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The human kidney maintains whole organism homeostasis through tightly regulated filtration, transport, and endocrine functions that are subserved by a wide array of specialised cell types with distinct developmental origins and anatomical organisation. Amongst these cells lies a network of tissue-resident immune cells with incompletely characterised roles in tissue homeostasis, repair, and defence. Furthermore, different regions of the kidney are affected by distinct immunologically relevant insults - ascending bacterial infection in the medullary-pelvic region, and immune complex-mediated inflammation within the cortex. We hypothesised that the kidney-resident immune network is adapted to respond to and counter these immunological challenges. Recently the advent of high-throughput single-cell genomics methods has permitted the atlasing of human organs in unprecedented molecular detail. These techniques permit the unbiased characterisation of cell types and cell states, the prediction of functional programmes, and the mapping of intercellular interaction networks underpinning tissue organisation. In this thesis, we used single-cell RNA sequencing to generate the first detailed and spatio-temporally resolved atlas of the human kidney in development and maturity. This study has identified the transcriptional signatures of mature kidney parenchymal cell types from nephron epithelial, vascular, and stromal compartments, and allowed the reconstruction of the developmental trajectories of early nephrogenesis. Within the immune compartment, we charted the landscape of immune cells, and the seeding of these cells through early development, identifying a macrophage subset in mature kidneys with transcriptional similarity to macrophages seeded early in fetal life, and likely representing a long-lived self-renewing subset. This work also identified a transcriptional signature mediating antimicrobial defence in the pelvis of the kidney. Here pelvic epithelial cells express antimicrobial peptides, and neutrophil- and monocyte-recruiting chemokines, orienting the immune response towards the dominant infectious threat. We later optimised and refined the single-cell genomics approach to the human kidney, generating high quality data on a larger panel of donors in a compartmentally-resolved fashion. In this work we were able to assess the immune composition of glomerular and tubulointerstitial compartments, finding a strikingly divergent immune landscape, with glomeruli occupied by monocytes, dendritic cells, and NK cells, and the tubulointerstitial compartment housing populations of resident macrophages, T cells, and B cells. Finally we have begun to explore the dynamic transcriptional responses of kidney immune and parenchymal cells to IgG immune-complex stimulation using an ex vivo normothermic normoxic perfusion model. These experiments have uncovered cell type-specific signalling responses in stimulated monocytes, NK cells, and glomerular endothelial cells, with evidence for abundant chemokine and cytokine signalling coordinating the early response to this highly disease-relevant perturbation.

Chronic kidney disease (CKD) has a rapidly rising global prevalence, affecting as many as one third of the population over the age of 75 years. CKD is a well-known risk factor for cardiovascular disease and in particular, there is a strong association with stroke. Cohort studies and trials indicate that reduced glomerular filtration rate increases the risk of stroke by about 40% and that proteinuria increases the risk by about 70%. In addition, CKD is also strongly associated with subclinical cerebrovascular abnormalities, vascular cognitive impairment, and dementia. There are however a number of outstanding questions regarding the relationship between CKD and stroke. Firstly, the mechanisms underpinning this relationship are currently unclear. CKD is associated with traditional risk factors such as hypertension, diabetes mellitus, and atrial fibrillation, but non-traditional risk factors such as uraemia, oxidative stress, mineral and bone abnormalities, and dialysis-related factors such as changes in cerebral blood flow or cardiac structure are also purported mechanisms. Hypertension is the leading modifiable risk factor for stroke in the general population and is highly prevalent in CKD, but its role has not been systematically examined. Secondly, it is not known which stroke subtypes are most closely associated with CKD. Thirdly, although CKD is associated with greater short- and long-term mortality post-stroke, its impact on initial stroke severity, recovery, and recurrence risk are less clear. Fourthly, it has been hypothesized that cerebral small vessel disease (SVD) and CKD may be part of a multisystem small-vessel disorder, but their association may simply be confounded as a result of shared risk factors (e.g. hypertension) rather than represent a systemic susceptibility to premature SVD. Finally, CKD has also been associated with cognitive dysfunction but it is not known whether this association is independent of cerebrovascular disease. The aim of my thesis therefore was to determine the role of hypertension in the relationship between CKD and stroke risk. Using the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classification, I also aimed to describe which transient ischaemic attack (TIA) and ischaemic stroke subtypes occur most frequently in patients with CKD. I sought to determine whether CKD is associated with worse initial stroke severity and recovery, and whether CKD is independently predictive of recurrent stroke. I aimed to study the age-specific associations of CKD and the overall burden of SVD (total SVD score), as well as individual SVD markers. Lastly, I investigated the association between CKD and dementia before and after TIA and stroke. The work of this thesis forms two parts. For the first part, I undertook two large and complex systematic reviews and meta-analyses to better describe what data were already available on the relationship between renal function, proteinuria, and stroke. For these reviews, I developed a search strategy, identified >10,000 studies for title and abstract review, and then >400 studies for full text review. I selected studies and extracted study, patient, stroke characteristics, and outcome data according to a pre-defined protocol. I assessed the quality of included studies, and performed all statistical analysis including meta-analysis, sub-analysis, meta-regression and funnel plots for publication bias. I categorized all studies according to a hierarchy of hypertension adjustment. For the second part of my thesis, I have collected, collated and analysed data from the Oxford Vascular Study (OXVASC), a population-based prospective cohort study. In particular I worked as one of the Clinical Research Fellows at OXVASC and was involved in regular recruitment, assessment and follow up of study patients. I studied a panel of 16 blood biomarkers related to inflammation, thrombosis, atherogenesis, and cardiac or neuronal cell damage in 1297 patients with TIA or ischaemic stroke. Biomarker levels were log-transformed and correlated with eGFR, adjusted for age. All ischaemic events were classified by TOAST subtypes (cardioembolism, large artery disease, small vessel disease, undetermined, multiple, other aetiology, or incompletely investigated). I used logistic regression analysis to determine the relationship between CKD and TIA/stroke subtypes in 2969 patients who presented between April 1, 2002 to March 31, 2017. I also studied initial stroke severity and early recovery measured using the National Institutes of Health Stroke Scale (NIHSS) and modified Rankin scale (mRS), respectively, in ordinal regression analyses in the same cohort of patients. I used Cox proportional hazard models to determine if CKD was an independent predictor of stroke and other vascular event recurrences. For the third part of my thesis, I focused on the relationship between CKD, subclinical cerebrovascular disease, and dementia in phenotyped cohorts within OXVASC. I investigated the age-specific associations of CKD and total SVD burden (total SVD score) adjusting for age, sex, and vascular risk factors in 1939 consecutive patients with TIA or ischaemic stroke who underwent cerebral magnetic resonance imaging (MRI) from 2004 to 2018. Finally, I examined associations between pre-event dementia and CKD using logistic regression, and between post-event dementia and CKD using both Cox and competing risk regression models (to account for the competing risk of death), adjusted for age, sex, education, cerebrovascular burden (stroke severity, prior stroke, white matter disease), diabetes mellitus, and dysphasia. There are several clinically relevant findings in this thesis. Firstly, I have shown that the association between CKD and stroke appears to be highly dependent on the method of adjustment for hypertension. The apparently independent relationship between CKD and stroke may be confounded by their shared association with long-term blood pressure burden. Secondly, I have demonstrated that proteinuria is strongly and independently associated with incident stroke risk even after robust adjustment for hypertension, possibly indicating a shared renal and cerebral susceptibility to vascular injury in this subset of patients of kidney disease that is not fully explained by traditional vascular risk factors. Thirdly, I found that correlations between biomarkers related to inflammation and thrombosis with renal dysfunction in the setting of cerebrovascular events were generally modest after adjustment for age, suggesting that putative risk factors such as chronic inflammation or coagulopathy are unlikely to be important stroke mechanisms in patients with CKD. Fourthly, I have shown that there were no independent positive associations between CKD and specific TOAST subtypes which suggests that renal-specific risk factors are unlikely to play an important role in the aetiology of particular subtypes. Fifthly, I have highlighted that CKD is associated with severity of cerebrovascular events (stroke vs TIA; initial NIHSS; 1-month mRS) and that it is independently predictive of stroke recurrence, particularly early recurrence. Sixthly, I have shown how the association of CKD and cerebral SVD is attenuated with adjustment for shared risk factors at older ages, but remains at younger ages, consistent with a shared susceptibility to premature vascular disease. Finally, in patients with TIA and stroke, CKD was not independently associated with either pre- or post-event dementia, suggesting that age, sex, education, and cerebrovascular burden may play a more important role in the aetiology of dementia in CKD than renal-specific neurodegenerative mechanisms.

