Diabetic nephropathy (DN) is a complication associated with diabetes and is characterized by proteinuria and a progressive loss of kidney function. The disease morphologically manifests as an increase in the extracellular matrix (ECM) produced by kidney cells including specialized mesangial cells found in the kidney glomeruli. The mesangial cells undergo increased proliferation and hypertrophy, produce ECM components at an elevated rate and in turn the ECM itself is broken down at a reduced rate. This leads to fibrosis, or the scarring of the glomeruli. The process of fibrosis is known to be promoted by pro-fibrotic factors such as transforming growth factor beta-1 (TGF-β1), which is activated by various proteins including thrombospondin-1 (TSP-1). Both of these proteins are known to have an increased rate of expression and activation in a high glucose environment and in the kidneys of diabetic patients. Glucose-regulate protein 78 (GRP78) is another protein altered by high glucose, as it is translocated to cell surface in DN (cell surface GRP78, csGRP78). In this study, we investigate the role csGRP78 has in the regulation of TSP-1 and downstream signaling by high glucose, using primary rat mesangial cell cultures. Our results confirm that TSP-1 protein levels are increased in the cell lysate and in the ECM of cells treated with high glucose. We further show that inhibitors of csGRP78 and downstream PI3K/Akt reduce the high glucose-induced increase in TSP1 at both protein and transcript levels, and attenuate TGF-β1 signaling. / Thesis / Master of Science (MSc) / Diabetic nephropathy is a condition that is associated with a gradual loss of kidney function as well as the presence of protein in the urine. As the name implies, diabetic nephropathy occurs as a result of diabetes mellitus. The disease causes the mesangial cells in the kidney to produce excess extracellular matrix leading to scarring in the kidney, a process called fibrosis. One of the key fibrotic proteins is called transforming growth factor beta-1 (TGF-β1), stored in a latent form. A major activator of TGF-β1 is thrombospondin-1 (TSP-1). Our results demonstrate that the cell surface localization of glucose regulated protein 78 (GRP78) is required for the upregulation of TSP-1 in a high glucose environment, leading to activation of profibrotic pathways that are well known to perpetuate the fibrotic phenotype seen in diabetic nephropathy.

Diabetic foot ulceration (DFU) is a known major complication of diabetes mellitus which contributes to lower extremities amputation. This study aimed to investigate the use of interventional devices either as a preventative or therapeutic strategy to improve clinical management of this pathology, as well as investigating the impaired function of endothelial progenitor cells in the diabetic condition. The first element targeted plantar callus formation among diabetic neuropathic (NRP) patients through the use of a SurroSenseRxTM biofeedback device. Reducing foot pressure with improved walking strategy in the 6 months study in diabetic neuropathy patients (n=20) appeared to minimise the size of non-ulcerative plantar callus (p < 0.05), potentially reducing future ulcer recurrence. The 2nd study focused on the use of a GekoTM electrical stimulation device to enhance DFU healing in 24 patients. Wounds were characterised as being neuroischaemic (NRI) or neuropathic (NRP) based on standard parameters adopted in the Manchester diabetes clinic. The device was worn by 11 intervention subjects and compared to 13 controls without any electrical stimulus. Results suggested healing and wound closure have potentially increased in participants with electrical stimulation. In addition, Neuropathy Disability Score (NDS) was improved among intervention patients compared to control (p < 0.0001). The 3rd, in vitro and mechanistic study focuses on the outgrowth of endothelial cells (OECs), abnormal angiogenic responses and inflammatory microenvironment which could contribute to impaired wound healing in diabetic patients. OECs were isolated from diabetic patients and healthy controls (HCs), characterised by immunohistochemistry and Polymerase Chain Reaction (PCR). The functions of the three OEC groups from NRI, NRP diabetic patients and healthy controls respectively were compared using in vitro proliferation, transwell migration and wound healing scratch assays, together with matrigel tube formation assays. Scratch assays showed 100% closure in HCs over 24 hours, while 86.6% closure was apparent in NRI vs 38.1% in NRP. Seahorse mitochondrial stress test was conducted and demonstrated mitochondrial dysfunction in NRP vs NRI vs HCs (p < 0.05). Western blot analysis showed a lack of ERK phosphorylation by NRP OECs and an up-regulation of plasma inflammatory cytokines (TNFa and IL-6) in diabetic samples vs HC (p < 0.0001), while the angiogenic factors ang-2, FGF-2, VEGF-D, HGF and IL-8, and nitric oxide bioavailability were all significantly reduced in diabetic samples vs HC (p < 0.05). The functional defects of the diabetic OECs were partially restored through glycomimetic (synthesis compounds for endothelial damage protection) treatment (p < 0.05). In summary, this study has highlighted areas worthy of future development both in terms of preventative and therapeutic strategies. With improvements in digital technology and the need to empower patients to take responsibility of their health and well-being as well as greater understanding of the cellular and molecular biological repair processes that may be exploited, there may be potentials to reduce the risk of future ulceration among patients using these novel approaches in the future.

