Kidney transplantation is the gold standard treatment for end-stage renal disease. Renal ischemia-reperfusion injury is an unavoidable consequence of the transplantation procedure and is responsible for delayed graft function and poorer long-term outcomes. Pharmacological inhibition of heat shock protein 90 is a preconditioning strategy that has previously been shown to reduce renal ischemia-reperfusion injury. However, the clinical application of heat shock protein 90 inhibitors is limited by their toxicity profile and the exact mechanisms of protection conferred are unknown. The aims of this thesis were to establish mechanisms of protection offered by these drugs and investigate a less toxic analogue that has the potential to be safely translated into human studies. AT13387 is a novel small molecule heat shock protein 90 inhibitor with a low toxicity profile, which is being evaluated in phase II studies in oncology and therefore has excellent translational potential in the context of transplantation. Heat shock protein 90 inhibition up-regulates protective heat shock proteins (especially heat shock protein 70) and potentially down-regulates NF-ҡB activity by disruption of the IҡB kinase complex. Toll-like receptor 4 is a further regulator of NF-ҡB activity and studies have suggested that Toll-like receptor 4 plays a dominant role in mediating kidney damage following ischemia-reperfusion injury. To explore potential molecular mechanisms of protection, human embryonic kidney cells were pre-treated with AT13387 and exposed to endotoxin-free hyaluronan to stimulate sterile Toll-like receptor 4-specific NF-ҡB activation. AT13387-treatment resulted in breakdown of IҡB kinase, which abolished Toll-like receptor 4-mediated NF-ҡB activation by hyaluronan. Inhibition of autophagy prevented IҡB kinase-α degradation by heat shock protein 90 inhibition and resulted in regain of NF-ҡB activity by hyaluronan. In subsequent investigations, AT13387 decreased pro-inflammatory cytokine release following hyaluronan stimulation and increased cell viability in an in vitro model of oxidative stress. In mice, AT13387 induced heat shock protein 70 expression in the kidney. AT13387 pre-treatment then significantly reduced kidney injury following renal ischemia-reperfusion injury. In contrast, in severe combined immunodeficient mice, AT13387 no longer reduced kidney injury from renal ischemia-reperfusion injury. This emphasises the potential importance of the adaptive immune system in the protective effect of this agent. This resonates with reports of heat shock protein 70 up-regulation in the context of heat preconditioning, which leads to renal protection from renal ischemia-reperfusion injury that is lymphocyte-dependent. Secondary lung injury is an additional consequence of renal ischemia-reperfusion injury. In further experiments, pre-treatment with AT13387 again did not reduce kidney injury following renal ischemia-reperfusion injury in severe combined immunodeficient mice. However, AT13387 did reduce secondary lung injury. This lung protective effect may have been related to heat shock protein 70 up-regulation in the lungs by AT13387. A rationale for enhancing recovery, following renal ischemia-reperfusion injury, by inhibiting heat shock protein 90 was then sought. This investigation was undertaken in order to broaden the range of the available therapies to a wider group of patients including renal transplant recipients. AT13387 pre-treatment of the recipient mice preceded an isograft renal transplantation with a kidney harvested from a treatment naive mouse and cold stored for 4 hours. Although a significant reduction in tubular necrosis was not demonstrated following AT13387 treatment, the feasibility of the treatment strategy was demonstrated and interestingly lung injury secondary to transplantation was reduced. This thesis therefore highlights AT13387 as a new agent with the potential of reducing kidney injury and secondary lung injury following renal ischemia-reperfusion injury. The findings also demonstrate that the mechanisms of protection offered by this drug may involve the adaptive immune system. In addition to the induction of heat shock protein 70 expression in the kidney and repression of Toll-like receptor 4-mediated NF-ҡB signalling through breakdown of IҡB kinase.