Background Hyporesponsiveness to erythropoiesis-stimulating agents (ESA) and its association with adverse cardiovascular outcomes remains a considerable problem in patients with end-stage renal disease (ESRD) undergoing haemodialysis. Pentoxifylline has been shown to have some beneficial effect on ESA Hyporesponsiveness by reducing inflammation in ESRD patients. Methods We conducted a single centre, double-blind, placebo-controlled randomised trial to study the effect of Pentoxifylline on Erythropoietin stimulating agent (ESA) requirement of stable haemodialysis patients. Inclusion criteria were equivalent ESA dose of greater than or equal to 6000 International Units (I.U) per week or ESA resistance index greater than or equal to 6.5 I.U /kg/wk/Hb (g/dl) and stable Hb between 9 to 12 g/dl. The primary study endpoint was ESA requirement relative to Haemoglobin (Hb) level at the end of study period of 6 months. Secondary endpoints included safety analysis, Hb values, ESA dose and cardiovascular imaging biomarkers such as vascular PET CT and cardiac MRI scan. Cytokine profile was also analysed during the study. Results A total of 69 patients underwent randomisation. At the end of the study period, there was no statistically significant (p value= 0.26) difference in ESA /Hb ratio between pentoxifylline and placebo group (Mean (SD) 3.98 mcg/gm/dl (3.09) versus 4.91 mcg/gm/dl (3.49) respectively). The secondary outcomes did not show any statistically significant change between pentoxifylline and placebo group. There were no concerns regarding the safety of pentoxifylline in haemodialysis patients. The cytokine profile showed a reduction in inflammatory cytokines titres and rise in anti-inflammatory cytokines in the entoxifylline group analysed as slopes of cytokine variability longitudinally. Conclusions Pentoxifylline did not improve the ESA requirement in ESA hyporesponsive, stable haemodialysis patients over six months period. There was no statistically significant change in cardiovascular imaging biomarkers between Pentoxifylline and placebo group. Cytokine profile showed a favourable response to Pentoxifylline therapy.

Intra-dialytic haemodynamic instability is a common and disabling problem which may lead to morbidity and mortality though repeated organ ischaemia, but it has proven difficult to link any particular blood pressure threshold with hard patient outcomes. The relationship between blood pressure and downstream organ ischaemia during haemodialysis has not been well characterised. Previous attempts to predict and prevent intra-dialytic hypotension have had mixed results, partly due to patient and event heterogeneity. Using the brain as the indicator organ, we aimed to model the dynamic relationship between blood pressure, real-time symptoms, downstream organ ischaemia during haemodialysis, in order to identify the most physiologically grounded, prognostic definition of intra-dialytic decompensation. Following on from this, we aimed to predict the onset of intra-dialytic decompensation using personalised, probabilistic models of multivariate, continuous physiological data, ultimately working towards an early warning system for intra-dialytic adverse events. This was a prospective study of 60 prevalent haemodialysis patients who underwent extensive, continuous physiological monitoring of haemodynamic, cardiorespiratory, tissue oxygenation and dialysis machine parameters for 3-4 weeks. In addition, longitudinal cognitive function testing was performed at baseline and at 12 months. Despite their use in clinical practice, we found that blood pressure thresholds alone have a poor trade off between sensitivity and specificity for predicting downstream tissue ischaemia during haemodialysis. However, the performance of blood pressure thresholds could be improved by stratification for the presence or absence of cerebral autoregulation, and personalising thresholds according to the individual lower limit of autoregulation. For patients without autoregulation, the optimal blood pressure target was a mean arterial pressure (MAP) of 70mmHg. A key finding was that cumulative intra-dialytic exposure to cerebral ischaemia, but not to hypotension per se, corresponded to change in executive cognitive function over 12 months. Therefore we chose cerebral ischaemia as the definition of intra-dialytic decompensation for predictive modelling. We were able to demonstrate that the development of cerebral desaturation could be anticipated from earlier deviations of univariate physiological data from the expected trajectory for a given patient, but sensitivity was limited by the heterogeneity of events even within one individual. The most useful phys- iological data streams included peripheral saturation variance, cerebral saturation variance, heart rate and mean arterial pressure. Multivariate data fusion techniques using these variables created promising personalised models capable of giving an early warning of decompensation. Future work will involve the refinement and prospective testing of these models. In addition, we envisage a prospective study assessing the benefit of autoregulation-guided blood pressure targets on short term outcomes such as patient symptoms and wellbeing, as well as longer term outcomes such as cognitive function.

Intradialytic Hypotension (IDH) is the commonest complication of maintenance haemodialysis and is associated with increased morbidity and mortality. However, there is no standardised definition of IDH, making comparisons between studies difficult. This observational study with a total of 80 patients and over 600 dialysis sessions showed a poor correlation between symptoms and hypotension. Importantly, patients experienced low blood pressure without symptoms, so continuous intradialytic blood pressure monitoring is required to identify this asymptomatic group. In light of these findings, a revised definition of IDH is suggested. This study also aimed to identify predictors of IDH that could be detected in sufficient time to allow a mitigating intervention. A novel non-invasive alternative for continuous blood pressure monitoring is introduced which uses intra-fistula pressure data from the sensors sited in the extracorporeal circuit of the dialysis machine. Results show that in the majority of patients, changes in intra-fistula pressure correlate with blood pressure measurements obtained by a standard oscillometric device. To investigate whether IDH can be predicted, a photoplethysmogram (PPG) waveform was obtained from a pulse oximeter attached to the finger or ear. Continuous PPG monitoring of patients with IDH during dialysis demonstrated that some IDH episodes were predictable using the variation in the PPG baseline with respiration as a surrogate for low blood volume. Additionally, the area under the curve of the PPG waveform can be used as a surrogate for cardiac output and peripheral vascular tone, resulting in a reasonable predictor for potentially critical changes in blood pressure during dialysis. Individually, the novel metrics described here are limited in their identification of IDH in all patients affected, but in combination they may be used to develop a multi-parameter predictive model. The relative merits of personalised versus population-based models are explored and a conclusion is drawn that personalised multi-parameter data fusion modelling for haemodialysis patients would be an important area for future work.