Background/aim. The incidence of microvascular complications has been reported to be unchanged the last decades. However, in randomized clinical trials it has been shown that improved metabolic control can reduce the development of long term complications. It has been debated whether it is possible to achieve the same results in an unselected population. In a previous study we found a decreased incidence of overt nephropathy, but unchanged incidence of severe laser treated retinopathy in a population of patients with type 1 diabetes diagnosed in childhood. The aim of the present study was to investigate the incidence 10 years later in the same population and to analyse the importance of possible risk factors. In another previous study we found a high prevalence of subclinical neuropathy among young diabetic patients despite intensive insulin therapy since diagnosis. The aim of the present study was to examine if intensive treatment is more effective in preventing early diabetic complications other than neuropathy. The incidence of type 1 diabetes has doubled in Sweden the last decades. The reason must be environmental factors. These, as well as more intensive insulin regimens from onset of diabetes, might also lead to different disease process. We wanted to analyse if clinical characteristics at onset had changed the last 25 years and if there was any secular trend of C-peptide secretion. We also intended to investigate if longer persistence of C-peptide secretion could be of importance for prevention of long term complications. Methods. The whole study population consisted of all 478 patients with type 1 diabetes diagnosed before the age of 15 during the years 1961 - 2000, living in the catchment area of the Paediatric Clinic, University Hospital, Linköping, Sweden. For the statistical analysis the population was divided into five–year cohorts according to time of onset of diabetes. The cumulative proportion of severe retinopathy and overt nephropathy in 269 patients with onset of diabetes 1961 - 1985 was computed with survival analysis. Multivariable regression models were used to analyse the importance of metabolic control, diabetes duration, blood pressure, smoking, BMI, lipids and persisting C-peptide secretion. The prevalence of all grades of retinal changes, nephropathy and neuropathy, defined as abnormal nerve conduction, was estimated in the late 1990s in a subgroup of 80 children and adolescents with mean 13 years of diabetes duration. Clinical characteristics at onset, duration of partial remission and regularly measurements of fasting and stimulated C-peptide secretion the first five years after onset were analysed in 316 patients with onset of diabetes 1976 - 2000. Results. The cumulative proportion of severe laser treated retinopathy showed a significant declining trend the last decades. The decrease was significant between the oldest cohort with diabetes onset 1961 - 1965 and the cohorts with diabetes onset 1971 - 1975 and 1976 - 1980. The cumulative proportion of overt nephropathy also declined with a significant decrease between the oldest cohorts and all the following cohorts. After 25 years of diabetes duration it was 30% and 8% in the two oldest cohorts respectively and remained largely unchanged after 30 years. Diabetes duration and long term HbA1c were the only significant independent risk factors for both retinopathy and nephropathy. The risk of overt nephropathy increased substantially when HbA1c was above 8.5%, while the risk of severe retinopathy increased already when HbA1c exceeded 7.5%. The prevalence of neuropathy was 59%, of retinopathy 27% and of nephropathy 5% in the population of young patients after mean 13 years of diabetes duration. During the last 25 years the clinical characteristics at onset were unchanged as well as duration of partial remission and magnitude and persistence of C-peptide secretion. Conclusions. In this unselected population the cumulative proportion of severe retinopathy and overt nephropathy decreased over the last decades. Diabetic nephropathy has probably been prevented and not just postponed. Good glycaemic control was the most important factor to avoid complications, with the necessity of a lower level of HbA1c to escape retinopathy than nephropathy. Intensive insulin regimens from diabetes onset was not sufficient to entirely escape early diabetic complications after mean 13 years of diabetes duration, even if the prevalence of retinopathy and especially nephropathy was lower than usually reported. The clinical picture at onset of diabetes was unchanged the last 25 years. There was no secular trend of partial diabetes remission or C-peptide secretion during the first years after diagnosis.