Tyrimo tikslas: ištirti kavos rūgšties fenetilo esterio (CAFE) poveikį išemijos paveiktų žiurkės inkstų mitochondrijų funkcijoms. Uždaviniai: įvertinti tiesioginį in vitro CAFE poveikį inkstų mitochondrijų funkcijoms; CAFE poveikį išemijos (20 min) in vitro paveiktų inkstų mitochondrijų funkcijoms; kvėpavimo grandinės I komplekso aktyvumui; mitochondrijų gebėjimui kaupti Ca2+. Metodai. Wistar veislės žiurkių patinėliai buvo skirstomi į 4 grupes: kontrolinė grupė, 20 min trukmės išemijos, CAFE 22 mg/kg ir CAFE 34 mg/kg. CAFE buvo leidžiamas 1,5 h prieš sukeliant išemiją. Mitochondrijos buvo išskiriamos diferencinio centrifugavimo būdu. Mitochondrijų kvėpavimo greitis mitochondrijoms oksiduojant I ir II komplekso substratus buvo registruojamas poliarografiškai Klarko tipo elektrodu. Ca2+ sugėrimas buvo matuojamas fluorimetriškai. Mitochondrijų kvėpavimo grandinės I komplekso aktyvumas buvo tiriamas spektrofotometriškai. Rezultatai: CAFE (0,7 – 4,5 M) didina mitochondrijų kvėpavimo greitį 2-oje metabolinėje būsenoje nuo 15 % iki 34 % ir neveikė mitochondrijų kvėpavimo greičio 3-ioje metabolinėje būsenoje (VADP). Didesnės koncentracijos (5,2 - 6,5 µM) slopina mitochondrijų kvėpavimo greitį VADP (16 % ir 43 % atitinkamai). Tiriant CAFE poveikį 20 min išemijos in vitro paveiktų mitochondrijų funkcijoms, nustatyta, jog 22 mg/kg ir 34 mg/kg CAFE, intraperitonealinė injekcija 1,5 h prieš sukeliant inkstų išemiją, 20 % (p<0,05) pagerino mitochondrijų kvėpavimo greitį VADP bei... [toliau žr. visą tekstą] / The aim of investigation: to analyse the effect of caffeic acid phenethyl ester (CAPE) on kidney mitochodrial functions. Objectives: to evaluate direct in vitro effect of CAPE on kidney mitochondrial functions; the impact of CAPE on ischemia (20 min) in vitro affected kidney mitochondrial functions; on the mitochondrial respiratory chain complex I activity and on the mitochondria capability to accumulate Ca2+. Methods. Wistar rats were pretreated intraperitoneal with 22 mg/kg and 34 mg/kg of CAPE. Animals were divided into 4 groups: control group, 20 min of ischemia, CAPE 22 mg/kg group and CAPE 34 mg/kg group. Mitochondria were isolated by means of differential centrifugation. The mitochondrial respiration rate while oxidizing complex I and II dependent substrates was registered polarographically with Clark-type electrode. Ca2+ accumulation was measured fluorometrically. Activity of complex I was measured spectrophotometrically. Results: the results shown that CAPE 0,7 - 4,5 µM increases mitochondrial State 2 respiration rate by 15 - 34 % and has no effect on the State 3 respiration rate. Higher concentrations (5,2 - 6,5 µM) decreased mitochondrial State 3 respiration rate by 16% and 43%, respectively. Pretreatment with CAPE (22 mg/kg and 34 mg/kg) increased (by 20%) the ischemia suppressed mitochondrial State 3 respiration rate and respiratory control index. CAPE had no effect on succinate oxydation. Pretreatment with CAPE (22 mg/kg and 34 mg/kg) increased Ca2+... [to full text]

Indiana University-Purdue University Indianapolis (IUPUI) / Knowledge of the molecular and cellular mechanisms of ischemic injury is necessary for understanding acute kidney injury and devising optimal treatment regimens. The cortical actin cytoskeleton in the proximal tubule epithelial cells of the kidney nephron, playing an important role in both the establishment and maintenance of cell polarity, is drastically disrupted by the onset of ischemia. We found that in LLC-PK cells (a porcine kidney proximal tubule epithelial cell line), cortactin, an important regulator of actin assembly and organization, translocated from the cell cortex to the cytoplasmic regions upon ischemia/ATP-depletion. Meanwhile both the tyrosine phosphorylation level of cortactin and cortactin’s interaction with either F-actin or the actin nucleator Arp2/3 complex were down-regulated upon ischemia/ATP-depletion or inhibition of Src kinase activity. These results suggest that tyrosine phosphorylation plays an important role in regulating cortactin’s cellular function and localization in the scenario of kidney ischemia. The Rho GTPase signaling pathways is also a critical mediator of the effects of ATP depletion and ischemia on the actin cytoskeleton, but the mechanism by which ATP depletion leads to altered RhoA and Rac1 activity is unknown. We propose that ischemia and ATP depletion result in activation of AMP-activated protein kinase (AMPK) and that this affects Rho GTPase activity and cytoskeletal organization (possibly via TSC1/2 complex and/or mTOR complex). We found that AMPK was rapidly activated (≤5 minutes) by ATP depletion in S3 epithelial cells derived from the proximal tubule in mouse kidney, and there was a corresponding decrease in RhoA and Rac1 activity. During graded ATP-depletion, we found intermediate levels of AMPK activity at the intermediate ATP levels, and that the activity of RhoA and Rac1 activity correlated inversely with the activity of AMPK. Activation of AMPK using two different drugs suppressed RhoA activity, and also led to morphological changes of stress fibers. In addition, the inhibition of AMPK activation partially rescued the disruption of stress fibers caused by ATP-depletion. This evidence supports our hypothesis that the activation of AMPK is upstream of the signaling pathways that eventually lead to RhoA inactivation and cytoskeletal dysregulation during ATP-depletion.