Hydroxyfasudil-mediated inhibition of ROCK1 and ROCK2 improves kidney function in rat renal acute ischemia-reperfusion injury

Renal ischemia-reperfusion (IR) injury (IRI) is a common and important trigger of acute renal injury (AKI). It is inevitably linked to transplantation. Involving both, the innate and the adaptive immune response, IRI causes subsequent sterile inflammation. Attraction to and transmigration of immune cells into the interstitium is associated with increased vascular permeability and loss of endothelial and tubular epithelial cell integrity. Considering the important role of cytoskeletal reorganization, mainly regulated by RhoGTPases, in the development of IRI we hypothesized that a preventive, selective inhibition of the Rho effector Rho-associated coiled coil containing protein kinase (ROCK) by hydroxyfasudil may improve renal IRI outcome. Using an IRI-based animal model of AKI in male Sprague Dawley rats, animals treated with hydroxyfasudil showed reduced proteinuria and polyuria as well as increased urine osmolarity when compared with sham-treated animals. In addition, renal perfusion (as assessed by (18)F-fluoride Positron Emission Tomography (PET)), creatinine- and urea-clearances improved significantly. Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, (18)F-fluordesoxyglucose-microautoradiography, Evans Blue, and real-time PCR analysis. We conclude from our study that ROCK-inhibition by hydroxyfasudil significantly improves kidney function in a rat model of acute renal IRI and is therefore a potential new therapeutic option in humans.     

Transport Mechanisms and Their Pathology-Induced Regulation Govern Tyrosine Kinase Inhibitor Delivery in Rheumatoid Arthritis

Background

Tyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions.

Methodology/Principal Findings

The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1β and IL-6, which all decreased the expression of hMATE1-mRNA and protein.

Conclusion/Significance

The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.     

The COP9 signalosome counteracts the accumulation of cullin SCF ubiquitin E3 RING ligases during fungal development

Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.     

Coordination of secondary metabolism and development in fungi: the velvet family of regulatory proteins

Filamentous fungi produce a number of small bioactive molecules as part of their secondary metabolism ranging from benign antibiotics such as penicillin to threatening mycotoxins such as aflatoxin. Secondary metabolism can be linked to fungal developmental programs in response to various abiotic or biotic external triggers. The velvet family of regulatory proteins plays a key role in coordinating secondary metabolism and differentiation processes such as asexual or sexual sporulation and sclerotia or fruiting body formation. The velvet family shares a protein domain that is present in most parts of the fungal kingdom from chytrids to basidiomycetes. Most of the current knowledge derives from the model Aspergillus nidulans where VeA, the founding member of the protein family, was discovered almost half a century ago. Different members of the velvet protein family interact with each other and the nonvelvet protein LaeA, primarily in the nucleus. LaeA is a methyltransferase-domain protein that functions as a regulator of secondary metabolism and development. A comprehensive picture of the molecular interplay between the velvet domain protein family, LaeA and other nuclear regulatory proteins in response to various signal transduction pathway starts to emerge from a jigsaw puzzle of several recent studies.     

Apoptotic, antioxidant and antiradical effects of majdine and isomajdine from Vinca herbacea Waldst. and kit

In the present study, apoptotic, antioxidant and antiradical effects of majdine and isomajdine from Vinca herbacea Waldst. and Kit were studied. For testing the possible apoptotic effects of majdine and isomajdine from V. herbacea, DNA fragmentation assay was conducted on the rat brain cortical tissue homogenates, in vitro. Also their possible effects on mitochondrial activity were tested by using the same tissue samples of rats. In addition, the antioxidant activity of isomajdine and majdine was determined using various in vitro antioxidant assays, including 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS(?+)) radical scavenging and N,N-dimethyl-p-phenylenediamine (DMPD(?+)) radical scavenging, ferric ions (Fe(3+)) and cupric ions (Cu(2+)) reducing abilities and ferrous ions (Fe(2+)) chelating activity. On the other hand, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were used as reference antioxidants.     

Evaluation of the Fas/FasL signaling pathway in diabetic rat testis

We investigated the role of the Fas/Fas ligand (FasL) signaling pathway in diabetic male infertility. Male rats were divided into two groups: a control group and a streptozotocin induced diabetic group. Thirty days after induction of diabetes, samples of testes were harvested and fixed in 10% formalin for light microscopy. Germ cell apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end-labeling (TUNEL) and immunostaining of caspase 8 and active caspase 3. We also investigated the expressions of Fas and FasL using immunohistochemistry. Streptozotocin-induced diabetes caused severe histopathological damage and increased apoptotic tubule and apoptotic cell indices, caspase 8 and caspase 3 expressions, and Fas and FasL-immunopositive cells in the rat testes. We suggest that the Fas/FasL signaling pathway may play a role in male infertility caused by diabetes.     

TGF-Mediated Dynamics in a System of Many Coupled Nephrons

This paper presents a mathematical model of a system of many coupled nephrons branching from a common cortical radial artery, and accompanying analysis of that system. This modeling effort is a first step in understanding how coupling magnifies the tendency of nephrons to oscillate owing to tubuloglomerular feedback. Central to the present work is the single nephron integral model (as in Pitman et al., The IMA Volumes in Mathematics and Its Applications, vol. 129, pp. 345–364, 2002 and in Zaritski, Ph.D. Dissertation, 1999) which is a simplification of the single nephron PDE model of Layton et al. (Am. J. Physiol. 261, F904–F919, 1991). A second principal idea used in the present model is a coupling of model nephrons, generalizing the work of Pitman et al. (Bull. Math. Biol. 66, 1463–1492, 2004) who proposed a model of two coupled nephrons. In this study, we couple nephrons through a nearest neighbor interaction. Speaking generally, our results suggest that a series of similar nephrons coupled to their nearest neighbors are more prone to be found in an oscillatory mode, relative to a single nephron with the same properties. More specifically, we show analytically that, for N coupled identical nephrons, the region supporting oscillatory solutions in the time delay–gain parameter plane increases with N. Numerical simulations suggest that, if N nephrons have gains and time delays that do not differ by much, the system is, again, more prone to oscillate, relative to a single nephron, and the oscillations tend to be approximately synchronous and in-phase. We examine the effect of parameters on bifurcation. We also examine alternative models of coupling; this analysis allows us to conclude that the increased propensity of coupled nephrons to oscillate is a robust finding, true for several models of nephron interaction.     

Fungal development and the COP9 signalosome

The conserved COP9 signalosome (CSN) multiprotein complex is located at the interface between cellular signaling, protein modification, life span and the development of multicellular organisms. CSN is required for light-controlled responses in filamentous fungi. This includes the circadian rhythm of Neurospora crassa or the repression of sexual development by light in Aspergillus nidulans. In contrast to plants and animals, CSN is not essential for fungal viability. Therefore fungi are suitable models to study CSN composition, activity and cellular functions and its role in light controlled development.     

The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts

Arthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra-terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA-seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT-qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFβ1-induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA-mediated depletion of BRD4. Together, these data reveal that BRD4-mediated epigenetic events support TGFβ1-mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.