No description available.

My PhD research studies, described in this thesis, were designed to document the temporal development of mechanical allodynia, a hallmark symptom of painful diabetic neuropathy (PDN), as well as opioid hyposensitivity using two different rat models of diabetes mellitus (DM). Specifically, the studies were conducted using the streptozotocin (STZ)-diabetic rat model of chemically-induced Type 1 diabetes in two different rat strains, as well as the Zucker Diabetic Fatty (ZDF) rat genetic model of Type 2 diabetes. Additionally, a longitudinal investigation of the effect of basal insulin replacement therapy to restore euglycaemia from 7-days post-STZ administration, on the development of mechanical allodynia in the hindpaws of the STZ-diabetic Wistar rat model of PDN, was conducted. The studies herein also included a longitudinal study to document the temporal development of mechanical allodynia and opioid hyposensitivity in the ZDF rat, which also examined the influence of dietary composition on the time course for the development of mechanical allodynia in the hindpaws, together with opioid hyposensitivity in these animals. In the final section of this thesis, the experiments were designed to examine possible mechanisms that may contribute to the development of opioid hyposensitivity in ZDF diabetic rats. These experiments involved the quantification of opioid receptor messenger ribonucleic acid (mRNA) gene expression as well as μ-opioid receptor (MOP-r) functional responses in tissues collected from 29-wk old diabetic ZDF rats relative to 7-wk old pre-diabetic control ZDF animals. In Chapter One, diabetes mellitus and more specifically its longterm complication, PDN, the focus of this doctoral research program, has been reviewed. Specifically, possible pathogenic mechanisms underlying mechanical allodynia, the relevant diabetic rodent models of PDN, use of insulin replacement therapy in diabetic rodents and its impact on hallmark symptoms of PDN, role of opioid pharmacology, the comparative efficacy of opioids in the treatment of PDN, and possible mechanisms that may underpin the development of opioid hyposensitivity in PDN, including the impact of altered excitatory neurotransmitters, have been reviewed. In Chapter Two, a preliminary study was conducted to investigate the efficacy of 4-wks treatment with Linplants (subcutaneous (s.c.) sustained-release bovine insulin implants) alone and in combination with ActRapid® (s.c. human insulin; 0.05 U to 3.5 U/100 g/day) with respect to glycaemic control in STZ-diabetic Wistar rats, and on acute diabetes characteristics for a 5-wk post-STZ administration period. Briefly, STZ-diabetic rats were divided into three groups: (1) rats which received no insulin treatment, (2) rats which were implanted with one s.c. Linplant at Day 7 post-STZ administration, and (3) rats which received one s.c. Linplant plus a once-daily injection of ActRapid® once diabetes was confirmed at 7-days post-STZ administration. The findings were that following implantation of a single Linplant at Day 7 post-STZ administration, euglycaemia was achieved in 50% of STZ-diabetic rats, with glycaemic control maintained for up to 4-wks post-implantation. Furthermore, once-daily injection of ActRapid™ to animals whose blood glucose levels (BGLs) were not well-controlled through use of Linplants alone, failed to achieve euglycaemia. It is possible that the ActRapid™ doses administered were not sufficient to achieve euglycaemia, and that increasing the doses may provide more effective glycaemic control. However, doubling the mean ActRapid™ dose from 1.63 (+ 0.3) U administered at Day 28 to 2.56 (+ 0.6) U administered at Day 34 post-STZ administration effectively only reduced BGLs by 1.3 mM to 11.6 + 1.6 mM. This suggests that although administering additional large doses of ActRapid™ to STZ-diabetic rats may eventually achieve euglycaemia, this method would presumably not be a more efficient method in achieving euglycaemia compared with the use of dosage-adjustable s.c. Linplants. Group (1) STZ-diabetic