Functions of N- and C-terminal protein domains for assembly, subcellular localization and physiology of TRP ion channels in Drosophila photoreceptors

In den Photorezeptorzellen von Drosophila melanogaster sind die Kationen-Kanäle TRP und TRPL für die Erzeugung des Rezeptorpotentials verantwortlich. Es ist bekannt, dass der TRPL-Ionenkanal lichtabhängig seine subzelluläre Lokalisation verändert, während TRP so-wohl in dunkel- als auch in hell-adaptierten Augen im Rhabdomer lokalisiert ist. Des Weiteren gilt als gesichert, dass diese Ionenkanäle als Tetramere vorliegen. In der wissenschaftlichen Literatur wird jedoch kontrovers diskutiert, ob TRP und TRPL nur als Homomultimere oder auch als Heteromultimere auftreten. In der vorliegenden Arbeit wurde mittels Co-Immunopräzipitationen (Co-IPs) von unmarkierten TRP- oder TRPL-Kanälen gezeigt, dass die tetrameren Kanäle in den Photore-zeptoren von Drosophila ausschließlich aus Homomultimeren bestehen. Co-IPs von eGFP-markierten TRP- oder TRPL-Kanälen zeigten, dass sich die Tetramere aus eGFP-markierten und den entsprechenden endogenen Kanaluntereinheiten zusammensetzen. Um die biochemischen und physiologischen Eigenschaften der cytosolischen N- und C-Termini von TRP und TRPL untersuchen zu können, wurden im Rahmen dieser Arbeit eGFP-markierte chimäre TRP/TRPL-Ionenkanäle konstruiert und in den Photorezeptorzellen R1-R6 von Drosophila exprimiert. Speziell wurde die Auswirkung dieser Termini auf die Translokation der Ionenkanäle zwischen Rhabdomer und Zellkörper sowie auf die Assemb-lierung der Tetramere untersucht. Untersuchungen zur Translokation eGFP-markierter Chimären zeigten, dass die Chimäre mit den TRP-Transmembranbereichen und TRPL N- sowie C-Termini eine TRPL-typische, je-doch zeitlich schnellere, Translokation durchführt. Der Austausch von einem der beiden TRPL-Termini gegen TRP bewirkte, dass die Chimären vorwiegend im Zellkörper lokalisiert waren. Diese Lokalisation entspricht weder der Translokation von TRPL noch der rhabdomerischen Lokalisation von TRP. Sequenzmotive, die die subzelluläre Lokalisation der Drosophila TRP-Kanäle bestimmen, sind daher offenbar sowohl im N- als auch im C-Terminus von TRPL lokalisiert und nur im Zusammenspiel beider Termini wirksam. Co-IPs dieser eGFP-markierten Chimären zeigten, dass für eine Interaktion mit dem TRPL-Kanal der C-Terminus von TRPL wichtiger zu sein scheint als der N-Terminus. Für die Inter-aktion mit dem TRP-Kanal scheint ebenfalls der C-Terminus von TRP wichtiger zu sein als der N-Terminus. Für beide Fälle konnte in einer Chimäre aber auch eine Co-IP von TRPL bzw. TRP über den N-Terminus beobachtet werden. Im Gegensatz zu den Termini werden die Transmembranbereiche beider Kanäle für eine Interaktion nicht benötigt. Unter der Annahme, dass eine Interaktion zwischen einer eGFP-markierten Chimäre und einer endogenen Kanaluntereinheit eine Fehllokalisation dieser endogenen Kanaleinheit ver-ursachen könnte, wurden immuncytochemische Querschnitte durch die Augen dunkel- und hell-adaptierter Fliegen angefertigt. Die Lokalisation der eGFP-markierten Chimären wurde mit Hilfe der eGFP-Fluoreszenz bestimmt, während die Lokalisation der endogenen Kanäle durch spezifische Antikörper-Markierungen ermittelt wurde. Es zeigte sich, dass diejenigen Chimären, die eine starke Interaktion mit den endogenen Kanaluntereinheiten in den Co-IPs zeigen, eine Fehllokalisation der jeweiligen endogenen Kanäle verursachen. Diese Arbeit klärte auf, dass TRP und TRPL in den Photorezeptoren von Drosophila aus-schließlich Homomultimere bilden. Darüber hinaus wurde deutlich, über welche Termini von TRP und TRPL die Homomultimerisierung erfolgt. Des Weiteren wurden Bereiche des TRPLs identifiziert, welche für die TRPL Translokation benötigt werden.In the photoreceptor cells of Drosophila melanogaster, the cation channels TRP and TRPL are responsible for generating the receptor potential. Previous publications have shown that the TRPL ion channel changes its subcellular localization depending on light conditions, while TRP is located in the rhabdomeres irrespective of light conditions. TRP and TRPL form tetramers. However, it is under debate in the scientific literature if TRP and TRPL form ho-momultimers only or also heteromultimers. In the present study, co-immunoprecipitations (Co-IPs) of untagged TRP or TRPL channels demonstrated that the tetrameric channels in the photoreceptors of Drosophila are composed of homomultimers exclusively. Co-IPs of eGFP-tagged TRP or TRPL channels showed that the tetramers consist of eGFP-tagged and the corresponding untagged channel subunits. To study the biochemical and physiological properties of the cytosolic N- and C-termini of TRP and TRPL, eGFP-tagged chimeric TRP/TRPL ion channels were generated and ex-pressed in the photoreceptor cells R1-R6 of Drosophila. The effect of these termini on trans-location of channels between the rhabdomere and the cell body and on ion channel assembly was studied. Studies of the subcellular localization of eGFP-tagged chimeras showed that a chimera com-posed of the transmembrane regions of TRP and both the N- and the C-terminus of TRPL displayed a light-dependent translocation behavior like TRPL. Interestingly, the translocation of this chimera was much faster than the TRPL-eGFP translocation. The exchange of either the N-terminus or the C-terminus of TRPL with the respective termini of TRP caused a locali-zation of these chimeras mainly in the cell body. This localization neither corresponded to the translocation of TRPL nor to the rhabdomeric localization of TRP. Therefore, motifs inducing light-dependent translocation of TRPL must be located in both termini and are only effective in concert. Co-IPs of the eGFP-tagged chimeras demonstrated that the C-terminus of TRPL appears to be more important than the N-terminus of TRPL. For interaction with the TRP channel the C-terminus of TRP also seems to be more important than the N-terminus. TRPL-TRPL or TRP-TRP interaction via the N-terminus could only be observed by Co-IPs in certain chimeras. In contrast to the termini, the transmembrane regions of both ion channels are not necessary for interaction. Assuming that an interaction between the eGFP-tagged chimera and endogenous channel subunits might cause a mislocalization of the endogenous subunit, immunocytochemical stu-dies were carried out. On cross sections through the eyes of dark and light adapted flies ex-pressing eGFP-tagged chimeras the localization of these eGFP-tagged channels was visua-lized by the eGFP fluorescence, while the localization of the endogenous subunits was de-termined by labeling with specific antibodies. It was found that those chimeras which show a strong interaction with the endogenous channels in Co-IPs cause a mislocalization of the corresponding endogenous channels. This thesis clarified that TRP and TRPL exclusively form homomutlimers in the photorecep-tors of Drosophila. Furthermore, it could be shown, which termini are responsible for the TRP and TRPL homomultimerization and which regions of the TRPL ion channel are necessary for TRPL translocation