Hypoxia regulates many hundreds of genes with important roles in ischemic and neoplastic disorders. Central to this response are the hypoxia inducible transcription factors (HIF). This work aimed to better understand the direct transcriptional response to HIF by mapping HIF-binding sites across the genome using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq). ChIP-seq for HIF in MCF-7 breast cancer cells under hypoxic conditions revealed more than 400 high-stringency HIF-binding sites genome-wide. Each member of the HIF heterodimer was present with near complete concordance. Binding of the two principle isoforms revealed a high degree of overlap with no differences in the DNA-binding motif. HIF-binding was associated with upregulation, but not downregulation of genes indicating that it functions as a transcriptional activator but not as a repressor. HIF-binding occurred preferentially at gene promoters, but was also present at promoter-distant sites, which were also associated with gene regulation, implicating long-range interactions in hypoxic gene activation. HIF-binding was associated with markers of open chromatin and active enhancers that were present in normoxia, indicating that HIF-binding sites are already “prepared” to bind HIF before the hypoxic stimulus. Analysis of normoxic and hypoxic RNA pol2 and H3K4me3 signals revealed distinctive hypoxia-inducible changes unique to HIF-binding genes. Comparable numbers of HIF-binding sites were observed in a second cell line (von Hippel-Lindau defective 786-O renal cancer cells) as in MCF-7 breast cancer cells, although approximately 65% were unique to 786-O cells. These unique sites were more frequently promoter-distant. Correlation with expression analyses from renal tumours indicated that many HIF-binding genes were upregulated in renal cancer. One such RCC unique promoter-distant HIF-binding site was identified at an intergenic locus on chromosome 11q13.3 that has been associated with renal cancer in Genome-Wide Association Studies. The HIF-binding site was in high linkage disequilibrium with the disease associated SNP and had the epigenetic hallmarks of an enhancer. Analysis of pan-genomic expression analyses identified the cell-cycle regulator cyclin D1 as highly HIF-regulated, and a physical association between the HIF-binding site and the CCND1 promoter could be determined. Furthermore, in a renal cancer cell line heterozygous at this locus, the RCC-protective allele disrupted HIF-binding leading to an allelic imbalance in cyclin D1 expression.

Tacrolimus, the most common immunosuppressant for organ transplant, has a narrow therapeutic range and is metabolized by CYP3A4/5. Up to 50% of the variability in tacrolimus exposure is explained by variants in the gene CYP3A5. Given equivalent doses, CYP3A5 expressers (*1 allele carriers) demonstrate faster tacrolimus metabolism than non-expressers. In chapter 1, we describe the effect of CYP3A5 gene, age, and concomitant fluconazole on the tacrolimus concentration. We hypothesize that CYP3A5 expressers will need more dose adjustments compared to CYP3A5 non-expressers in the 8 weeks following tacrolimus initiation. Our aim is to study pediatric patients within the first 8 weeks after kidney transplant to examine the effect of CYP3A5 genotype and other clinical factors (i.e., age and fluconazole) on tacrolimus trough concentrations and TDM outcomes. We analyzed the electronic health records of 93 patients. Banked DNA was collected, and genotyping was performed for CYP3A5. Included patients received a kidney transplant between Jan 2010 to April 2021. The tacrolimus target range was 10-15 ng/mL in the first four weeks and 7-10 ng/mL in the next four weeks. We found that CYP3A5 expressers (n=21) had fewer measurements in range than non-expressers (n=72), (32.4% vs 39.6%, P=0.029). Expressers had more dose adjustments (8 vs 6, p=0.0234), and they needed more than 170% of the required daily dose compared to the non-expressers. The concentration/dose (C0/D) ratio was influenced by age and concomitant fluconazole (p=0.0003, P=0.034, respectively). In chapter 2, our aim is to study the association between tacrolimus concentration and toxicity in the first 8 weeks after tacrolimus initiation in pediatric kidney transplant. We hypothesize that the trough concentration of tacrolimus is associated with its toxicity. Adverse reactions to tacrolimus present as CNS toxicity, gastrointestinal toxicity, and other toxicities like muscle cramps, edema, and rashes. A global consensus report recommends that the target in pediatric kidney transplant patients be 10- 20 ng/mL during the first 2 months after transplantation. We investigated toxicity using a trough threshold of 15ng/mL because it is the upper limit of the target range at our center; we found that Patients who ever had troughs over 15 ng/mL were more likely to develop tremors and CNS toxicity compared to patients with troughs never over 15 ng/mL.In chapter 3, our hypothesis is that adenine is an aquaretic agent that could be used to correct hyponatremia. We aimed to study the effect of different doses of adenine on kidney handling of salt and water. Adenine when given orally in high dose as 0.5% (wt/wt) has a massive water depletion effect in rats during the first 24 hours of treatment. In our study, we investigate the dose-response and the time-response of Adenine in doses of 0.15, 0.20 and 0.25%. We found that adenine at 0.25%, at least in 1 and 3 weeks, acts as an aquaretic agent that increases water excretion without affecting salt excretion or food intake. Therefore, it prevents hyponatremia in a rat model of SIADH (Syndrome of inappropriate antidiuretic hormone secretion).

INTRODUCTION: The use of drugs can lead to renal adverse effects prompting the need to include warnings and precautions on medication labels. Studies evaluating renal warnings and precautions need to be improved. This study assessed the characteristics of FDA renal toxicity warnings and precautions included in the labels of drugs approved by the FDA and marked in the US. The study also evaluated the potential factors of including a boxed warning on a drug label. METHODS: We extracted the labels of human prescription drugs with renal toxicity boxed warnings and warnings and precautions from the “FDA Label: Full-Text Search of Drug Product Labeling” database, FDA regulatory information from Drugs@FDA as of September 31, 2022. We extracted the therapeutic classification from the World Health Organization (WHO) Anatomical Therapeutic Chemical (ATC) Classification System. We conducted descriptive and logistic regression analyses. RESULTS: The FDA drug label database listed 611 drugs and combinations with renal warnings or boxed warnings for 511 active ingredients as of September 1, 2022. Metformin hydrochloride, hydrochlorothiazide, dextrose, potassium chloride, and amlodipine besylate were the drugs with the highest number of renal warnings and precautions. The greatest number of renal warnings occurred in the 2000-2019 decade. There were 85 drugs, including 3 with emergency use authorization for COVID-19, with a boxed warning mentioning renal toxicity. The largest therapeutic classes with renal toxicity boxed warnings were anti-infectives for systemic use, antineoplastic and immunomodulating agents, and alimentary tract and metabolism. The most common boxed warnings were renal impairment, nephrotoxicity, and nephrogenic systemic fibrosis, with some warnings referring to potential problems for patients with kidney transplants. CONCLUSIONS: Many FDA-approved drugs have renal warnings and precautions, with preventable adverse drug events being the most common type of warning. Antineoplastic and immunomodulating agents have the highest incidence of renal warnings among therapeutic classes. The FDA approval year and some therapeutic classes are associated with the presence of renal boxed warnings, but other factors may also play a role. Further research is needed to improve drug safety for patients. Patients receiving medication, particularly those receiving drugs associated with renal warnings and precautions, require ongoing monitoring and management.

Currently, rural hospitals account for thirty percent of all hospitals in the United States (U.S). However more of these facilities have experienced closure in the last decade than any time in U.S. history. Typical reasons for this occurrence are financial distress stemming from shrinking patient volume, physician departure, and reimbursement reductions. Currently the state of Tennessee has experienced several rural hospital closures to date. Finding opportunities to grow in-patient census while expanding substantial revenue generation is paramount to slow the closure trend. Covid-19 has caused a recent rise and demand for telehealth services in the healthcare industry. Is now a time for rural hospitals to improve telehealth resources to capture greater patient volume and potential improvement in revenue reducing financial burdens? A descriptive causal design utilizing secondary data from four rural hospitals in east Tennessee will determine if telehealth has increased in-patient volume over a four-year period of time. Diagnosis related groups (DRG) will be tested with Pearson’s Chi-square with year over year group comparisons for improvement before, during, and since the pandemic. As telehealth use has grown during Covid- 19, have rural hospitals leveraged benefits to care for specialty diseases (i.e., neurology, nephrology, pulmonology) that before may have not been an option? Can comparing hospitals to understand if less or greater telehealth is performed in each studied facility influence in-patient volume? Using Medicare payment codes consideration for this research maybe used in other geographic regions to perform similar analysis in the future.

