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Green juices are an example of a modern food innovation that has developed partly by itself in response to general nutritional advice in the media imploring people to consume more fruits and vegetables. Unfortunately, the risks of developing acute oxalate nephropathy is high following the consumption of some green juices and fruit juices. This study investigates the occurrence and levels of oxalates in green juices made from common fruit and vegetables available in New Zealand. The influence of the type of juicer used to prepare the juices, and the use of treatments and/or additions to reduce the oxalate content of these mixed juices, was also investigated. A Design of Experiments multi-factor approach was used to investigate the extraction of oxalates from a green juice. Fresh spinach samples were juiced in a high speed blender and the oxalate contents were extracted using seven levels of pH, eight levels temperature and six extraction times. A statistically significant quadratic model with an overall level of significance of P < 0.0002 was achieved by using 20 experimental runs performed over one day. The optimum conditions for extracting all the oxalate from the model green spinach juice was at pH 0.93, with a temperature of 65°C and at any time greater than 15 minutes. The least amount of oxalic acid was extracted at pH 4.59 with a temperature of 25°C. To investigate the levels of oxalates found in homemade green juices, five green juice recipes containing between three and nine ingredients were made. Fresh spinach was the common component, and ranged from 20.1% to 37.9% w/w. The oxalate content ranged from 90.34 to 152 and 13.0 to 82.85 mg/100 g fresh weight (FW) for total and soluble oxalates, respectively. The percentage of soluble oxalate varied from 11.9 to 67.8%. The juices were made using a masticating juicer (MJ), which separates out a waste pulp fraction that is normally discarded. This fraction contained large amounts of oxalic acid, 134.6 to 348.09 mg/100 g FW in total. The waste pulp also had a higher concentration of minerals of all 11 minerals present, apart from Al, compared to the green juice. It is proposed there is an interaction between the oxalate, calcium and the pulp fraction of the green juice. A 200 g glass of green juice containing a mix of apple, celery and spinach, contained 165.7 mg of soluble oxalate; this is a considerable amount of soluble oxalate to ingest. In the home, juicing commonly uses two basic types of juicer, a high speed blender (HSB) or a masticating juicer (MJ); the MJ separates out a pulp fraction while the HSB does not. High and low spinach containing juices were made using both juicers. The MJ had a concentrating effect during processing, resulting in 528.41 mg total oxalate/100 g FW compared to the HSB, at 369.47mg total oxalic acid/100 g FW. They both produced green juices that had similar ratios of soluble oxalate, for both the high and low spinach juices. A significant amount of the oxalate and minerals were separated out into the waste pulp fraction from the juice made using the MJ. The waste pulp contained 238.10 to 55.67 mg total oxalate/100 g FW and 61.7 to 74.7% of the calcium content of the original spinach leaves. Two strategies to lower the soluble oxalate content of green juices were investigated; direct addtion of a food grade calcium salt, and soaking the raw materials used to make the fresh green juice. Four calcium salts tested. Calcium chloride was the most effective, reducing the soluble oxalate by 98.3% after the addtion of 500 mg/100 g to the fresh juice. Fresh spinach leaves were soaked in either tap water, a 1% w/v/ NaCl or a 1%w/v CaCl solution, prior to processing into a juice. The tap water and 1% w/v NaCl showed no loss of soluble oxalate but the 1% w/v CaCl treatment showed a 50% reduction in soluble oxalate and a 28.% increase in insoluble oxalate. This treatment had the added benefit of increasing the bioavailabilty of calium in the soaked spinach leaves. This study has shown that there are high levels of oxalate in green juices and, if consumed, this could pose a risk for diet induced nephropathy.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder affecting approximately 2.5 million people worldwide. It is the number one, life-threatening monogenic disorder known, caused by mutations in either the Pkd1 or Pkd2 genes resulting in numerous fluid-filled cysts within the kidneys. ADPKD presents in the 2nd decade of life and presents with progressive renal decline and eventually to end stage renal disease (ESRD) in the 5th to 6th decade of life for half of all patients. Currently no treatments exist to ameliorate or cure ADPKD, creating a large financial burden on both the health care system, the patient, and their care-givers. Treatment options once patients reach ESRD are limited to dialysis and kidney transplant. Since the discovery of the genetic link to ADPKD, there has been intense research to understand what role the Pkd1/2 genes play in the formation of cysts and the progression of ADPKD. The slow progression of ADPKD and the difference in disease severity between individuals implies that there may be environmental factors acting as causative agents in promoting cyst formation. Animal models of ADPKD appear to be more susceptible to cyst formation following injury with nephrotoxic chemicals, ischemia, and partial or total unilateral nephrectomy. These data demonstrate that the injured state is a driver of cyst formation once Pkd1/2 have been mutated, but these experimental approaches are not representative of normal environmental injuries to the