Master of Science / Department of Clinical Sciences / Gregory F. Grauer / Hyperthyroidism is the most common endocrinopathy of geriatric cats and has physiologic effects on almost every organ in the body. It specifically affects the kidneys by increasing renal blood flow and glomerular filtration rate. In addition, activation of the renin angiotensin aldosterone system (RAAS) is increased and ultimately leads to efferent glomerular arteriole constriction and potentially glomerular hypertension. The classic treatment modalities for feline hyperthyroidism (anti-thyroid medication, radioiodine or surgery) have been evaluated for their overall effects on renal function. Studies have demonstrated that glomerular filtration rate (GFR) declines and serum creatinine increases with hyperthyroid treatment independent of the treatment modality. Hill’s® Prescription Diet® y/d® Feline, a relatively new dietary treatment modality for feline hyperthyroidism with controlled iodine concentrations, reduced phosphorus and protein, and increased omega-3 fatty acids, has been shown to significantly decrease thyroid hormone levels. The research provided in this report is the first evaluating the posttreatment effects of y/d® Feline on renal function. In agreement with previous studies, our research found that y/d® Feline resulted in a significant decrease in thyroid hormone levels. However, in contrast to other treatment modalities, y/d® Feline did not result in a significant decline in GFR, and it did result in a significant decline in mean serum creatinine concentration. These data indicate that y/d® Feline, as a treatment for feline hyperthyroidism, does not have a negative effect on renal function.

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.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O gengibre possui em sua composiÃÃo inÃmeras substÃncias volÃteis e nÃo volÃteis. Dentre as substÃncias nÃo-volÃteis destacam-se, principalmente, os gingerols, sendo o 6-gingerol o composto mais abundante e o responsÃvel pela grande maioria das atividades farmacolÃgicas descritas, como a anti-hipertensiva. Neste trabalho, foram investigados os efeitos renais, vasculares e em cultura de cÃlulas tubulares renais do tipo MDCK (Madin-Darby Canine Kidney) causados pelo 6-gingerol. Foram utilizados ratos Wistar machos pesando entre 250 e 300g, cujos rins foram isolados e perfundidos com SoluÃÃo de Krebs-Hanseleit contendo 6%p/v de albumina bovina previamente dialisada. Foram investigados os efeitos do 6-gingerol (3 μM, 10 μM, 30 μM; n=6) sobre a PressÃo de PerfusÃo (PP), ResistÃncia Vascular Renal (RVR), Fluxo UrinÃrio (FU), Ritmo de FiltraÃÃo Glomerular (RFG), Percentual de Transporte Tubular de SÃdio (%TNa+), de PotÃssio (%TK+) e de Cloreto (%TCl-). O 6-gingerol foi adicionado apÃs 30 minutos de controle interno. As cÃlulas MDCK foram cultivadas em meio de cultura RPMI 1640 suplementado com 10% v/v de Soro Bovino Fetal e entÃo avaliadas na presenÃa do composto em diversas concentraÃÃes em dois perÃodos de incubaÃÃo, 6 (seis) e 24 (vinte e quatro) horas. ApÃs esses perÃodos, foram realizados ensaios de viabilidade celular. Foi avaliada a resposta do 6-gingerol em diversas concentraÃÃes na pressÃo arterial mÃdia de ratos wistar normotensos anestesiados. Os resultados encontrados na pressÃo arterial dos animais foi uma queda acentuada de maneira dose-dependente na pressÃo arterial destes animais. Em relaÃÃo à perfusÃo renal, o 6-gingerol mostrou-se um potente diurÃtico e com baixÃssimos danos renais tanto nos dados encontrados no perfil histolÃgico, como nos experimentos de avaliaÃÃo de viabilidade celular em cÃlulas MDCK o que està em consonÃncia com o conhecimento da medicina tradicional. Os resultados encontrados nÃo foram totalmente abolidos pelo inibidor especÃfico do receptor TRPV1 utilizado no estudo, demonstrando que o 6-gingerol possui outras vias renais a serem exploradas em estudos posteriores.

All inbred strains of mice carry the Ren-1 structural gene, which encodes the renin-1 isozyme, the classical renin activity found in kidneys. In addition, some strains carry a second renin structural gene, Ren-2, which encodes the predominantly expressed SMG rennin isozyme, renin-2. Ren-1 and Ren-2 exhibit markedly different patterns of tissue- specific expression. In an effort to understand the molecular basis for this differential expression, a detailed analysis of the transcripts originating from these loci was undertaken. S1 analysis of SMG and kidney RNA populations indicated that the majority of transcripts initiate at one major site on Ren-1 and Ren-2. Interestingly a minor fraction of transcripts in the SMG initiate at two upstream sites. These transcripts encode an upstream ORF which is in frame with that of the renin precursor. The precise tissue-specificities of Ren-1 and Ren-2 were also examined: in the kidney, SMG, and also in several extrarenal tissues, since there is increasing evidence of renin expression in a number of extrarenal sites. To distinguish between the two highly homologous transcripts, an assay was developed exploiting established base sequence differences between Ren-1 and Ren-2 mRNA's by extension of a primer downstream of such a base difference in the presence of the appropriate ddNTP. Using this assay, Ren-1 and Ren-2 were found to be equally well expressed in the kidney, whereas in the SMG only Ren-2 is efficiently expressed. Interestingly in the other extrarenal tissues examined, testis, liver and heart, it is the Ren-1 allele that is preferentially expressed. The assay was also able to demonstrate the similarity in response of Ren-l/Ren-2 to certain physiological stimuli, such as sodium depletion. Thus, this study of the regulation of mouse renin gene expression has demonstrated further striking differences in the tissue-specific expression of Ren-l/Ren-2, and added to the increasingly compelling evidence of extrarenal renin gene expression.