Donor kidneys are damaged upon circulatory death before being preserved with machine perfusion until transplantation. During machine perfusion, blood is pumped back into the vasculature of the kidney, causing damage called ischemic reperfusion injury. The two forms of machine perfusion are hypothermic machine perfusion and normothermic machine perfusion. Different methods of treatment can be used during either hypothermic machine perfusion or normothermic machine perfusion to prevent ischemic reperfusion injury. Each of the treatment methods that were reviewed showed potential for preventing ischemic reperfusion injury, with hypothermic treatments having higher average levels of feasibility than normothermic treatments. The effect of the analyzed treatments on graft function after transplantation is likely positive, so a conclusion can be drawn that undergoing any one of the treatment methods will lead to positive results after transplantation.

Background: Elevated urine microalbumin to urine creatinine (UMC) ratios have been associated with the progression of acute kidney injury (AKI) to chronic kidney disease. Urine neutrophil gelatinase-associated lipocalin (uNGAL) has demonstrated a better association with proteinuria compared to AKI in neonates. We aimed to identify if the direct relationship between uNGAL and proteinuria persisted in infants following non-cardiac surgical procedures. Methods: Infants admitted to the Level IV NICU, =150 ng/mL by 48 hours. Proteinuria (>=0.8 mg/mg) was present in 83% of samples. Elevated uNGAL procedures were associated with increased urine concentrations of protein (UPC) and UMC (UPC: 2.2 mg/mg [IQR: 1.4, 3.4] vs. 1.3 mg/mg [IQR: 0.9, 2.0], p= 0.0009; UMC: 251 mg/g [132, 757] vs. 168 mg/g [109, 295], p=0.0012). Conclusion: Proteinuria and microalbuminuria are common in neonates undergoing non-cardiac surgical procedures and are associated with postoperative elevated urine NGAL >=150 ng/mL by 48 hours.

Background: Acute kidney injury (AKI) is associated with increased mortality, morbidity, length of hospital stay, and in-hospital cost. Timely intervention is needed to prevent disease progression, but the delay in diagnostic process often results in delayed treatment. Recent studies show a significant increase in novel urine biomarkers (UB), TIMP-2 and IGFBP7, has been linked to an increased risk of AKI. Objectives: The purpose of this study was to examine the relationships among participants’ sociodemographic and clinical characteristics, including select biomarkers, care management processes, treatments, and patient outcomes in a sample of hospitalized critically ill patients in southern California. Findings: Study results from 383 critically ill adult patients demonstrated almost 40% of the study participants presented with AKI (22.5% AKI stage 1 and 17.2% AKI stage 2-3). During inpatient hospital stay, an additional 26.6% developed AKI, resulting in a total of 66.3% participants are affected by AKI, either upon admission or during hospitalization. Urine biomarkers had significant association with AKI status. Patients with high risk for AKI based on initial, repeat, and peak UB values had a higher incidence of developing AKI stage 2-3 at discharge. Patients with UB peak showing high risk developed AKI stage 2-3 at discharge ten times more (44%) than those with low risk (4.4%). The incidence of AKI stage 2-3 in this study population is almost double when compared to other clinical studies. Mortality was significantly higher for patients with AKI stage 2-3 (55.9%). In contrast, hospital LOS for patients with AKI stage 2-3 (9.76 days) were significantly shorter than those with AKI stage 1 (12.24 days) or no AKI (13.43 days). Implications: Nurses at the bedside play an important role in detecting changes in patient condition. Significant associations between UB and select factors that contribute to or predispose the patient to develop AKI indicate UB have utility in the early detection of AKI in the clinical setting and can aid in the escalation process of patients’ care, guide treatment, and provide an objective measure of the effectiveness of treatments. If left undetected and untreated, AKI can progress to advanced stages, multi-organ failure, and even death.

Background: The kidneys play a crucial role in maintaining homeostasis of serum potassium levels (K+). Patients with advanced chronic kidney disease (CKD) are at a higher risk of experiencing dyskalemia events (i.e. hyper- and hypokalemia; especially the former) and thus future adverse outcomes. Currently, there is a dearth of literature on prediction models for hyperkalemia and the effects of dyskalemia on outcomes such as incidence of ischemic stroke and short-term hospital/emergency room (ER) utilization in an advanced CKD population transitioning to dialysis. Objectives: Using a nationally representative sample of US veterans with advanced CKD transitioning to dialysis, in the pre-dialysis period we studied the following aims: Aim 1) Develop and validate a prediction model for predicting hyperkalemia in individual patients; Aim 2) Examine the association of dyskalemias with time to first ischemic stroke; Aim 3) Examine the association of dyskalemias with time to short-term hospital/ER utilization. Methods: A retrospective cohort analysis of the Transition of Care in Chronic Kidney Disease cohort (n=102,477), a nationally representative sample of US veterans with advanced CKD transitioning to dialysis between October 1, 2007 through March 31, 2015 identified from the United States Renal Data System was conducted. Across the three study aims, we identified patients with an initial selection criterion (prior to dialysis initiation) of two estimated glomerular filtration rate (eGFR) of /min/1.73m2 90-365 days apart (second eGFR as index); at least one-year each of baseline period (prior to index) and follow-up period (following index but prior to dialysis initiation); and at least one K+ measurement each in the baseline and follow-up period. For each study aim, further inclusion criteria were used to yield a final sample size of 21,654, 21,357, and 21,366 for aim 1, aim 2, and aim 3, respectively. For Aim 1 (Chapter 2), we compared the performance (area under the receiver operating curve [AUROC]) of different machine learning methods including logistic regression (LR), random forest, extreme gradient boosting, and support vector machines using geographical splitting (for creating training and test set) with 10-fold cross validation to predict the outcome of hyperkalemia (K+ >5.5 mEq/L). The method that yielded the best performance was used to build a reduced model with 10 predictors to develop a patient-level hyperkalemia risk score. For Aim 2 (Chapter 3), we assessed the association of baseline time-averaged K+ levels (distant exposure) and time-updated K+ levels (acute exposure) (both categorized as hypokalemia [K+ 5.5 mEq/L] and referent [3.5 mEq/L ≤ K+ ≤ 5.5 mEq/L]) with time to first ischemic stroke using Cox regression models. Finally, for Aim 3 (Chapter 4), we assessed the association of time-updated outpatient K+ levels (categorized as hypokalemia [K+ 5.5 mEq/L] and referent [3.5 mEq/L ≤ K+ ≤ 5.5 mEq/L]) with hospital/ER utilization (as separate events) using generalized estimating equations. Across all the three study aims (Aim 1, 2, and 3) several different sensitivity analyses were conducted to test the robustness of the results. Results: Across the analytic samples (Aim 1, 2, and 3), the mean age was 69 years, ~98% were males; ~28% were African Americans, ~69% had diabetes mellitus, and the one-year baseline averaged K+ was 4.5 mEq/L. In aim 1 (n=21,654), the LR model yielded the best performance with an average AUROC (95% confidence interval [CI]) of 0.765 (0.756-0.774) (training set) and 0.763 (0.753-0.771) (test set) using the geographical splitting with 10-fold cross validation. Using the LR method, the top 10 predictors identified were K+ value prior to index, age, having at least 1 K+ >5.5 mEq/L in the baseline, index eGFR, baseline averaged SBP, baseline averaged HCO3-, number of K+ counts, thiazide use, number of outpatient visits, and NSAIDs use in baseline. The LR parameter estimates for the above listed predictors were used to develop a patient-level risk score for predicting hyperkalemia. In aim 2 (n=21,357), hypokalemia (distant exposure) was associated with higher risk of ischemic stroke (hazard ratio [HR]; 95 % CI: 1.35, 1.01-1.81). Conversely, hyperkalemia (acute exposure) was associated with a lower risk of ischemic stroke (HR; 95% CI: 0.82, 0.68-0.98). Finally, in aim 3 (n=21,366) using outpatient K+ levels, both hyperkalemia (odds ratio [OR]; 95% CI: 2.04; 1.88-2.21) and hypokalemia (OR; 95% CI: 1.66; 1.48-1.86) were associated with higher risk of hospital visit within 2 calendar days of outpatient K+ measurement. Similarly, both hyperkalemia (OR; 95% CI: 1.83; 1.65-2.03) and hypokalemia (OR; 95% CI: 1.24; 1.07-1.44) were associated with higher risk of ER visit within 2 calendar days of outpatient K+ measurement. Across all the three study aims, the results were robust to various sensitivity analysis. Conclusion: In an advanced CKD population transitioning to dialysis, in the pre-dialysis period, we developed an internally valid model for predicting hyperkalemia. We observed that hypokalemia as a chronic exposure is associated with higher risk of ischemic stroke and hyperkalemia as an acute exposure is associated with lower risk of ischemic stroke. Finally, both hyper- and hypokalemia are associated with higher risk of short-term hospital/ER visits. Further studies are needed to externally validate the hyperkalemia risk prediction model; understand the mechanisms underlying the association of dyskalemias with stroke; and expand on the association of dyskalemias with short-term hospital/ER visits by including cost as an outcome.

