Bayram Edemir

Organic cation transporter 2 mediates cisplatin-induced oto- and nephrotoxicity and is a target for protective interventions.

The use of the effective antineoplastic agent cisplatin is limited by its serious side effects, such as oto- and nephrotoxicity. Ototoxicity is a problem of special importance in children, because deafness hampers their language and psychosocial development. Recently, organic cation transporters (OCTs) were identified in vitro as cellular uptake mechanisms for cisplatin. In the present study, we investigated in an in vivo model the role of OCTs in the development of cisplatin oto- and nephrotoxicity. The functional effects of cisplatin treatment on kidney (24 hours excretion of glucose, water, and protein) and hearing (auditory brainstem response) were studied in wild-type and OCT1/2 double-knockout (KO) mice. No sign of ototoxicity and only mild nephrotoxicity were observed after cisplatin treatment of knockout mice. Comedication of wild-type mice with cisplatin and the organic cation cimetidine protected from ototoxicity and partly from nephrotoxicity. For the first time we showed that OCT2 is expressed in hair cells of the cochlea. Furthermore, cisplatin-sensitive cell lines from pediatric tumors showed no expression of mRNA for OCTs, indicating the feasibility of therapeutic approaches aimed to reduce cisplatin toxicities by competing OCT2-mediated cisplatin uptake in renal proximal tubular and cochlear hair cells. These findings are very important to establish chemotherapeutical protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.

Individual PKC-Phosphorylation Sites in Organic Cation Transporter 1 Determine Substrate Selectivity and Transport Regulation

To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after site-directed mutagenesis of putative PKC phosphorylation-sites were compared with wild-type (WT) rOCT1 using microfluorometric measurements with the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). Either substitutions of single (S286A, S292A, T296A, S328A, and T550A) or of all five PKC-sites (5x-PKC) with alanine suppressed PKC-induced stimulation of ASP(+) uptake, whereas regulation by p56(lck) tyrosine kinase was conserved in all mutants. Remarkably, the apparent affinities for TEA(+), TPA(+), and quinine were changed differently in each mutant (EC(50) in WT, S286A, S292A, T296A, S328A, T550A, and 5x-PKC in mumol: TEA(+): 105, 153, 56, 1135, 484, 498, 518; TPA(+): 0.1, 2.1, 0.3, 1.0, 43, 0.3, 2.2; quinine: 1.5, 3.0, 2.5, 4.8, 81, 7.6, 8.9, respectively). After mutations, no effects of PKC activation on apparent affinity of rOCT1 for these substrates could be detected, in contrast to what was observed in WT. PKC activation had no significant effect on rOCT1 trafficking from intracellular pools to the cell membrane. Substitution of all PKC sites suppressed PKC-induced phosphorylation of rOCT1. In conclusion, it was found that the presence of all five potential PKC phosphorylation sites is necessary for the PKC-induced stimulation of rOCT1. The different effects on the EC(50) values by the different mutations suggest that the large intracellular loop participates in building the substrate binding pocket of rOCT1 or specifically modulates its structure.

Atrial natriuretic peptide and nitric oxide signaling antagonizes vasopressin-mediated water permeability in inner medullary collecting duct cells

AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An ANP- and nitric oxide (NO)/soluble guanylate cyclase (sGC)-induced insertion of AQP2 into the plasma membrane is reported from different models. However, functional data on the insertion of AQP2 is missing. We used primary cultured inner medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein-quenching measurements, and immunofluorescence and Western blotting to analyze the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression, and water permeability. While AVP led to acceleration in osmotically induced swelling, ANP had no effect. However, in AVP-pretreated cells ANP significantly decreased the kinetics of cell swelling. This effect was mimicked by 8-bromo-cGMP and blunted by PKG inhibition. Stimulation of the NO/sGC pathway or direct activation of sGC with BAY 58-2667 had similar effects to ANP. In cells treated with AVP, AQP2 was predominantly localized in the plasma membrane, and after additional incubation with ANP AQP2 was mostly localized in the cytosol, indicating an increased retrieval of AQP2 from the plasma membrane by ANP. Western blot analysis showed that ANP was able to reduce AVP-induced phosphorylation of AQP2 at position S256. In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a decreased localization of AQP2 in the plasma membrane which is mediated by cGMP and PKG.