Chemical-induced nephrotoxicity is a major cause of acute kidney injury. My dissertation reveals that all-trans-retinoic acid (ATRA) affords cytoprotection against renal cell injury. Pretreatment with ATRA (25 μM, 24 hr) affords selective cytoprotection against p-aminophenol (PAP), iodoacetamide (IDAM), and 2-(glutathion-S-yl)-hydroquinone-induced necrosis. In contrast, pretreatment of cells with ATRA provides no protection against cisplatin-induced apoptosis. Inhibition of protein synthesis blunts ATRA-mediated cytoprotection, suggesting that critical cell survival signaling pathways are activated prior to toxicant exposure. Oxidative stress is a major contributor to cellular damage. To investigate the mechanism(s) by which ATRA affords cytoprotection, we determined its effects on ROS generation using a DCFDA assay. ATRA did not alter PAP or MGHQ-induced ROS levels. Moreover, ATRA had no effect on GSH levels nor Nrf2 expression, suggesting that other cytoprotective mechanisms are engaged by ATRA. Elevated ROS disrupt endoplasmic reticulum protein folding guided by the molecular chaperone Grp78. During ATRA-mediated pretreatment, the ER stress proteins Grp78 and p-eIF2α were induced (2-fold) in a time-dependent manner (24 and 4 hr respectively). In addition to influencing organelle stress proteins in the ER, ATRA rapidly (15 min) induced levels of the cellular stress kinases p-ERK and p-AKT with maximum levels achieved at 30 min. Moreover, induction of these stress kinases was observed at concentrations of ATRA (10 and 25 μM) required for cytoprotection. Inhibition of p-ERK with PD98059 reduced the ability of ATRA to provide protection against PAP toxicity, implying a role for p-ERK and downstream target genes in the protective effects of ATRA. Gene ontology analysis of a microarray experiment of cells treated with ATRA revealed that ATRA rapidly (0.5, 1 hr) induced growth factors and genes involved in cell proliferation, with subsequent (4, 8, 12 hr) induction of genes involved in ribosome biogenesis, DNA replication and repair, and cell cycle regulation. Complementary data from a cell stress protein array and western blot analyses indicated that ATRA induced HIF1α 3-fold at 8 hr. Furthermore, the microarray data indicated the HIF1α target gene BHLHE40 (which encodes a basic-helix-loop-helix protein involved in cell differentiation) was increased 3-fold. As ATRA induced genes that were associated with cell proliferation, related assays were employed. ATRA had a small effect on cell cycle distribution demonstrated by an increase in the population of cells in the S and G2 phases between 8 and 24 hr. In addition, ATRA markedly increased total DNA content and cell number at 24 hr suggesting that mitogenic/proliferative effects contribute to ATRA cytoprotection. The present studies indicate that a signaling cascade of proteins downstream of p-ERK associated with mitogenesis work cooperatively to afford ATRA protection against renal cell injury. Understanding the mechanism of ATRA-mediated cytoprotection will provide insights into the development of novel therapeutic strategies for renal pathological conditions.

Rheological measurements on intact fresh snap beans (Phaseolus vulgaris v.) and purees made from raw beans were used to assess the effect of variety and sieve size on the textural characteristics of green snap beans. Seed length, percentage dry matter and physical fiber measurements were used as textural quality indicators. Four varieties of green snap beans (Tendercrop, Rainier, Harvester and BBL 290) were tested in 1971 and 1972. Each variety was harvested five times in each year. Adverse environmental conditions in 1971 caused bean textural quality to be higher in 1972 than in 1971. The four varieties tested showed significant differences with Rainier exhibiting the best textural quality while Harvester generally showed the poorest quality. Tests involving the resistance to shearing of intact bean pods were carried out using the Ottawa Texture Measuring System and the Food Technology Corporation's Texture Test System (formerly the Kramer shear press). Viscosity tests were performed on purees composed of macerated raw green bean tissue and water. Results were obtained from spread test using a simplified Adams-type consistometer and from rotating coaxial cylinder tests using a Brookfield RVT Synchro-Lectric Viscometer fitted with a small sample adapter. The Brookfield data were then fitted to the power-law equation. Rheological parameters showed highly significant interrelationships in most instances. Viscous properties of purees (spread, m, n and yield stress) were highly correlated with percentage dry matter of the beans. Peak force readings of the Kramer shear press and the Ottawa Texture Measuring System were significantly correlated with all textural quality and viscometric parameters. / Land and Food Systems, Faculty of / Graduate