Nephrotic syndrome (NS) is the second leading cause of chronic kidney disease (CKD) presenting under the age of 30. NS presents in children with edema and severe proteinuria, caused by the effacement of podocyte foot processes within the glomerular filtration barrier. Patients with steroid-resistant NS (SRNS) frequently develop end-stage renal disease (ESRD). Additionally, renal biopsies from these patients often reveal focal segmental glomerulosclerosis (FSGS). Pathogenic mutations in known monogenic disease genes have been found in 11-45% children with FSGS/SRNS. Notably, most Mendelian etiologies exhibit recessive inheritance, while dominant vertical inheritance with incomplete penetrance is observed in the remainder. The role of de novo variants (DNVs) in NS necessitates further investigation. Tripartite motif containing 8, TRIM8, is an E3 ubiquitin ligase. De novo TRIM8 variants were previously implicated in a syndromic disease consisting of neurodevelopmental delay, epilepsy, cerebral atrophy, and nephrotic syndrome. In this study, we recapitulate the patient-specific mutations in inducible overexpression cell lines and in CRISPR/Cas9-generated mouse models. N-terminal MYC or GFP-tagged TRIM8 inducible cell lines were generated and characterized using the pInducer21 system. Western blot and immunofluorescence data show that MYC- and GFP-TRIM8 were induced by doxycycline in immortalized podocyte cell lines. Candidate interactors for TRIM8 from the literature and stratified using kidney single cell mRNA sequencing expression were cloned into mammalian expression vectors. Finally, a Trim8 knockout allele (c. 56_162del; p.H20Qfs*124 and c.367_463+304delins46) was generated and bred to yield an allelic series of wildtype, heterozygous and homozygous animals. These mice exhibited normal survival and did not demonstrate proteinuria through three to four months of life. Overall, further studies are ongoing with regards to the continued monitoring of proteinuria and kidney dysfunction, as well as the potential interactor cloning and cell line characterization.

Nephrotoxicity is a dose-limiting side effect of gentamicin that accounts for a significant portion of clinical acute kidney injury (AKI). The mechanism of gentamicin-induced nephrotoxicity is uncertain and effective therapy for gentamicin-induced renal cell injury is limited by incomplete mechanistic insight. To address this challenge, we hypothesized that RNAi signal pathway screening can identify both a unifying mechanism of gentamicin-induced cell injury and a therapeutic that ameliorates its toxicity. Dual shRNA screens of 5,000 individually barcoded signal pathway genes were performed in gentamicin-exposed human proximal tubule cell lines and differentially expressed shRNAs were analyzed by Ingenuity Pathways Analysis (IPA) software. Computational analysis of RNAi signal screens identified the Cross-Organelle Stress Response (CORE), the Unfolded Protein Response (UPR), and cell chaperones as key injury targets of gentamicin-induced proteotoxicity. To validate these injury mechanisms, we assessed the effect of gentamicin on the CORE, UPR and cell chaperone function, and tested the therapeutic efficacy of enhancing cell chaperone content. Early gentamicin exposure disrupted the CORE, evidenced by a rise in the ATP:ADP ratio, mitochondrial-specific H2O2 accumulation, Drp-1 associated mitochondrial fragmentation, and endoplasmic reticulum-mitochondrial dissociation. CORE disruption preceded measurable increases in whole cell oxidative stress, misfolded protein content, transcriptional UPR activation and its untoward downstream effects: CHOP expression, PARP cleavage and cell death. Geranylgeranylacetone, a therapeutic that increases cell chaperone content, prevented mitochondrial H2O2 accumulation, preserved the CORE, reduced the burden of misfolded proteins and CHOP expression, and significantly improved survival in gentamicin-exposed cells. We identify CORE disruption as an early and remediable cause of gentamicin proteotoxicity that precedes downstream cytosolic UPR activation and cell death. Preserving the CORE is associated with improved renal cell survival likely by reducing organelle-specific proteotoxicity during gentamicin exposure.

Chronic kidney disease (CKD) is associated with high healthcare costs, poor health outcomes and low quality of life (Donald et al., 2018). The US CKD prevalence is 15%, costing Medicare $79 billion in 2016 (United States Renal Data System, 2018). Self-management could reduce CKD burden (Jeddi, Nabovati, and Amirazodi, 2017). Mobile technology offers a low-cost, ease-of-access platform for chronic disease self-management (Whitehead & Seaton, 2016), potentially slowing disease progression and improving health outcomes (Jeddi, 2017). The purpose of this project was to utilize a CKD-specific mobile application, CARELogiQ, to facilitate symptom management; increasing patient satisfaction and decreasing hospitalizations and ER visits over three months. A five-database literature review yielded six high-quality articles; three level I, two level II, and one level III evidence based on JHNEBP (Dang & Dearholt, 2017). Evidence supports a multifaceted approach to effectively use a CKD-specific mobile application for patient and provider communications, appointment and biometric check reminders, education, and telemonitoring of BP and weight. The Stetler Model guided the project (Stetler, 2001). In a Northwest Indiana nephrology clinic, CARELogiQ was used to report symptoms, communicate with patients and providers, and record medical appointments. DaVita’s Kidney Smart© educational class provided education. Participants recorded weekly BP and weights manually in a log. Weekly calls collected BP and weight results, reminded participants to use CARELogiQ to report symptoms, encouraged medication adherence, and continued telemonitoring. Aggregate and person-to-person hospitalization/ER visit comparisons, and a post-intervention satisfaction-of-care survey were completed. Healthcare utilization was less in the intervention group (N = 10, M = 0.00, SD = 0.00) compared to the non-intervention group (N = 32, M = 0.125, SD = 0.42) (t(40) = .930, p = .36) . Rate of healthcare utilization in the intervention group (N = 10) did not change from pre (M = 0.00, SD = 0.00) to post-intervention (M = 0.00, SD = 0.00). Participants were satisfied with use of CARELogiQ. CARELogiQ has the potential to reduce healthcare utilization by improving self-management, thus effectively impacting CKD burden. Significant outcomes may be attainable for future CARELogiQ projects by increasing sample size, lengthening implementation time, increasing functionality of CARELogiQ, and providing a smart device to participants.

