Adhesion-GPCR Gpr116 (ADGRF5) is a Regulator of Urine Acidification and Surface Expression of the Vacuolar-type H+-ATPase in Renal α-Intercalated Cells

The G protein-coupled receptor (GPCR) superfamily is among the largest in the human genome. Their diversity and nearly universal expression underlie their significance in many physiologic processes. GPCRs are a common target of pharmaceutical drug development, and uncovering the function of understudied GPCRs in the kidney represents a wealth of untapped therapeutic potential. We previously identified Gpr116, an adhesion-class GPCR, as one of the most highly expressed GPCRs in the kidney. In the present study, we confirm the localization of Gpr116 to the luminal membrane of acid-secreting α-intercalated cells (αICs) in the nephron using both imaging and functional studies, where we demonstrate in situ receptor activation using an agonist peptide unique to Gpr116. Additionally, kidney-specific knockout (KO) of Gpr116 caused a significant reduction to urine pH. Notably, the loss of acid in the urine is accompanied by a small, but significant, increase in blood pH, and a small, but significant, decrease in pCO2 compared to wild-type littermates. Results from transmission electron micrographs show greater accumulation of V-ATPase proton pumps at the surface of αICs in KO mice, suggesting a possible role for Gpr116 in the regulation of V-ATPase trafficking. We conclude that loss of Gpr116 from the nephron causes a primary loss of acid in the urine which results in a mild metabolic alkalosis (“renal tubular alkalosis”) due to reabsorption of HCO3- by αICs. This study establishes a significant physiologic role of the previously understudied Gpr116 in the murine kidney and demonstrates the scientific potential of future investigations into novel GPCRs

Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells.

Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection

The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis

Objectives: Obesity is characterized by excessive fat mass and is associated with serious diseases such as type 2 diabetes. Targeting excess fat mass by sustained lipolysis has been a major challenge for anti-obesity therapies due to unwanted side effects. TLQP-21, a neuropeptide encoded by the pro-peptide VGF (non-acronymic), that binds the complement 3a receptor 1 (C3aR1) on the adipocyte membrane, is emerging as a novel modulator of adipocyte functions and a potential target for obesity-associated diseases. The molecular mechanism is still largely uncharacterized. Methods: We used a combination of pharmacological and genetic gain and loss of function approaches. 3T3-L1 and mature murine adipocytes were used for in vitro experiments. Chronic in vivo experiments were conducted on diet-induced obese wild type, β1, β2, β3-adrenergic receptor (AR) deficient and C3aR1 knockout mice. Acute in vivo lipolysis experiments were conducted on Sprague Dawley rats. Results: We demonstrated that TLQP-21 does not possess lipolytic properties per se. Rather, it enhances β-AR activation-induced lipolysis by a mechanism requiring Ca2+ mobilization and ERK activation of Hormone Sensitive Lipase (HSL). TLQP-21 acutely potentiated isoproterenol-induced lipolysis in vivo. Finally, chronic peripheral TLQP-21 treatment decreases body weight and fat mass in diet induced obese mice by a mechanism involving β-adrenergic and C3a receptor activation without associated adverse metabolic effects. Conclusions: In conclusion, our data identify an alternative pathway modulating lipolysis that could be targeted to diminish fat mass in obesity without the side effects typically observed when using potent pro-lipolytic molecules. Author Video: Author Video Watch what authors say about their articles Keywords: Adipocyte, Complement 3a receptor, Ca2+, MAPK/ERK, β-AR, Isoproterenol, VG

Kinetics of IFN gene expression in response to IAV infection of primary pAECs.

<p>Primary pAECs were infected with IAV pH1N1 CA/09 (circle) or H1N1 IL/08 (square) at 1 MOI and the total cellular RNA was extracted at indicated h p.i.. Interferon (IFN)- λ1, λ3, and β gene expression was analyzed by RT-qPCR. The ΔCT values were normalized to β-actin gene expression and represented as fold change (2^-ΔΔCT) over mock infected cells. The experiment was performed in duplicate using cells derived from two donor animals. *p <0.05.</p

Replication of IAV in pAECs.

<p>(A) Primary pAECs were infected with the indicated virus at MOI of 0.01 without exogenous trypsin and the supernatants were collected at 0, 4, 8, 12, 24, 48, 72, and 96 h p.i.. Virus titers were determined by TCID₅₀ assay in MDCK cells in the presence of 0.5 μg/ml TPCK-trypsin. (B) Titer of IAV pH1N1 CA/09 and H1N1 IL/08 in the culture supernatant of ipAECs 72 h p.i in the presence and absence of exogenous TPCK-trypsin. Data are average of 2 independent experiments (n = 2).</p

Conditioned medium from Poly I:C stimulated cultures differentially inhibits IAV replication in pAECs.

<p>(A) Titers of IAV pH1N1 CA/09 and H1N1 IL/08 in pAECs pretreated with conditioned medium. Cells were treated with indicated concentrations of poly I:C conditioned medium (poly I:C CM) or mock CM for 8 h prior to infection with IAV (0.01 MOI) as described in Materials and Methods. Culture supernatant collected 24 h p.i. were assessed by TCID₅₀ assay on MDCK cells. Data is expressed as the mean ± SEM of two independent experiments performed in duplicate. ND; not detected. (B) The percent reduction in virus titer was calculated from data in (A) and expressed as percent of mock CM treated control. (C) Analysis of IFNs in pAECs treated with poly I:C 1 h after infection with H1N1 IL/08. pAECs were infected with H1N1 IL/08 at 3 MOI and 50 μg/ml poly I:C was added 1 h p.i. Total RNA was extracted at 12 h p.i. and the expression of IFNs was analyzed by RT-qPCR. Data are presented as the mean ± SEM of two independent experiments performed in triplicate. *p <0.05.</p