in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer treated with the CXCL12 inhibitor NOX-A12 and preliminary safety in combination with PD-1 checkpoint inhibitor pembrolizumab

Abstract Background: NOX-A12 (olaptesed pegol) is a novel inhibitor of the chemokine CXCL12, ligand for CXCR4 and CXCR7, for treatment of solid tumors. Binding of the CXCL12 ligand by NOX-A12 prevents receptor engagement and also blocks the ability of CXCL12 to create a chemotactic concentration gradient by neutralization of the anchor domain. CXCL12 in complex with NOX-A12 has an increased half-life relative to unbound CXCL12, and as such increased CXCL12 levels are a marker of inhibition. The OPERA study (NCT03168139) is a phase 1/2 open-label clinical study to evaluate pharmacodynamic effects and safety of monotherapy with NOX-A12 and safety and efficacy of a combination of NOX-A12 with pembrolizumab in advanced MSS, metastatic colorectal and pancreatic cancer with liver metastasis. The study comprises two weeks of NOX-A12 monotherapy followed by repeated 21-day cycles of NOX-A12 plus pembrolizumab. Here we present pharmacodynamic biomarker data from for monotherapy phase with NOX-A12 as well as safety for the combination with pembrolizumab. Methods: Patients received 300 mg NOX-A12 by i.v. infusion on days 1, 4, 8, and 11 of the monotherapy phase. Needle biopsies were taken from suitable liver metastases before treatment and on day 14, peripheral blood was drawn at the same time points. Collected tumor samples were assessed for immune cell infiltration by IHC and cytokine signature using multiplex protein analysis. Results: At the time of abstract submission, 19 out of 20 patients have been recruited, 10 with colorectal and 9 with pancreatic cancer. Serial biopsies at baseline and end of monotherapy suitable for immunohistochemistry (IHC) and cytokine analysis were obtained from 12 out of 19 patients. CXCL12 levels were found to be increased in blood, but also in post-treatment tumor biopsies, suggesting penetration of NOX-A12 into the tumor. The comparison of cytokine levels in the currently available baseline tumor biopsies with those taken after two weeks of NOX-A12 monotherapy revealed changes in markers that are consistent with a Th1-like immune response in multiple patients. In particular IFN-γ, IL-2 and TNF-β were increased, accompanied by an increase of CXCL9 and/or CXCL10 indicating simultaneous T-cell attraction, which indicates a tissue response to treatment with NOX-A12. However, there were also increased levels of IL-1α, IL-1β and IL-6 in a number of patients, indicating potential myeloid counter regulation. IHC analysis is ongoing, and results for all patients will be presented. Treatment with NOX-A12 monotherapy and in combination with pembrolizumab was generally safe and well tolerated (139 AEs in total, thereof 45% grade 1; 37% grade 2; 17% grade 3; no grade 4 and 1 % grade 5). Conclusions: NOX-A12 penetrates into the tumor where it binds and neutralizes CXCL12. Changes in the cytokine signature suggest that NOX-A12 modulates the tumor microenvironment and induces an immune-stimulatory Th1-like signature in multiple patients. The safety profile of NOX-A12 combined with pembrolizumab is consistent with that of pembrolizumab in advanced cancer patients. Citation Format: Niels Halama, Ulrike Prüfer, Anna Froemming, Diana Beyer, Dirk Eulberg, Jarl Ulf Jungnelius, Aram Mangasarian. Evaluation of tumor biomarkers in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer treated with the CXCL12 inhibitor NOX-A12 and preliminary safety in combination with PD-1 checkpoint inhibitor pembrolizumab [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B206.

Pathophysiological aspects of nephropathy caused by non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used medications associated with nephrotoxicity, especially when used chronically. Factors such as advanced age and comorbidities, which in themselves already lead to a decrease in glomerular filtration rate, increase the risk of NSAID-related nephrotoxicity. The main mechanism of NSAID action is cyclooxygenase (COX) enzyme inhibition, interfering on arachidonic acid conversion into E2 prostaglandins E2, prostacyclins and thromboxanes. Within the kidneys, prostaglandins act as vasodilators, increasing renal perfusion. This vasodilatation is a counter regulation of mechanisms, such as the renin-angiotensin-aldosterone system works and that of the sympathetic nervous system, culminating with compensation to ensure adequate flow to the organ. NSAIDs inhibit this mechanism and can lead to acute kidney injury (AKI). High doses of NSAIDs have been implicated as causes of AKI, especially in the elderly. The main form of AKI by NSAIDs is hemodynamically mediated. The second form of NSAID-induced AKI is acute interstitial nephritis, which may manifest as nephrotic proteinuria. Long-term NSAID use can lead to chronic kidney disease (CKD). In patients without renal diseases, young and without comorbidities, NSAIDs are not greatly harmful. However, because of its dose-dependent effect, caution should be exercised in chronic use, since it increases the risk of developing nephrotoxicity.