A laboratory manual of kidney perfusion techniques

An organ such as the kidney naturally functions best in a healthy organism. Once a kidney has been removed from its normal context, it is crucial to find the optimal compromise between the demands of the problem at issue and the experimental model to be adopted. An isolated kidney is, in effect, a “dying kidney”, although it may continue to function for between 1 and 4 hours, depending on one's definition of normal. The basic reason for the progressive deterioration in function can be found in the stresses set up by the transition from perfusion by whole blood to the use of a more or less artificial perfusing medium. Indeed, blood is a “liquid organ”, a highly complex cocktail that is steadily being reconditioned by the actions of other organs, and this complexity explains why all efforts to maintain kidney function in isolation are ultimately doomed to failure. If we want to approach its fantastic properties, then we should leave an organ as the kidney within the body. As Mephisto remarks in Goethe's drama “Faust”, Studierzimmer II: “Blut ist ein ganz besondrer Saft”: “blood is a quite special juice”.<br

Laborhandbuch Nierenperfusion

Die beste und normale Funktion hat ein Organ wie die Niere nur im gesunden Organismus. Sobald man die Niere experimentell isoliert, muss man die richtige Balance finden zwischen Fragestellung, der dazu passenden Variante des experimentellen Modells und der Antwort, die man von diesem Experiment erwarten kann. Eine isolierte Niere ist eine "sterbende Niere", auch wenn man über 1-4 h eine Funktion aufrechterhalten kann, je nach Definition und den Ansprüchen an eine normale Funktion. Der Grund für die Begrenzung der Überlebensfähigkeit eines isolierten Organs mag in der Übergangsphase liegen zwischen der in vivo Vollblutperfusion zu der Perfusion mit einer mehr oder weniger künstlichen Perfusionslösung als Blutersatz: Blut ist als "flüssiges Organ" eine hochkomplexe Mischung die permanent rekonditioniert wird durch eine Reihe von Organen und wenn wir an diese phantastischen Eigenschaften herankommen wollen, dann sollten wir ein Organ wie die Niere besser im Organismus belassen. Goethe lässt im Drama Faust I, Studierzimmer II Mephisto sagen: "Blut ist ein ganz besondrer Saft"

Expression und Funktion des Proteins KIBRA im Podozyten

Im Rahmen dieser Doktorarbeit konnte die Interaktion zwischen dem wenig charakterisierten Protein KIBRA und dem Polaritätsprotein PATJ bestätigt und auf bestimmte Interaktionsmodule eingegrenzt werden. Um die Funktion von KIBRA im Podozyten zu untersuchen, wurde eine KIBRA knock down Podozyten-Zelllinie generiert. Bei der Durchführung von Wound Healing- und Adhäsions-Assays mit KIBRA knock down Podozyten zeigte sich, dass eine reduzierte KIBRA Expression wesentlichen Einfluss sowohl auf die Fähigkeit der Zellen nimmt, eine gesetzte Wunde durch gerichtete Migration zu schließen als auch sich durch die Ausbildung fokaler Kontakte wieder an einer Kollagen-beschichteten Matrix anzuheften. Da gezeigt werden konnte, dass die Funktion des Aktin-Netzwerks und die Ausbildung von Zellpolarität im Podozyten unmittelbar von KIBRA beeinflusst werden, darf man annehmen, dass KIBRA zwischen Zellpolarität und Aktin-Zytoskelett für die Morphologie der hoch differenzierten Podozyten von zentraler Bedeutung ist

Characterization of a short isoform of the kidney protein podocin in human kidney

BACKGROUND: Steroid resistant nephrotic syndrome is a severe hereditary disease often caused by mutations in the NPHS2 gene. This gene encodes the lipid binding protein podocin which localizes to the slit diaphragm of podocytes and is essential for the maintenance of an intact glomerular filtration barrier. Podocin is a hairpin-like membrane-associated protein that multimerizes to recruit lipids of the plasma membrane. Recent evidence suggested that podocin may exist in a canonical, well-studied large isoform and an ill-defined short isoform. Conclusive proof of the presence of this new podocin protein in the human system is still lacking. METHODS: We used database analyses to identify organisms for which an alternative splice variant has been annotated. Mass spectrometry was employed to prove the presence of the shorter isoform of podocin in human kidney lysates. Immunofluorescence, sucrose density gradient fractionation and PNGase-F assays were used to characterize this short isoform of human podocin. RESULTS: Mass spectrometry revealed the existence of the short isoform of human podocin on protein level. We cloned the coding sequence from a human kidney cDNA library and showed that the expressed short variant was retained in the endoplasmic reticulum while still associating with detergent-resistant membrane fractions in sucrose gradient density centrifugation. The protein is partially N-glycosylated which implies the presence of a transmembranous form of the short isoform. CONCLUSIONS: A second isoform of human podocin is expressed in the kidney. This isoform lacks part of the PHB domain. It can be detected on protein level. Distinct subcellular localization suggests a physiological role for this isoform which may be different from the well-studied canonical variant. Possibly, the short isoform influences lipid and protein composition of the slit diaphragm complex by sequestration of lipid and protein interactors into the endoplasmic reticulum

Evaluation of multi-exponential curve fitting analysis of oxygen-quenched phosphorescence decay traces for recovering microvascular oxygen tension histograms

Although it is generally accepted that oxygen-quenched phosphorescence decay traces can be analyzed using the exponential series method (ESM), its application until now has been limited to a few (patho)physiological studies, probably because the reliability of the recovered oxygen tension (pO2) histograms has never been extensively evaluated and lacks documentation. The aim of this study was, therefore, to evaluate the use of the ESM to adequately determine pO2 histograms from phosphorescence decay traces. For this purpose we simulated decay traces corresponding to uni- and bimodal pO2 distributions and recovered the pO2 histograms at different signal-to-noise ratios (SNRs). Ultimately, we recovered microvascular pO2 histograms measured in the rat kidney in a model of endotoxemic shock and fluid resuscitation and showed that the mean microvascular oxygen tension, 〈pO2〉, decreased after induction of endotoxemia and that after 2 h of fluid resuscitation, 〈pO2〉 remained low, but the hypoxic peak that had arisen during endotoxemia was reduced. This finding illustrates the importance of recovering pO2 histograms under (patho)physiological conditions. In conclusion, this study has characterized how noise affects the recovery of pO2 histograms using the ESM and documented the reliability of the ESM for recovering both low- and high-pO2 distributions for SNRs typically found in experiments. This study might therefore serve as a frame of reference for investigations focused on oxygen (re)distribution during health and disease and encourage researchers to (re-)analyze data obtained in (earlier) studies possibly revealing new insights into complex disease states and treatment strategies

In Vivo, In Vitro, and In Silico Characterization of Peptoids as Antimicrobial Agents

Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system