Background. Ethanol (EtOH) consumption is known to affect multiple organs; this is unsurprising, as the concentration of EtOH in the blood at relevant doses reaches the millimolar range. The overarching goal of this dissertation was to elucidate mechanisms of alcohol-induced organ injury, specifically the effects of alcohol on the hepatic extracellular matrix (ECM) proteome, the alcoholic hepatitis (AH) plasma peptidome, and the effects of alcohol on the renal cortex proteome and transcriptome. Methods. Mice were pair-fed ethanol-containing liquid diet chronically, and then some mice were administered lipopolysaccharide (LPS). Liver sections from these mice were processed in a series of increasingly rigorous extraction buffers to separate proteins by ‘age’ and crosslinking. Extracted proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the AH study, a workflow was developed for the peptidomic analysis of plasma from healthy participants or AH patients. AH severity was stratified by MELD score as mild (19; n=37). The peptidome in AH and control plasma was analyzed with LC-MS/MS. For the kidney study, renal cortex proteins were extracted in lysis buffer, and RNA was also isolated. Extracted proteins were identified using LC-MS/MS, and RNA sequencing (i.e. transcriptomics) identified transcripts. Results. Chapter III introduced a new proteomic approach for characterizing the hepatic matrisome, which demonstrated that the hepatic matrisome responds dynamically to both acute (LPS) and chronic (ethanol) stresses, long before more dramatic fibrotic changes to the liver. Chapter IV demonstrated that AH causes detectable changes in the plasma ECM degradome/peptidome of patients, and that the LC-MS/MS analysis of the plasma peptidome is a novel, minimally-invasive method for prognosis stratification in patients with AH. Finally, Chapter V revealed that chronic, moderate ethanol consumption affects renal cortical oxidant response pathways at the protein and transcript level. Conclusions. The work presented in this dissertation has, in conclusion, revealed that the hepatic ECM responds dynamically to stress, plasma peptides, including ECM peptides, change with AH severity, and chronic ethanol consumption affects renal cortical oxidant response pathways.

Chronic kidney disease (CKD), defined as a decrease in renal function, is a global issue. The treatment of CKD and its comorbidities imparts a costly burden on the American healthcare system, therefore the need for therapeutics that prevent the progression of chronic kidney disease is urgent. Microarray studies have shown that the serine protease inhibitor clade A member 3 (SERPINA3) is transcriptionally upregulated in kidney injury. SERPINA3 is an extracellular protease inhibitor that maintains the homeostasis of extracellular matrix proteins. Our lab hypothesizes that SERPINA3 might not only be a transcriptional biomarker for kidney injury, but the SERPINA3 protein might act as a key upstream regulator in the advancement of renal inflammation and fibrosis. Our research characterizes the expression patterns of SERPINA3 in models of acute and chronic kidney injury through immunoblotting and immunohistochemistry. Our unilateral ureteral obstruction (UUO) model of chronic renal injury displays significant glomerular localization of SERPINA3. The adenine diet model of chronic kidney injury and the renal ischemic reperfusion injury (RIRI) model of acute kidney injury both display tubular upregulation of SERPINA3. The DOCA-salt hypertension model of chronic kidney injury was imposed on two strains of mice, C57BL/6 and 129/sv, both of which display tubular and glomerular upregulation of SERPINA3. However, the C57BL/6 strain, which is known for its resistance to glomerular sclerosis, displays higher renal localization of SERPINA3 when exposed to DOCA-salt hypertension, than does the 129/sv strain. In conclusion, our data suggests that SERPINA3 protein is upregulated in both acute and chronic kidney injury. The role of SERPINA3 in these models remains unknown, however, our lab theorizes that SERPINA3 protein may be renoprotective in certain instances of kidney injury. Functional assays must be performed to elucidate the role of SERPINA3 in these models of kidney injury. Characterizing the function of SERPINA3 in chronic and acute kidney injury might aid in the development of novel therapeutics to prevent the advancement of CKD.

Optimal anemia management strategies for end-stage kidney disease patients treated with hemodialysis are unknown, with controversies over how best to utilize erythropoiesis-stimulating agents (ESA) and intravenous iron to support hemoglobin levels and minimize adverse events. With large randomized trials rare in nephrology, it is thus crucial that research questions are clearly defined, study designs are appropriately selected, and analytic techniques are properly implemented when using observational data. The three aims of this dissertation attempt to address current controversies in anemia management using innovative statistical methods, leveraging data from the Dialysis Outcomes and Practice Patterns Study (DOPPS), an international prospective cohort study of hemodialysis patients. Aim 1 focused on anemia management during the transition period to hemodialysis. Among patients who initiated hemodialysis with hemoglobin < 10 g/dL, the highest (vs. low) doses of ESA and intravenous iron were each associated with elevated mortality. To assess the impact of pre-dialysis anemia treatment, a seemingly counterintuitive design – restricting to patients who achieved target hemoglobin (>=10 g/dL) four months later – was used to limit inclusion of patients whose low hemoglobin at hemodialysis initiation was likely confounded by poor health status. Even in this subset, anemia at hemodialysis initiation was common and associated with elevated mortality. A more proactive approach to anemia management prior to end-stage kidney disease may thus avoid aggressive correction of hemoglobin levels during the early dialysis period and improve survival. Aim 2 focused on how hemoglobin response to ESA therapy may be blunted by inflammation. Hemoglobin and ESA doses were compared over the 3 months before and after detection of new inflammation, defined as an acute C-reactive protein increase from < =5 to >10 mg/L. Confounding due to baseline characteristics, whether measured (age, sex, comorbidity history) or unmeasured (genetic or environmental factors), was avoided by this longitudinal self-matched design. Patients experiencing new inflammation had both higher ESA doses and lower hemoglobin (vs. pre-inflammation levels), supporting the hypothesis that inflammation increases resistance to ESA treatment. Quicker recognition of new inflammation in hemodialysis patients could help identify the cause of worsening anemia and guide ESA and intravenous iron dosing decisions more proactively. Aim 3 focused on applying the parametric g-formula, an extension of standardization to longitudinal data, to replicate a randomized trial using observational data. DOPPS data were used to compare iron supplementation strategies, with the goal of mimicking the recently published PIVOTAL randomized trial. Comparing the proactive high-dose vs. reactive low-dose strategy, 1-year mortality risk was 20% greater under the parametric g-formula simulation, but similar in the PIVOTAL trial. Simulated differences for all secondary outcomes were directionally consistent but of lesser magnitude than in the PIVOTAL trial. Success in mimicking the PIVOTAL trial was mixed, and potential explanations for the divergent results include model misspecification and/or differences in the study populations. This example illustrates the potential of the parametric g-formula to evaluate many variations of complex interventions across different populations, which could prove enormously informative in the age of big data. This dissertation outlines critical gaps in the literature on anemia management in hemodialysis patients, and describes three studies that utilize innovative designs and complex statistical analyses to address these gaps. These studies attempt to advance both the optimization of anemia management strategies in hemodialysis patients and the use of causal inference principles to guide epidemiologic research using observational data.