Organic cation transporters OCT1, 2, and 3 mediate high-affinity transport of the mutagenic vital dye ethidium in the kidney proximal tubule

The positively charged fluorescent dyes ethidium (Et(+)) and propidium (Pr(2+)) are widely used as DNA and necrosis markers. Et(+) is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (< or =500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCT1/2 are involved. In mouse kidney, perfused Et(+) accumulated predominantly in the S2/S3 segments of the PT, but not Pr(2+). In cells stably overexpressing human OCTs (hOCTs), Et(+) uptake was observed with K(m) values of 0.8 +/- 0.2 microM (hOCT1), 1.7 +/- 0.5 microM (hOCT2), and 2.0 +/- 0.5 microM (hOCT3), whereas Pr(2+) was not transported. Accumulation of Et(+) was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP(+)), cimetidine, and tetraethylammonium (TEA(+)). For hOCT1 and hOCT2, the IC(50) values for MPP(+), TEA(+), and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et(+) uptake by MPP(+) and cimetidine was shown to be competitive. Et(+) also inhibited transport of 0.1 microM [(3)H]MPP(+) by all hOCT isoforms with IC(50) values between 0.4 and 1.3 microM, and the inhibition of hOCT1-mediated uptake of MPP(+) by Et(+) was competitive. In Oct1/2(-/-) mice, Et(+) uptake in the PT was almost abolished. The data demonstrate that Et(+) is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et(+) and their strong expression in various organs, strict safety guidelines for Et(+) handling should be reinforced.

Translational 18F-FDG PET/CT Imaging to Monitor Lesion Activity in Intestinal Inflammation

In patients with inflammatory bowel disease (IBD) and in murine IBD models, mucosal disease activity is routinely assessed by endoscopy and histologic evaluation. This information is valuable for monitoring treatment response, with mucosal healing being a major treatment goal. The aim of this study was to evaluate the translational potential of noninvasive 18F-FDG PET/CT for the assessment of mucosal damage in murine dextran sodium sulfate (DSS) colitis and human IBD. Methods: After induction of DSS colitis, 18F-FDG uptake was serially assessed from colonic volumes of interest defined on PET/CT scans and intraindividually correlated to histologic findings and to infiltrating cell types. In addition, 18F-FDG PET/CT scans of 25 Crohn disease patients were analyzed, and colonic 18F-FDG uptake was correlated to endoscopically assessed damage. Results: At days 4 and 7 after DSS induction, colonic 18F-FDG uptake was significantly increased, with a distinct peak in the medial colon. 18F-FDG uptake strongly correlated with histologic epithelial damage. Additionally, 18F-FDG uptake increased in the bone marrow in the course of the disease, correlating with an increase in intestinal 18F-FDG uptake. Histology and fluorescence-activated cell sorting analysis of the bone marrow of DSS mice revealed an increased number of immature neutrophils, whereas mucosal polymerase chain reaction suggested a correlation of 18F-FDG uptake to T cell infiltration. In accordance with the results of 18F-FDG PET/CT in DSS colitis, an increased 18F-FDG uptake was found in 87% of deep mucosal ulcerations in IBD patients, whereas mild endoscopic lesions were detected only by 18F-FDG PET/CT in about 50% of patients assessed. Conclusion: 18F-FDG PET/CT is a noninvasive method for evaluation of both experimental colitis and Crohn disease patients and thereby offers promising translational potential.

Acute Rejection After Rat Renal Transplantation Leads to Downregulation of Na + and Water Channels in the Collecting Duct

Renal transplantation is associated with alterations of tubular functions and of the renin–angiotensin–aldosterone system. The underlying cellular and molecular mechanisms are unclear. We used an allogeneic rat renal transplantation model of acute rejection with and without immunosuppression by cyclosporine A (CsA) and a syngeneic model as control. Uninephrectomized Lewis or Lewis–Brown‐Norway (LBN) rats received a kidney from LBN‐rats. Renal transporters and receptors were analyzed by immunohistochemistry, semiquantitative RT‐PCR and Western‐blot analysis. Intracellular Na+ was analyzed microfluorimetrically in isolated cortical collecting ducts. mRNA expression and function of the epithelial Na+‐channel (ENaC) and mRNA and protein expression of the water‐channel AQP2 were downregulated in transplanted kidneys undergoing rejection. Expression of the serum‐ and glucocorticoid‐kinase (Sgk1) was decreased and that of the ubiquitin–protein ligase Nedd4‐2 was increased. These changes were absent under CsA‐therapy and in syngeneic model. Expression and function of the Na+–K+‐ATPase, expression of the secretory K+‐channel and of the mineralocorticoid receptor remained unchanged. Reduced ENaC function is likely due to decreased Sgk1‐ and increased Nedd4‐2 mRNA expression leading to reduced ENaC expression in the membrane. These acute downregulations of ENaC and AQP2 may be triggered to reduce energy consumption in the distal nephron to protect the kidney immediately after transplantation.