Peritoneal Expression of SGLT-2, GLUT1, and GLUT3 in Peritoneal Dialysis Patients

Introduction!#!In peritoneal dialysis (PD) patients, the peritoneal membrane is affected by glucose-based solutions used as peritoneal dialysate fluids. This exposure leads to changes of the membrane which may eventually culminate in fibrosis and method failure. In vitro or animal studies demonstrated that glucose transporters are upregulated upon exposure to these solutions. Expression studies of glucose transporters in human peritoneum have not been reported yet.!##!Methods!#!Expression of SGLT-2, GLUT1, and GLUT3 in human peritoneal biopsies was analyzed by real-time polymerase chain reaction and Western blot analysis. The localization of these glucose transporters in the peritoneum was evaluated by immunohistochemistry using a Histo-Score.!##!Results!#!Peritoneal biopsies of patients (healthy controls, uremic, PD, and encapsulating peritoneal sclerosis [EPS]) were analyzed. We found evidence of SGLT-2, GLUT1, and GLUT3 expression in the peritoneal membrane. Protein expression of SGLT-2 increases with PD duration and is significantly enhanced in EPS patients. All transporters were predominantly, but not exclusively, located adjacent to the vessel walls of the peritoneal membrane.!##!Conclusion!#!Our study showed that SGLT-2, GLUT1, and GLUT3 were regularly expressed in the human peritoneum. SGLT-2 was particularly upregulated in PD patients with EPS, suggesting that this upregulation may be associated with pathological changes in the peritoneal membrane in this syndrome. Since preclinical studies in mice show that SGLT-2 inhibitors or downregulation of SGLT-2 ameliorated pathological changes in the peritoneum, SGLT-2 inhibitors may be potentially promising agents for therapy in PD patients that could reduce glucose absorption and delay functional deterioration of the peritoneal membrane in the long term