Abstract: Chloride intracellular channel (CLIC) proteins comprise six members CLIC1–CLIC6 in mammals and mediate functions not fully known. Their founding member was isolated from bovine kidney using the chloride channel inhibitor indanyloxyacetic acid-94, so CLICs were assumed to be typical anion channels. However, CLIC proteins possess several properties discordant with classical channels, and there are doubts regarding their capacity to form the integral transmembrane (TM) architecture of α- and β-type ion channels. Chief among such properties are their structural dimorphism. CLICs assume multiple conformational folds, and the transitions between them accompany their translocation to their diverse subcellular locales. Though several X-ray structures of their soluble form have been solved, the signaling pathways which mediate their transition in cells have not yet been identified, nor has any membrane-associated structure been solved. In comparison to the diverse expression of most CLICs, CLIC5A is highly enriched in glomerular tissue within podocytes and endothelial cells and is frequently found in actin-rich projections of the plasma membrane (PM) in a highly polarized distribution. At the apical PM, CLIC5A is localized in clusters containing phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2), where it promotes PM–actin cytoskeleton linkages by stimulating the activation of the cross-linking ezrin-radixin-moesin (ERM) proteins, in part by Rac1-induced activation of phosphoinositide kinases. Given that these signaling events are centralized at and near the PM, it is important to discern the cellular processes translocating CLIC5A from the cytosol to this site. The primary focus of this thesis is to investigate the translocation process of CLIC5A using structural and cellular approaches, and to challenge widely-held notions that CLICs form TM channels. NMR spectra of truncated N-terminal CLIC5A mutants containing the putative transmembrane domain were obtained to reveal that CLIC5A adopts multiple conformations in solution. Next, CLIC5A was assayed in transfected COS-7 cells under membrane permeabilizing and intact conditions, which revealed that this protein was detected only in the former condition, strongly suggesting CLIC5A is confined in the intracellular space with no appreciable extracellular domains. Furthermore, subcellular fractions extracted by differential detergent fractionation revealed that overexpressed CLIC1, CLIC4, and CLIC5A were predominantly cytosolic and not detectable in membrane fractions. However, Calyculin A-induced phosphatase inhibition significantly increased the retrieval of CLIC4, CLIC5A, and to a much lesser extent CLIC1, in membrane fractions. For CLIC5A, the phosphorylation-driven translocation to membranes was sensitive to Staurosporine, suggesting a protein kinase C (PKC) as a likely candidate mediating this process. Multifactorial prediction analyses reveal that CLIC5A is likely to be phosphorylated at two C-terminal Thr residues during this process. Overall, this study demonstrates for the first time that CLIC4 and CLIC5A are translocated to membranes in a phosphorylation-driven process which for CLIC5A is mediated by a PKC. Further, our results corroborate earlier claims that CLIC5A does not form an integral TM protein and thus unlikely to function as a classical ion channel.

The mitochondrial permeability transition pore (mPTP) contributes to pathological outcomes in various contexts of tissue injury and disease involving mitochondrial perturbation, including in the kidneys. The prominent stressors leading to formation of the mPTP include increased cellular calcium influx and increased reactive oxygen species (ROS) production or accumulation. Subsequent opening of the mPTP can be a key contributor to poor outcome through several possible mechanisms including a decreased ability of cells to produce ATP and the induction of necrotic and apoptotic cell death. Here, we prevented expression of a critical modulator of the mPTP, namely Cyclophilin D (CypD), in mice that develop chronic kidney disease (CKD) by crossing mice with CypD deficiency to those carrying the Col4a3-/- mutation modeling Alport nephropathy. The mouse model of Alport nephropathy exhibits increased ROS production and tubule epithelium cell death in the kidney, suggesting that mPTP opening may be occurring and driving disease progression. In the present study, we found that human and murine kidneys with CKD exhibited alterations indicative of mitochondrial dysfunction possibly associated with mPTP formation. CypD deletion, however, did not improve survival or renal function in Alport mice nor did it reduce histopathology. Together, these results demonstrate that inhibition of CypD activity as an approach to limit mPTP formation does not appear to be a viable pharmacologic target for the treatment of Alport nephropathy.

In 2015, 224 infants in the United States were diagnosed with end-stage renal disease (ESRD). Infants with ESRD often require renal replacement therapy (RRT), commonly known as dialysis, for treatment. Although they are a minute subset of children, like other children with medical complexity (CMC), infants who require renal replacement therapy (RRT) are medically complex and need high levels of professional and home care. Although treatments are proving more promising, infants with ESRD have historically poor outcomes. Medical decision making for parents of infants with end stage renal disease can be a process fraught with conflict, uncertainty, discord, and stress. Questions of which treatment to pursue or whether or not to forego life-sustaining treatment, weigh heavily. Knowing which decision to make is not a simple task. To aid in decision making, healthcare professionals have promoted Shared Decision Making (SDM) as a model, however, the vast majority of individuals who become patients, still are not supported with the SDM model. The SDM model and others have not been adequately studied from the parent and patient perspective. Parents of infants with ESRD and other CMC, are affected emotionally, financially, physically and relationally by the everyday parenting and care of their child. Decision making is another task that increases the burden to caregivers. This dissertation will provide an explanation of the experience of parents making decisions for infants with ESRD, the meaning made for mothers within that experience, situating both in current research. The dissertation the provides the implications of experience and meaning findings--to social work practice, policy, and education. Article 1 describes the experience of mothers, resulting in four essential themes: (a) information and communication are important, (b) I want to give my baby every chance, (c) it's hard to know for certain what to do, and (d) mothers and fathers take different roles in care giving and decision making. Article 2 provides an explanation of the meaning that mothers of infants with ESRD make of their decision making and caregiving experience: 1) Information has its limits, and 2) Mothers pull the load. Understanding the broader experience and meaning of decision-making for parents, and specifically, mothers of infants with CMC, may increase the effectiveness of decision-making techniques, interventions, and tools developed for parent caregivers in the future. In addition this dissertation and the articles included in it provide a close look at author reflexivity in qualitative study as well as a template for studying the broad experience of parent decision makers that includes beginning with experience, adding meaning in order to reveal a practical purpose and meaningful implications for social workers, social work reserachers and other healthcare professionals.

Introduction: Limited data exist regarding outcomes of patients with outpatient acute kidney injury (AKI). To determine whether outpatient AKI is associated with increased mortality and chronic kidney disease (CKD), we conducted a retrospective cohort study utilizing an electronic health record in Minnesota. Methods: All adult patients receiving primary care through Fairview Health Services were included. All outpatient Cr values during an 18 month exposure period were used to define five comparator groups as follows: No outpatient AKI (reference group), outpatient AKI with recovery, outpatient AKI without recovery, outpatient AKI without repeat Cr, or no Cr. A Cox proportional hazard model was utilized to assess whether outpatient AKI was associated with an increase in mortality, CKD stage 4 and secondary outcomes including hospitalization and recurrent AKI. Results: The cohort consisted of 384,869 patients and 51% had at least one Cr measured during the exposure period. Outpatient AKI occurred in 1.4% of patients during the 18 month exposure period and 37.8% recovered while 26.5% had no repeat Cr. Mortality was 3.2% over an average follow-up of 5.3 years. Outpatient AKI was associated with an increased risk of mortality (aHR 1.90, 95% CI 1.76-2.06) and CKD stage 4 (aHR 1.33, 95% CI 1.11-1.59) including those that recovered from their AKI (mortality aHR 2.15, 95% CI 1.91-2.41; CKD aHR 1.73, 95% CI 1.37-2.19) and in those with stage 1 AKI (mortality aHR 1.90, 95% CI 1.74-2.07; CKD aHR 1.34, 95% CI 1.10-1.62). Outpatient AKI was also associated with with an increased risk of hospitalization (aHR 1.71, 95% CI 1.63-1.79), hospital AKI (aHR 2.14, 95% CI 1.93-2.37), and recurrent outpatient AKI (aHR 2.75, 95% CI 2.57-2.93). Conclusion: Outpatient AKI is common and is a risk factor for death, CKD, hospitalization, and recurrent AKI, including those with stage 1 AKI and those that recover.

