The effect of mycophenolate mofetil on podocytes in nephrotoxic serum nephritis.

Mycophenolate mofetil (MMF) is applied in proteinuric kidney diseases, but the exact mechanism of its effect on podocytes is still unknown. Our previous in vitro experiments suggested that MMF can ameliorate podocyte damage via restoration of the Ca2+-actin cytoskeleton axis. The goal of this study was to characterize podocyte biology during MMF treatment in nephrotoxic serum (NTS) nephritis (NTN). NTN was induced in three-week old wild-type mice. On day 3, half of the mice were treated with MMF (100 mg/kgBW/d p.o.) for one week. On day 10, we performed proteomic analysis of glomeruli as well as super-resolution imaging of the slit diaphragm. For multiphoton imaging of Ca2+ concentration ([Ca2+]i), the experimental design was repeated in mice expressing podocyte-specific Ca2+ sensor. MMF ameliorated the proteinuria and crescent formation induced by NTS. We identified significant changes in the abundance of proteins involved in Ca2+ signaling and actin cytoskeleton regulation, which was further confirmed by direct [Ca2+]i imaging in podocytes showing decreased Ca2+ levels after MMF treatment. This was associated with a tendency to restoration of podocyte foot process structure. Here, we provide evidence that MPA has a substantial direct effect on podocytes. MMF contributes to improvement of [Ca2+]i and amelioration of the disorganized actin cytoskeleton in podocytes. These data extend the knowledge of direct effects of immunosuppressants on podocytes that may contribute to a more effective treatment of proteinuric glomerulopathies with the least possible side effects.

Upregulation of RGS7 may contribute to tumor necrosis factor-induced changes in central nervous function.

The central nervous dysfunctions of lethargy, fever and anorexia are manifestations of sepsis that seem to be mediated by increased cytokine production. Here we demonstrate that tumor necrosis factor (TNF)-alpha, an essential mediator of endotoxin-induced sepsis, prevents the proteasome-dependent degradation of RGS7, a regulator of G-protein signaling. The stabilization of RGS7 by TNF-alpha requires activation of the stress-activated protein kinase p38 and the presence of candidate mitogen-activated protein kinase phosphorylation sites. In vivo, RGS7 is rapidly upregulated in mouse brain after exposure to either endotoxin or TNF-alpha, a response that is nearly abrogated in mice lacking TNF receptor 1. Our findings indicate that TNF-mediated upregulation of RGS7 may contribute to sepsis-induced changes in central nervous function.

Inhibition of the production of endothelium-derived hyperpolarizing factor by cannabinoid receptor agonists.

1. The endogenous cannabinoid, anandamide, has been reported to induce an 'endothelium-derived hyperpolarizing factor (EDHF)-like' relaxation in vitro. We therefore investigated the effects of cannabinoid CB1 receptor agonists; HU 210, delta9-tetrahydrocannabinol (delta9-THC) and anandamide, and a CB1 antagonist/inverse agonist, SR 141716A, on nitric oxide (NO) and EDHF-mediated relaxation in precontracted rings of porcine coronary, rabbit carotid and mesenteric arteries. 2. In rings of mesenteric artery HU 210 and delta9-THC induced endothelium- and cyclo-oxygenase-independent relaxations which were sensitive to SR 141716A. Anandamide (0.03-30 microM) induced a slowly developing, endothelium-independent relaxation which was abolished by diclofenac and was therefore mediated by cyclo-oxygenase product(s). None of the CB1 agonists tested affected the tone of precontracted rings of rabbit carotid or porcine coronary artery. 3. In endothelium-intact segments, HU 210, delta9-THC and anandamide did not affect NO-mediated responses but under conditions of continuous NO synthase/cyclo-oxygenase blockade, significantly inhibited acetylcholine and bradykinin-induced relaxations which are attributed to the production of EDHF. The effects of HU 210 and delta9-THC were not observed when experiments were performed in the presence of SR 141716A suggesting the involvement of the CB1 receptor. 4. In a patch clamp bioassay of EDHF production, HU 210 decreased the EDHF-mediated hyperpolarization of detector smooth muscle cells when applied to the donor segment but was without effect on the membrane potential of detector cells. The inhibition of EDHF production was unrelated to alterations in Ca2+ -signalling or cytochrome P450 activity. 5. These results suggest that the activation of endothelial CB1 receptors appears to be negatively coupled to the production of EDHF.

14-3-3 interacts with regulator of G protein signaling proteins and modulates their activity.

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) that stimulate the inactivation of heterotrimeric G proteins. We have recently shown that RGS proteins may be regulated on a post-translational level (Benzing, T., Brandes, R., Sellin, L., Schermer, B., Lecker, S., Walz, G., and Kim, E. (1999) Nat. Med. 5, 913-918). However, mechanisms controlling the GAP activity of RGS proteins are poorly understood. Here we show that 14-3-3 proteins associate with RGS7 and RGS3. Binding of 14-3-3 is mediated by a conserved phosphoserine located in the Galpha-interacting portion of the RGS domain; interaction with 14-3-3 inhibits the GAP activity of RGS7, depends upon phosphorylation of a conserved residue within the RGS domain, and results in inhibition of GAP function. Collectively, these data indicate that phosphorylation-dependent binding of 14-3-3 may act as molecular switch that controls the GAP activity keeping a substantial fraction of RGS proteins in a dormant state.