kidney. The focus of my research is to elucidate an environmental agent that can promote cystogenesis in an otherwise healthy individual. The kidney is responsible for filtering solutes from the blood which include ions and molecules that under the right circumstances can form precipitates in the urinary filtrate. It is our hypothesis that the endogenous kidney injury in ADPKD patients results via the precipitation of microcrystals and the inappropriate response to crystal clearance. The kidney is producing sub-clinical microcrystals daily that are excreted into the urine without incident. Individuals with ADPKD may be unable to respond appropriately in the clearance of these small crystals. Previously, our lab has shown that ADPKD cyst lining epithelial cells exhibit increased levels of mTOR and STAT3 activity. This same activation of mTOR and STAT3 can be seen following calcium oxalate crystal deposition in both wild-type mice and rats. In addition to the increase in STAT3 and mTOR activity, there is a concomitant increase in tubule dilation. This dilation persists until crystals are no longer detected in kidneys along with a cessation of mTOR and STAT3 activity. Additionally, we demonstrate that the Han:SPRD and pck rat models of PKD exhibit an exacerbated renal phenotype following an increase in kidney microcrystals. Cyst number and cystic index increase following crystal deposition demonstrating that the mechanism of cystogenesis is crystal dependent. The discovery that crystals act as a trigger for cyst formation is an important step in elucidating the causal mechanism of cystogenesis in ADPKD and a critical step in creating effective preventative and palliative strategies.

This dissertation describes the use of infrared and Raman microspectroscopic methods for the analysis of small mineral inclusions in kidney biopsies and efforts to quantitatively analyze components at the localized mineral/tissue interface. Chapter 1 provides a background on the current state of kidney stone disease research and also describes the instrumental methods utilized. Chapter 2 presents the use of attenuated total internal reflection (ATR) infrared spectroscopy to generate calibration curves for mixtures of powdered kidney stone components. This study demonstrates that reproducible quantitation can be achieved if the particle sizes of the components are small and comparable. ATR imaging was employed to analyze kidney biopsies with small mineral inclusions in Chapter 3. ATR imaging has several benefits over other infrared sampling methods (primarily transflection and transmission) for the analysis of kidney biopsies due to the smaller focused beam size achievable and the reduced optical pathlength that eliminates spectral artifacts. Chapter 4 describes an investigation into small particle analysis with transmission infrared microspectroscopy and ATR imaging. The results of the study show that spectral artifacts are dependent on the particle’s size and shape and it is anticipated that this research will provide a framework for the analysis of particles below the diffraction limit. Chapter 5 presents a comparative study of Raman microspectroscopy and ATR imaging for kidney biopsy analysis. Using both methods in unison allows the investigator to obtain a full spectroscopic picture of the sample. Because Raman interrogates a larger, more uncontrolled sample volume, ATR may be more beneficial for quantitative studies. Chapter 6 summarizes the presented research and discusses future work.

Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as global endocrine and physiological effectors. In recent years the zebrafish has been utilized as a favorable animal model as a step towards dissecting and better understanding these postembryonic developmental processes. One of the more powerful methods utilized in zebrafish has been the identification of new gene functions through the use of mutant screens. The nutria mutant was recovered from one such screen to identify postembryonic defects in pigment pattern, growth and metamorphosis. These mutants exhibited a pigment cell defect, touch unresponsiveness and severe growth retardation. Here I will discuss my work towards dissecting the underlying developmental processes governing the phenotypic changes in nutria mutants. I characterize gross alterations in skeletal development in nutria mutants that lead to accelerated endochondral ossification but delayed intramembranous ossification. I show that the nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and a kinase. I find trpm7 expression in the fish-specific, ion homeostasis-regulating gland known as the corpuscles of Stannius (CS), and in the mesonephric kidney. I show that mutants also develop kidney stones. Together these results suggest a role for trpm7 activity in regulation of physiological ion homeostasis. Next I confirm that role by identifying late-embryonic and early larval defects in the CS and the kidney, two organs that regulate physiological ion homeostasis. I demonstrate the early larval detection of kidney stones in trpm7 mutants and show that their appearance is presaged by decreased levels of total calcium and magnesium. Furthermore I establish a link between trpm7 function in the CS and stanniocalcin1 (stc1), a potent molecular regulator of calcium homeostasis. Finally, using transgenic overexpression and morpholino-oligonucleotide knockdown, I demonstrate that stc1 modulates calcium and magnesium levels in trpm7 mutant and wild-type backgrounds. Together these analyses establish postembryonic roles for trpm7 function in growth, skeletogenesis, kidney function, and physiological ion homeostasis.