Background and objectives: Studies have demonstrated higher parathyroid hormone (PTH) and lower 25-hydroxy Vitamin D in African-American children. There is minimal information on these differences in pediatric patients on dialysis.Design, setting, participants and measurements: Differences in the markers of mineral and bone metabolism (MBM) were assessed in 661 pediatric patients. Differences by race and ethnicity over the first year of dialysis treatment were assessed using linear mixed models.Results: African-American race predicted 23% higher serum PTH when compared to Caucasian patients and Hispanic ethnicity trended toward 14.8% higher PTH. Upon gender stratification, the differences in PTH were magnified in African-American and Hispanic females who had 38% and 28.8% higher PTH compared to Caucasian females, respectively.Conclusions: The markers of mineral and bone metabolism differ by race and ethnicity within the pediatric dialysis population. Therefore, consideration of these differences may impact the goal-targeted treatment of MBM disorders.

Cisplatin is a nephrotoxic chemotherapeutic that causes acute kidney injury (AKI) in 30% of patients. Although recovery can occur after one episode of cisplatin-induced AKI, studies have indicated multiple episodes may lead to the development of chronic kidney disease (CKD), an irreversible disease with no current treatments. The standard mouse model of cisplatin-induced AKI consists of one, high dose of cisplatin (> 20 mg/kg) that is lethal to the animal three days later. This model doesn’t accurately reflect the repeated dosing regimen patients receive, and doesn’t allow for long-term outcome studies of pathologies associated with CKD. We have developed a repeated dosing model of cisplatin (7mg/kg once a week for four weeks). This model allows for the long-term survival of mice, and the associated pathology is fibrosis-the hallmark of CKD. Thus, data indicate that the repeated dosing model can be used to study AKI to CKD progression.

Cardiac magnetic resonance (CMR) is a powerful tool to noninvasively image ventricular fibrosis. Late gadolinium enhancement (LGE) CMR identifies focal and, with T1 mapping, diffuse fibrosis. Despite prevalent cardiac fibrosis and heart failure, patients with end stage renal disease (ESRD) are excluded from LGE. Absence of a suitable diagnostic has limited the understanding of heart failure and obstructed development of therapies in the setting of ESRD. A quantitative, gadolinium free fibrosis detection method could overcome this critical barrier, propelling the advancement of diagnostic, monitoring, and therapy options. This project describes the development of a gadolinium free CMR technique and application for cardiac fibrosis measurement in patients with ESRD. Magnetization transfer (MT) occurs during standard cine balanced steady state free precession (bSSFP) CMR, where extracellular matrix protons exchange magnetization with water molecules. Extracellular water volume expansion, concomitant with fibrosis, reduces MT and subtly elevates signal intensity. Our technique, 2-pt bSSFP, extracts endogenous contrast sensitive to tissue fibrosis by obtaining pairs of high and low MT-weighted images and calculating normalized signal differences, denoted by ΔS/So. We tested 2-pt bSSFP in patients referred for CMR and found excellent agreement spatially with LGE and quantitatively with extracellular volume fraction. Diagnostic and clinical application of 2-pt bSSFP was comparable to LGE. We applied 2-pt bSSFP to patients with ESRD for multiscale comparison with correlates of fibrosis ranging from blood biomarkers to whole organ function. Patients with ESRD displayed hypertrophy with reduced contraction, but elevated ΔS/So and fibrosis. Some biomarkers correlated with both hypertrophy and fibrosis, highlighting the need to distinguish between hypertrophic and fibrotic remodeling. We monitored fibrosis over 1 year using 2-pt bSSFP in a cohort of patients with ESRD. ΔS/So and fibrotic burden increased substantially, despite minor changes in structure and function. Collectively these studies validate and apply 2-pt bSSFP for gadolinium free fibrosis CMR in patients with ESRD. While ventricular structure and function are commensurate with progression toward heart failure, it is now possible to specifically describe global and focal patterns of cardiac fibrosis in ESRD, along with comparisons to blood biomarkers which may lead to improved diagnostics and molecular treatment targets.

Mortality is high among incident hemodialysis patients especially during the first several months after initiation of dialysis and can be influenced by many factors. Age, vascular access, co-morbid conditions, serum levels of albumin, and dialysis modality may affect survival. We attempted to identify factors that influence survival of incident hemodialysis patients and to determine if changes in clinical practices could improve survival. We hypothesized that patients surviving at least 24 months differ in their characteristics from those who died and so indentifying those differences and making changes may improve survival in all hemodilaysis patients. Standardized mortality ratios (SMR) were calculated for each of the first 24 months on dialysis to determine the temporal pattern of mortality. Cox proportional hazard models were fitted to examine mortality at several a priory determined time periods for each predictor using case-mix adjustments. The associations between all-cause mortality and combined serum levels of albumin and nPCR were studied for each of first 8 quarters on dialysis. Changes in serum albumin and nPCR levels from the prior quarter were examined separately. Finally, marginal structural models (MSM) were fitted to analyze the association between mortality and dialysis modality during 24 months of treatment. Inverse probability weights were created to account for time varying confounding of quarterly measured laboratory values and changes in dialysis modality over the first 8 quarters. SMRs were the highest during the 3-6 months after starting dialysis. Use of central venous catheters and low levels of serum albumin at the time of dialysis initiation were associated with all-cause and infection-related mortality during the first 24 months of treatment. Volume overload was associated with cardio-vascular mortality especially in non-hypoalbuminemic patients. Decrease in albumin and nPCR was associated with mortality during 24 months on dialysis. Patients on peritoneal dialysis showed better survival compared to hemodialysis patients after adjusting for modality changes by using MSMs. Patients who changed modalities at least once showed a survival advantage over those who remained on initial modality during 24 months of dialysis treatment.

The optimal manner in which to train nephrologists has not been studied. The objectives of this research were to determine:- 1. The educational and historical basis underpinning the Australian nephrology training program. 2. The drivers surrounding a career choice in nephrology. 3. What constitutes an ‘ideal’ nephrologist and how nephrologists spend their work time, thereby identifying skills and attributes to be fostered in training. 4. Impediments to training, including examination of the available workforce. Five sub studies were undertaken, utilising a combined quantitative and qualitative approach (mixed methods): 1) A national Basic Physician Trainee (BPT) questionnaire, 2) a national nephrology workforce study, and in-depth interviews of: 3) nephrology patients, 4) nephrology trainees and 5) practicing nephrologists. New findings arising from this research reveal: doctors choose nephrology as a career if exposed to the specialty in a positive manner with good role models, however, there are a range of modifiable factors that make nephrology unattractive to many BPTs; workload is high, impacting negatively on training and trainee recruitment; Nephrologists spend most time in the management of dialysis and transplant patients but have a range of other roles in day to day practice, essential information to develop a competency based training program; availability of nephrologists for training is suboptimal and will likely worsen; Patients and doctors apply and weight parameters differently when defining an ‘ideal nephrologist’. Both groups believed that specialist knowledge remains an essential requirement but patients focused more on good communication skills. This research provides evidence that the training program should incorporate training in advanced communication and basic research skills and promotion of an holistic approach to patient care. There is no formal alignment of training with assessment. Trainees and nephrologists believe that feedback is critical to learning, yet the assessment process is not underpinned by sound educational principles. This can be rectified using the findings of this research in conjunction with curriculum development and performance assessment. This research should provide an approach to the examination of training that is applicable to many internal medicine specialties. Importantly, nephrology training can now be improved with sound educational principles, underpinned by the findings of this research.