Effect of the Monocyte Chemoattractant Protein-1/CC Chemokine Receptor 2 System on Nephrin Expression in Streptozotocin-Treated Mice and Human Cultured Podocytes

OBJECTIVE-Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes. RESEARCH DESIGN AND METHODS-Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting. RESULTS-In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin. CONCLUSIONS-These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes

Spiral ligament fibrocyte-derived MCP-1/CCL2 contributes to inner ear inflammation secondary to nontypeable H. influenzae-induced otitis media

<p>Abstract</p> <p>Background</p> <p>Otitis media (OM), one of the most common pediatric infectious diseases, causes inner ear inflammation resulting in vertigo and sensorineural hearing loss. Previously, we showed that spiral ligament fibrocytes (SLFs) recognize OM pathogens and up-regulate chemokines. Here, we aim to determine a key molecule derived from SLFs, contributing to OM-induced inner ear inflammation.</p> <p>Methods</p> <p>Live NTHI was injected into the murine middle ear through the tympanic membrane, and histological analysis was performed after harvesting the temporal bones. Migration assays were conducted using the conditioned medium of NTHI-exposed SLFs with and without inhibition of MCP-1/CCL2 and CCR2. qRT-PCR analysis was performed to demonstrate a compensatory up-regulation of alternative genes induced by the targeting of MCP-1/CCL2 or CCR2.</p> <p>Results</p> <p>Transtympanic inoculation of live NTHI developed serous and purulent labyrinthitis after clearance of OM. THP-1 cells actively migrated and invaded the extracellular matrix in response to the conditioned medium of NTHI-exposed SLFs. This migratory activity was markedly inhibited by the viral CC chemokine inhibitor and the deficiency of MCP-1/CCL2, indicating that MCP-1/CCL2 is a main attractant of THP-1 cells among the SLF-derived molecules. We further demonstrated that CCR2 deficiency inhibits migration of monocyte-like cells in response to NTHI-induced SLF-derived molecules. Immunolabeling showed an increase in MCP-1/CCL2 expression in the cochlear lateral wall of the NTHI-inoculated group. Contrary to the <it>in vitro </it>data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation <it>in vivo</it>. We demonstrated that targeting MCP-1/CCL2 enhances NTHI-induced up-regulation of MCP-2/CCL8 in SLFs and up-regulates the basal expression of CCR2 in the splenocytes. We also found that targeting CCR2 enhances NTHI-induced up-regulation of MCP-1/CCL2 in SLFs.</p> <p>Conclusions</p> <p>Taken together, we suggest that NTHI-induced SLF-derived MCP-1/CCL2 is a key molecule contributing to inner ear inflammation through CCR2-mediated recruitment of monocytes. However, deficiency of MCP-1/CCL2 or CCR2 alone was limited to inhibit OM-induced inner ear inflammation due to compensation of alternative genes.</p

Fault detection in sequential machines

Sequences and techniques useful in the design of checking experiments are examined. Necessary and sufficient conditions are formulated for a machine to possess distinguishing and synchronizing sequences. Least upper bounds are placed upon the length of repeated symbol distinguishing and repeated symbol synchronizing sequences. An improperly designed test sequence for the state identification phase of a checking experiment is given to illustrate a faulty machine which is not detected. The restrictions necessary to impose upon machines to make the testing problem tractable are discussed. It is shown these restrictions are not overly burdensome. Some useful properties of compound distinguishing sequences are proved. Characterizing sequences are examined in detail and a previous bound is reduced by 50 percent to obtain the least upper bound on their total length. The reduced bounds on characterizing sequences are used in the construction of locating sequences. A series of theorems puts the locating sequence concept on a firm theoretical foundation. The precise conditions needed to insure locating sequences always transfer a machine to the same state are delineated and proved. Better use is made of the strong connectedness of tested machines to achieve a 50 percent reduction over previous bounds on the length of transfer sequences. Both the reduced bounds on characterizing and on transfer sequences are used to derive a bound smaller than those published on the length of checking experiments for a machine possessing a simple I/O sequence and a valid homing sequence. A resettable machine with and without a unique output terminal to identify the reset state is examined to determine the effect of the reset feature upon the length of checking experiments for machines possessing valid homing, distinguishing, and locating sequences. It is shown that resettable machines possessing valid homing sequences achieve about a 20 percent reduction over bounds derived for non-resettable machines. Resettable machines possessing distinguishing sequences achieve about a 50 percent reduction over non-resettable machines, and resettable machines possessing only locating sequences achieve about a 95 percent reduction over the non-resettable machine. In each of the above classes of machines, no assumptions are made on the correct operation of the reset feature. Instead the machine's reset is thoroughly tested as an integral part of the checking experiment. The experiment design procedures and the upper bound derivations are clearly given to illustrate the testing advantages inherent in each of the classes of resettable machines examined.Electrical and Computer Engineering, Department o