Activation of counter-regulatory mechanisms in a rat renal acute rejection model

BackgroundMicroarray analysis provides a powerful approach to identify gene expression alterations following transplantation. In patients the heterogeneity of graft specimens, co-morbidity, co-medications and the challenges in sample collection and preparation complicate conclusions regarding the underlying mechanisms of graft injury, rejection and immune regulation.ResultsWe used a rat kidney transplantation model with strict transplant and sample preparation procedures to analyze genome wide changes in gene expression four days after syngeneic and allogeneic transplantation. Both interventions were associated with substantial changes in gene expression. After allogeneic transplantation, genes and pathways related to transport and metabolism were predominantly down-regulated consistent with rejection-mediated graft injury and dysfunction. Up-regulated genes were primarily related to the acute immune response including antigen presentation, T-cell receptor signaling, apoptosis, interferon signaling and complement cascades. We observed a cytokine and chemokine expression profile consistent with activation of a Th1-cell response. A novel finding was up-regulation of several regulatory and protective genes after allogeneic transplantation, specifically IL10, Bcl2a1, C4bpa, Ctla4, HO-1 and the SOCS family.ConclusionOur data indicate that in parallel with the predicted activation of immune response and tissue injury pathways, there is simultaneous activation of pathways for counter regulatory and protective mechanisms that would balance and limit the ongoing inflammatory/immune responses. The pathophysiological mechanisms behind and the clinical consequences of alterations in expression of these gene classes in acute rejection, injury and dysfunction vs. protection and immunoregulation, prompt further analyses and open new aspects for therapeutic approaches.

Renal Transplantation Modulates Expression and Function of Receptors and Transporters of Rat Proximal Tubules

Kidney transplantation often leads to disturbances of solute and volume maintenance in humans. To investigate underlying mechanisms, expression and function of renal transporters and receptors of the proximal tubule (PT) were analyzed in an acute rejection model of rat kidney transplantation. Semiquantitative RT-PCR and Western blot, histology, immunohistochemistry, and microfluorometry were performed on whole kidneys and isolated PT. With acute rejection, Na+/H+-exchanger type-3 (NHE-3) was markedly downregulated. Na+-HCO(3)(-)-cotransporter (NBC-1) and Na+-glucose transporter type-2 (SGLT2) were upregulated after transplantation. Expressions of Na+/H+-exchanger type-1 (NHE-1), Na+/K+-ATPase (NKA), angiotensin II (AngII) receptor (AT-1), or natriuretic peptide receptor (GC-A) were unaltered. Microfluorometric analyses of intracellular pH, Na+, and Ca2+ demonstrated a decrease in NHE-3 function and AngII-mediated stimulation of NHE-3. AngII-mediated inhibition of NHE-1 and function of all other transporters tested remained unaltered. Function of AT-1 and GC-A were unaffected. Reduced expression of NHE-3 was also confirmed by semiquantitative immunohistochemistry. These findings suggest that expression and function of transmembrane proteins involved in Na+-transport after transplantation and rejection is specifically modulated. The local renin-angiotensin-system is apparently not altered. Downregulation of NHE-3 may be a protective mechanism occurring in the graft.

Potential of Noninvasive Serial Assessment of Acute Renal Allograft Rejection by 18F-FDG PET to Monitor Treatment Efficiency

We propose 18F-FDG PET as a method to monitor acute rejection of allogeneic renal transplants in a rat model. Methods: Allogeneically transplanted (aTX) rats (binephrectomized Lewis–brown Norway to Lewis) served as the renal transplant model. aTX rats treated with cyclosporine A (CSA) served as a therapy monitoring group. Healthy control rats, rats with acute CSA nephrotoxicity, rats with acute tubular necrosis, syngeneically transplanted (sTX) rats, and aTX rats treated with CSA since postoperative day 0 served as controls. After surgery, renal glucose metabolism was assessed in vivo serially up to postoperative day 7 by performing small-animal PET 3 h after intravenous injection of 30 MBq of 18F-FDG. Mean radioactivity (cps/mm3 of tissue) was measured and the percentage injected dose calculated. Results were confirmed by histologic, functional, and autoradiographic analysis. Results: Renal 18F-FDG uptake was significantly elevated at postoperative day 4 in aTX rats, when compared with control, sTX, acute tubular necrosis, or CSA-treated rats (P < 0.05). In vivo 18F-FDG uptake correlated with the results of autoradiography and with inflammatory infiltrates observed on histologic examination. Notably, 18F-FDG PET assessed the response to therapy 48 h earlier than the time at which serum creatinine decreased and when histologic examination still showed signs of allograft rejection. In aTX rats, the CSA-susceptible graft infiltrate was dominated by activated cytotoxic T cells and monocytes/macrophages. Conclusion: 18F-FDG PET is an option to noninvasively assess early response to therapy in rat renal allograft rejection.

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 18F-fluoride Positron Emission Tomography (PET)), creatinine- and urea-clearances improved significantly. Moreover, endothelial leakage and renal inflammation was significantly reduced as determined by histology, 18F-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.