The mechanisms of osmotic and ionic regulation in marine mammals are of interest because of the apparent lack of "fresh" water in their environment. Previous investigation on the harbor seal, (Phoca vitulina, L.), generally indicated that the seal can obtain all the water it requires from its food. However, some dispute still exists as to whether the seal may ingest sea water along with its food and conserve water by concentrating ions and excreting them with a net water gain. The effects of a 16-hour period with no fluids and also of intubation with varying amounts of distilled water and varying amounts and concentrations of sea water were determined. The concentrations of sodium, chloride and potassium ions as well as the osmotic pressures of plasma and urine were measured for the periods before and after intubation. The results of this study are discussed with respect to published data and proposed mechanisms of osmotic and ionic regulation by the kidney. The findings indicate, as suggested by others, that the seal does not gain any substantial amount of water from sea water ingestion. / Science, Faculty of / Zoology, Department of / Graduate

Ischaemia-reperfusion injury (IRI) has been identified as a major contributor to both short and long term kidney transplant failure. Experimental evidence from the literature suggests that Erythropoietin (EPO) is tissue protective, reducing both inflammation and apoptosis following IRI. We performed a randomised, double blind, placebo controlled trial examining the tissue protective effect of high dose EPO (100,000iu over 3 days) in 39 recipients of an extended criteria donor kidney or a non-heart-beating donor kidney. The primary endpoints of the study were difference in plasma and urinary biomarker levels (NGAL, IL-18 and KIM-1) in addition to changes in gene expression. Secondary endpoints included safety, clinical data and differences in metabolomics profiles. There was no difference detected between the treatment groups in terms of biomarkers, gene expression, metabolomics profiling or clinical parameters. No adverse events related to EPO therapy were recorded. In addition, we developed a cell model of kidney transplantation using primary tubulo-epithelial cells and HMEC-1 cells, with which to confirm the protective effects of EPO. Treatment with 50U/ml one hour prior to undergoing cold hypoxia resulted in the maximum degree of tissue protection, as measured using an MTT and an LDH assay. No evidence of EPO toxicity was demonstrated. Tubulo-epithelial cells expressed EPOR mRNA and protein. No CD131 receptor could be demonstrated. In summary, EPO confers tissue protection in a cell model of kidney transplantation but this has not been shown to occur in a clinical trial using high dose EPO in recipients of marginal donor kidneys.

This work on polycystic disease of the kidney commenced while the author was engaged in general practice in Swellendam, in the South Western Cape from 1953-1956. Within a period of a year three patients, suffering from this disease, were seen. They were questioned about their family-relationship but they denied any such association. It was regarded as highly unlikely that three patients with a relatively rare disease, should be found in a population existing between those patients. The genealogy of each patient was worked out and when this information was bought together, it was found that they were fairly closely related. This was the first experience that the information obtained from a patient about his family may not be reliable, not even in a small, fairly closed community. As the family become known to the author the members were systematically investigated for polycystic kidney disease and an attempt was made to determine how many individuals were affected and i how many generations.

archives@tulane.edu / The kidney develops from the intermediate mesoderm from E10 to P4 in mice and weeks 5 to 34 in humans. The development relies on the physical and signaling interactions between the nephron progenitor cells (NPCs), the stroma progenitor cells, and the ureteric branching tip cells (UBTCs). Kidney development relies on signals that vary based on location and temporally with NPC recruitment order determining the part of the nephron they will form. Kidney organogenesis and nephrogenesis relies on signals from BMPs, growth factors, Wnt, cytokines, and autonomous and exogenous cell proliferation and survival signals. These signals lead into or are regulated by cell metabolism, environmental signals, and chromatin modifications. IUGR is an environmental condition known to cause hypertension, chronic kidney disease, and kidney failure. We hypothesized that disruption of metabolic homeostasis in the nephron progenitor cells in the IUGR fetus impairs nephrogenesis and is the direct link between the maternal environment and nephron endowment leading to adult hypertension and chronic kidney disease (CDK). IUGR from low protein diet caused small pups, small kidneys, increased kidney/body weight ratio. The changes begin at E13.5 with a 30% decrease in ureteric tip count, disorganized/smaller cap mesenchyme (CM) (37.5% decrease in Six2+ NPCs), and smaller kidneys. P0 NPCs show dysregulation to growth factors, Wnt, cell metabolism, and autonomous and exogenous cell proliferation and survival signals shown by bulk RNA-seq and immunofluorescence. Changes from LPD IUGR persist with delayed postnatal growth of skin, hair, body, and kidneys. P21 and adult IUGR show damage to kidneys and increased risk of developing hypertension, and CDK. IUGR LPD is the first hit in the multi-hit disease causation of CDK. The P0 NPCs had dysregulated metabolism and chromatin; postnatal development continues to be dysregulated despite removal of LPD environment. The LPD IUGR model produces a new tool for the study of multi-hit kidney disease. / 1 / Francesca Edgington-Giordano