Enhanced Oxidative DNA-Damage in Peritoneal Dialysis Patients via the TXNIP/TRX Axis

Peritoneal dialysis (PD) is an effective method of renal replacement therapy, providing a high level of patient autonomy. Nevertheless, the long-term use of PD is limited due to deleterious effects of PD fluids to the structure and function of the peritoneal membrane leading to loss of dialysis efficacy. PD patients show excessive oxidative stress compared to controls or chronic kidney disease (CKD) patients not on dialysis. Therefore, defense systems against detrimental events play a pivotal role in the integrity of the peritoneal membrane. The thioredoxin-interacting-protein (TXNIP)/thioredoxin (TRX) system also plays a major role in maintaining the redox homeostasis. We hypothesized that the upregulation of TXNIP negatively influences TRX activity, resulting in enhanced oxidative DNA-damage in PD patients. Therefore, we collected plasma samples and human peritoneal biopsies of healthy controls and PD patients as well. Using ELISA-analysis and immunohistochemistry, we showed that PD patients had elevated TXNIP levels compared to healthy controls. Furthermore, we demonstrated that PD patients had a reduced TRX activity, thereby leading to increased oxidative DNA-damage. Hence, targeting the TXNIP/TRX system as well as the use of oxidative stress scavengers could become promising therapeutic approaches potentially applicable in clinical practice in order to sustain and improve peritoneal membrane function

Under-recognition of Acute Kidney Injury after Cardiac Surgery in the ICU Impedes Early Detection and Prevention

Background!#!Acute kidney injury (AKI) is associated with high morbidity and mortality; therefore, prevention is important. The aim of this study was to systematically assess AKI incidence after cardiac surgery as documented in clinical routine compared to the real incidence because AKI may be under-recognized in clinical practice. Further, its postoperative management was compared to Kidney Disease: Improving Global Outcomes (KDIGO) recommendations because recognition and adequate treatment represent the fundamental cornerstone in the prevention and management of AKI.!##!Methods!#!This retrospective single-center study included n = 100 patients who underwent cardiac surgery with cardiopulmonary bypass. The coded incidence of postoperative AKI during intensive care unit stay after surgery was compared to the real AKI incidence. Furthermore, conformity of postoperative parameters with KDIGO recommendations for AKI prevention and management was reviewed.!##!Results!#!We found a considerable discrepancy between coded and real incidence, and conformity with KDIGO recommendations was found to be relatively low. The coded incidence was significantly lower (n = 12 vs. n = 52, p < 0.05), representing a coding rate of 23.1%. Regarding postoperative management, 90% of all patients had at least 1 episode with mean arterial pressure <65 mm Hg within the first 72 h. Furthermore, regarding other preventive parameters (avoiding hyperglycemia, stopping angiotensin-converting enzyme inhibitors/angiotensin receptor blockers, avoiding contrast media, and nephrotoxic drugs), only 10 patients (20.8%) in the non-AKI group and in 5 (9.6%) subjects in the AKI group had none of all the above potential AKI-promoting factors.!##!Conclusions!#!AKI recognition in everyday clinical routine seems to be low, especially in lower AKI stages, and the current postoperative management still offers potential for optimization. Possibly, higher AKI awareness and stricter postoperative management could already achieve significant effects in prevention and treatment of AKI

Enhanced expression of thioredoxin-interacting-protein regulates oxidative DNA damage and aging

The "free radical theory of aging" suggests that reactive oxygen species (ROS) are responsible for age-related loss of cellular functions and, therefore, represent the main cause of aging. Redox regulation by thioredoxin-1 (TRX) plays a crucial role in responses to oxidative stress. We show that thioredoxin-interacting protein (TXNIP), a negative regulator of TRX, plays a major role in maintaining the redox status and, thereby, influences aging processes. This role of TXNIP is conserved from flies to humans. Age-dependent upregulation of TXNIP results in decreased stress resistance to oxidative challenge in primary human cells and in Drosophila. Experimental overexpression of TXNIP in Hies shortens lifespan due to elevated oxidative DNA damage, whereas downregulation of TXNIP enhances oxidative stress resistance and extends lifespan