rats which were not treated with insulin developed diabetic signs including polydipsia, hyperphagia, decreased rate of body weight gain, and mechanical allodynia. Group (2) rats in which insulin treatment from 7-days post-STZ administration restored euglycaemia and reversed polydipsia and hyperphagia, were protected against the development of mechanical allodynia and reduced weight gain for the 5-wk study duration, while rats from Group (3) with incomplete glycaemic control developed levels of polydipsia, hyperphagia, reduced weight gain and mechanical allodynia intermediate between rats in Groups (1) and (2). These findings collectively suggest a direct correlation between the level of glycaemic control and the extent to which mechanical allodynia, a defining symptom of PDN, develops. In Chapter Three, the findings from the preliminary 5-wk study in Chapter Two were used to design a 24-wk longitudinal study of the temporal development of mechanical allodynia and opioid hyposensitivity in STZ-diabetic Wistar rats for comparison with the findings of a similar study previously undertaken by our laboratory using STZ-diabetic Dark Agouti rats (Nielsen et al, 2007). Additionally, this study examined the effects of tight glycaemic control achieved through the use of insulin implants as a means of potentially preventing the development of mechanical allodynia and opioid hyposensitivity for up to 24 weeks in STZ-diabetic Wistar rats. Briefly, STZ-diabetic rats were divided into 3 groups: (1) non-insulin treated STZ-diabetic Dark Agouti rats to provide comparison data with our laboratory’s previously published data in this rat strain (Nielsen et al, 2007), (2) non-insulin treated STZ-diabetic Wistar rats to examine possible between-species differences, and (3) STZ-diabetic Wistar rats which were treated with adjustable-dose s.c. Linplants from Day 7 post-STZ administration to maintain euglycaemia for the remainder of the 24-wk study period. In this 24-wk longitudinal study in STZ-diabetic rats, body weight, 24-hr water intake, paw withdrawal thresholds (PWTs) and BGLs were monitored at fortnightly intervals in all animals in order to document possible temporal changes in the development of diabetic signs and mechanical allodynia in the hindpaws respectively. STZ-diabetic rats underwent 6-wkly opioid antinociceptive testing, using single bolus doses of each of morphine and oxycodone with a 2-3 day washout period between individual opioids in order to assess the potential influence of both diabetes and glycaemic control on opioid potency in these animals. The findings demonstrate that non-insulin treated STZ-diabetic rats of both strains exhibited a decreased rate of body weight gain and polydipsia, as well as progressive development of mechanical allodynia in the hindpaws and loss of morphine potency. Importantly, STZ-diabetic Wistar rats which were treated with insulin to maintain euglycaemia from Day 7 post-STZ administration failed to develop these diabetic symptoms for the duration of the 24-wk study period, highlighting the importance of chronic hyperglycaemia in the development of mechanical allodynia and morphine hyposensitivity in the STZ-diabetic rodent model of PDN. The research described in Chapter Four involved a 22-wk longitudinal study of the development of diabetes and its longterm sensory nerve complications, viz mechanical allodynia and opioid hyposensitivity, in the ZDF rodent model of Type 2 diabetes commencing at 7-wks of age. This study also examined the influence of four different diets fed to separate groups of ZDF rats from 7-wks age, on the time course for the development of diabetes, mechanical allodynia in the hindpaws and opioid hyposensitivity in these animals. Briefly, ZDF rats were sub-divided into four dietary groups, each of which was fed one of the four following diets for 22-wks commencing at 7-wks of age, viz: (a) Purina 5008™, (b) a domestically-produced rat chow of similar composition to Purina 5008 (termed Purina Composition diet), (c) a Diabetogenic diet, or (d) Standard Rat Chow. All rats underwent