Role of Hepatic Organic Anion Transporter 2 in the Pharmacokinetics of <i>R</i>- and <i>S</i>‑Warfarin: In Vitro Studies and Mechanistic Evaluation

Interindividual variability in warfarin dose requirement demands personalized medicine approaches to balance its therapeutic benefits (anticoagulation) and bleeding risk. Cytochrome P450 2C9 (<i>CYP2C9</i>) genotype-guided warfarin dosing is recommended in the clinic, given the more potent <i>S</i>-warfarin is primarily metabolized by CYP2C9. However, only about 20–30% of interpatient variability in <i>S</i>-warfarin clearance is associated with <i>CYP2C9</i> genotype. We evaluated the role of hepatic uptake in the clearance of <i>R</i>- and <i>S</i>-warfarin. Using stably transfected HEK293 cells, both enantiomers were found to be substrates of organic anion transporter (OAT)­2 with a Michaelis–Menten constant (<i>K</i>_m) of ∼7–12 μM but did not show substrate affinity for other major hepatic uptake transporters. Uptake of both enantiomers by primary human hepatocytes was saturable (<i>K</i>_m ≈ 7–10 μM) and inhibitable by OAT2 inhibitors (e.g., ketoprofen) but not by OATP1B1/1B3 inhibitors (e.g., cyclosporine). To further evaluate the potential role of hepatic uptake in <i>R</i>- and <i>S</i>-warfarin pharmacokinetics, mechanistic modeling and simulations were conducted. A “bottom-up” PBPK model, developed assuming that OAT2–CYPs interplay, well recovered clinical pharmacokinetics, drug–drug interactions, and <i>CYP2C9</i> pharmacogenomics of <i>R</i>- and <i>S</i>-warfarin. Clinical data were not available to directly verify the impact of OAT2 modulation on warfarin pharmacokinetics; however, the bottom-up PBPK model simulations suggested a proportional change in clearance of both warfarin enantiomers with inhibition of OAT2 activity. These results suggest that variable hepatic OAT2 function, in conjunction with CYP2C, may contribute to the high population variability in warfarin pharmacokinetics and possibly anticoagulation end points and thus warrant further clinical investigation

<i>In vitro</i> studies with two human organic anion transporters: OAT2 and OAT7

<p>1.Penciclovir, ganciclovir, creatinine, <i>para</i>-aminohippuric acid (PAH), ketoprofen, estrone 3-O-sulfate (E3S), dehydroepiandrosterone 3-O-sulfate (DHEAS) and cyclic guanosine monophosphate (cGMP) were screened as substrates of human liver organic anion transporters OAT2 and OAT7.</p> <p>2.For OAT7, high uptake ratios (versus mock transfected HEK293 cells) of 29.6 and 15.3 were obtained with E3S and DHEAS. Less robust uptake ratios (≤3.6) were evident with the other substrates. OAT2 (transcript variant 1, OAT2-tv1) presented high uptake ratios of 30, 13, ∼35, ∼25, 8.5 and 9 with cGMP, PAH, penciclovir, ganciclovir, creatinine and E3S, respectively. No uptake was observed with DHEAS.</p> <p>3.Although not a substrate of either transporter, ketoprofen did inhibit transfected OAT2-tv1 (IC₅₀ of 17, 22, 23, 24, 35 and 586 μM; creatinine, ganciclovir, penciclovir, cGMP, E3S and prostaglandin F2α, respectively) and penciclovir uptake (IC₅₀ = 27 µM; >90% inhibition) by plated human hepatocytes (PHH).</p> <p>4.It is concluded that penciclovir and ketoprofen may serve as useful tools for the assessment of OAT2 activity in PHH. However, measurement of OAT7 activity therein will prove more challenging, as high uptake rates are evident with E3S and DHEAS only and both sulfoconjugates are known to be substrates of organic anion transporting polypeptides.</p