once-fortnightly measurement of BGLs, body weight, 24-hr water intake, and measurement of PWTs in the hindpaws. Additionally, ZDF rats underwent opioid antinociceptive testing, similar to that previously described for STZ-diabetic rats (Chapter Three), to investigate the influence of diabetes and dietary composition on the antinociceptive potency of single bolus doses of morphine and oxycodone administered at 6-weekly intervals over a 22-wk study period. The afore-mentioned data were compared with the respective data obtained from the pre-diabetic control group of ZDF rats that were euthanised at 7-wks of age prior to the development of hyperglycaemia. The results demonstrate that the ZDF rat develops mechanical allodynia in the hindpaws and opioid hyposensitivity in a temporal fashion, in a manner similar to that previously documented for the STZ-diabetic Wistar rat model of Type 1 diabetes (Chapter Three). For the four diets assessed, there did not appear to be significant differences between dietary groups with respect to the time course and extent of development of hyperglycaemia, mechanical allodynia or opioid hyposensitivity in the ZDF rat model of PDN. The study described in Chapter Five investigated the effect of both diabetes and dietary composition on opioid receptor mRNA expression in tissue samples collected from the five groups of ZDF rats used in the behavioural studies described in Chapter Four and outlined above. Briefly, mRNA expression for each of the - (MOP), - (DOP), and - (KOP) receptors were quantified in mid-brain and spinal cord tissues prepared from 29-wk old diabetic ZDF rats maintained on one of four diets from 7-wks age, and compared with the respective expression levels in samples prepared from pre-diabetic ZDF rats euthanised at 7-wks of age. Overall, the findings suggest that diabetes does not alter opioid receptor mRNA expression in the mid-brain or spinal cord of diabetic ZDF rats at 29-wks of age relative to the corresponding levels of mRNA expression in the mid-brain and spinal cord of pre-diabetic ZDF rats at 7-wks of age. Hence, the marked reduction in the anti-allodynic potency of morphine and oxycodone observed in diabetic ZDF rats at 29-wks of age relative to that observed in pre-diabetic ZDF rats at 7-wks of age (Chapter Four) does not appear to be associated with a decrease in opioid receptor mRNA expression. In Chapter Six, the effect of both advanced diabetes and dietary composition on opioid-agonist stimulated [35S]GTPγS binding was examined in spinal cord tissue membranes from the ZDF rat. Specifically, [35S]GTPγS binding assays were used to assess the ability of a -opioid ligand (DAMGO) to stimulate -opioid receptor coupling to inhibitory G proteins in homogenates prepared from spinal cord samples of 29-wk old ZDF rats maintained on one of four different diets from 7-wks age (Chapter Four), relative to [35S]GTPγS binding in homogenates prepared from spinal cord samples of pre-diabetic 7-wk old ZDF rats. As specific MOP agonist-stimulated [35S]GTPγS binding was significantly decreased in spinal cord homogenates from diabetic ZDF rats at 29-wks of age relative to that for pre-diabetic ZDF rats (7-wks), this may contribute, at least in part, to the morphine hyposensitivity observed in diabetic ZDF rats at 29-wks of age relative to the pre-diabetic ZDF group. However, closer examination of these data revealed that specific MOP agonist-stimulated [35S]GTPγS binding above basal did not differ significantly between the pre-diabetic group and the longterm diabetic group of ZDF rats. Instead, there was significantly lower basal [35S]GTPγS binding in the spinal cord of ZDF rats at 29-wks c.f. 7-wks of age. Together, the findings suggest that impaired basal G-protein function rather than impaired coupling of MOP-r to its inhibitory G-protein may, at least in part, underpin -opioid agonist hyposensitivity in 29-wk ZDF rats. Finally, Chapter 7 contains a brief description of the main conclusions and discussion of the relevance of this doctoral research project, including potential future research directions.

Type 2 diabetes mellitus (T2DM) is one of the leading causes of death from worldwide non-communicable diseases. The prevalence of diabetes in the Mexico (MX)-United States border states exceeds the national rate in both countries. The economic burden of diabetes, due to decreased productivity, disability, and medical costs, is staggering and increases significantly when T2DM-related complications occur. The purpose of this study was to use a modified behavioral risk factor surveillance system (BRFSS) to describe the T2DM self-management behaviors, diabetes care, and health perception of a convenience sample of adults with T2DM in Monterrey, MX. This cross-sectional study design, with convenience sampling, was conducted with a convenience sample (n = 351) of adults in the metropolitan area of Monterrey, MX who self-reported a diagnosis of T2DM. Potential participants were recruited from local supermarkets. Twenty-six diabetes and health-related items were selected from the BRFSS and administered in face-to-face interviews by trained data collectors. Data analysis was conducted using descriptive statistics. The mean age was 47 years, and the mean length of time with T2DM was 12 years. The majority was taking oral medication and 34% required insulin. Daily self-monitoring of feet was performed by 56% of the participants; however, only 8.8% engaged in blood glucose self-monitoring. The mean number of health-care provider visits was 9.09 per year, and glycated hemoglobin level (HbA1c) was assessed 2.6 times per year. Finally, only 40.5% of the participants recalled having a dilated eye exam. We conclude the modified BRFSS survey administered in a face-to-face interview format is an appropriate tool for assessing engagement in T2DM self-management behaviors, diabetes care, and health perception. Extension of the use of this survey in a more rigorous design with a larger scale survey is encouraged.

Patient nonadherence to physicians' prescribed therapeutic regimen is the greatest challenge in the effective treatment of patients with diabetes worldwide. Scientific evidence has revealed that nonadherence to prescribed medication could result in diabetic complications such as cardiovascular disease, retinopathy, nephropathy, and neuropathic diabetic foot ulcers. The purpose of this study was to explore predictive relationships between levels of adherence to antidiabetic medications, patient HbA1c levels, and diabetic complications among Jamaicans, an understudied population. The research question that guided this study was: Do the patient level of adherence and HbA1c levels have any predictive relationship with the severity of diabetic complications (cardiovascular disease, retinopathy, nephropathy and neuropathic foot ulcer) among Jamaicans after controlling for age and gender? The theory of planned behavior was used to guide the study. Data regarding diabetic complications were collected from 119 records during a cross-sectional review of patient dockets. Level of adherence was determined from an interviewer-administered Morisky 8-item adherence scale. A multiple regression analysis revealed that lower levels of patient adherence to treatment and higher HbA1c levels predicted greater severity of cardiovascular disease (p = .000; p = .000), retinopathy (p = .009; p =.090), nephropathy (p =.007; p =.001) and diabetic neuropathic foot ulcers (p =.027; p =.001). Findings from this study will contribute to the knowledge base on diabetic medication nonadherence and may encourage health care professionals to advocate for better medication adherence strategies among people with diabetes.

The early detection of acquired losses of colour vision can provide the ophthalmologist with a very sensitive indicator of visual dysfunction. As a result we have developed and tested an automated, CRT-based, chromatic discrimination system that allows us to measure acquired colour-vision deficits with great precision. This system, known as the Sussex Gratings Machine, can produce chromatic stimuli in any direction in equal luminance colour space. However, we have found that measurements made along a constant MIL-cone axis (Tritan) or a constant S-cone (Red/Green) confusion axis are most useful. Using this system we have investigated acquired colour vision deficits in diabetes ,. and thyroid eye disease. We have shown that tritan discrimination losses can be used to screen for severe diabetic retinopathy and also to predict those who are likely to develop it within 18 months. We have also confirmed that acquired tritan discrimination losses are a very useful indicator of optic-nerve compression in thyroid eye disease.

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy