- Title
- Understanding the pathogenesis of cystinuria
- Creator
- Bhatt, Nirmal Prasad
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2024
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Cystinuria is an autosomal recessive disorder characterised by excessive urinary excretion of cystine, resulting in recurrent cystine stones. Cystine stones typically present in children and young adults. Similar to other types of kidney stones, cystine crystals accumulate in the urine, can cause urinary tract obstruction and are a significant risk factor for the development of chronic kidney disease. Current treatments focus on reducing the cystine concentration in the urine and removing the stones, often with multiple invasive procedures. However, there is a lack of understanding of the mechanism of cystine stone formation and its consequences in the urinary system. Hence, there is a clear need for novel approaches to prevent stone recurrence and its deleterious consequences. Understanding the fundamental mechanisms underpinning the cystine stone formation and pathophysiology of urinary tract injury is critical for developing novel strategies for diagnosis and treatment. The body of the thesis consists of a systematic review of pharmacological interventional strategies of cystinuria and an experimental Slc7a9G105R mouse model of type B cystinuria. In a systematic review, it was found that the current pharmacological intervention approaches are insufficient to treat cystinuria patients and reduce cystine stone growth. In the experimental study, a novel mouse model was established and various interventions tested. The systematic review (Chapter 2) found that diuresis and alkalinisation methods were insufficient to prevent cystine stone formation. However, they effectively decrease cystine stone formation by increasing the urinary output and pH of the urine. Further, pharmacological thiol drugs were found to treat cystinuria patients before and after surgical interventions. However, these thiol interventions were limited to slowing down the cystine stone growth by increasing cystine solubility by chelating the cysteine, avoiding their dimerization. However, this study lacked sufficient biochemical parameters that could assist in monitoring the condition and provide proper treatments to reduce cystine stone formation. Identifying these limitations of pharmacological approaches to treat cystine stones suggests an urgent need to identify novel treatment avenues to prevent cystine stone formation in humans. In this thesis's experimental mouse chapter, the Slc7a9G105R mouse model of type B cystinuria was initially characterised for the optimal time point of cystine stone formation and its consequences. Results revealed that male and female homozygote Slc7a9G105R mice displayed urinary bladder cystine stones at 9 weeks; however, heterozygote and wild-type controls did not get cystine stones at the same age. Interestingly, female homozygote Slc7a9G105R mice showed bigger and fewer cystine stones than males. Furthermore, the histopathological assessment showed renal tubular damage and bladder epithelium remodelling in homozygote Slc7a9G105R mouse kidney and bladder, respectively. Moreover, mRNA expression analysis revealed that the kidneys showed up-regulated cytokines and chemokines in homozygote Slc7a9G105R mice compared to heterozygotes. mRNA expression could be associated with promoting immune cell and inflammatory responses induced by cystine stones. Next, spironolactone, an aldosterone antagonist, intervened in homozygote Slc7a9G105R mice. Previous studies have shown that males have more cystine stones than females, as sex dimorphism may cause this difference due to different hormones present in males and females. Several studies demonstrated that spironolactone effectively protects against vascular calcification, alleviates myocardial fibrosis, and protects against diabetic cardiomyopathy. However, little is known about spironolactone's effects on cystine stones or other types of stones. Results demonstrated that cystine stones were unchanged, although serum testosterone levels were reduced by spironolactone treatment. Similarly, no change in cystine stone-induced kidney and bladder damage was identified with histopathological assessment. Further experiments, such as mRNA expression analysis and immunohistochemistry, could be required to understand the effect of spironolactone on cystine stone formation in homozygote Slc7a9G105R mice. The microbiome is known to impact other kidney stones, but its role in cystinuria is unknown. As cystinuria is a metabolic disorder, it may be interesting to see the role of gut microbiota on cystine stone formation in homozygote Slc7a9G105R mice. A microbiome study achieved through two experimental protocols was used to investigate the effect of gut microbiota on cystine stone development by co-housing homozygote Slc7a9G105R mice with wild-type mice and cocktails of antibiotic treatment to administered to homozygote Slc7a9G105R mice. Results revealed that bacterial load was reduced by antibiotic treatment suggesting antibiotic treatment may be sufficient to reduce the gastrointestinal microbiota. However, both cohoused and antibiotic-treated mice had no change in cystine stones. Further, no change in the kidney and bladder histopathology with either co-housing or antibiotics treatment was observed. While gene expression analysis was only performed in the antibiotic study, it was found that cytokines and chemokines were down-regulated by antibiotic treatment compared to controls. These studies still require additional experiments to identify the microbiome's role in cystine stone formation, by profiling the microbiota, analysing urine metabolites and immune cell analysis. Finally, the effect of alpha-lipoic acid (ALA) was observed in homozygote Slc7a9G105R mice for changes in cystine stone and tissue injury. Previously, ALA supplementation reduced cystine stone growth in Slc3a1 knockout mice of type A cystinuria. Results showed that ALA supplementation significantly reduced cystine stones and their consequences in the urinary tract. Further, histopathology of the kidney and bladder, displaying tissue injury, was reduced with the ALA supplementation compared to the control diet. Finally, comprehensive mRNA expression analysis was performed in kidneys to determine whether ALA impacts gene expression induced by cystine stone formation. Interestingly, cytokines and chemokines were down-regulated by the ALA supplement. Moreover, Chemokine (C-C motif) ligand 2 (Ccl2), Chemokine (C-C motif) ligand 5 (Ccl5), (C-X-C motif) ligand-1(Cxcl1), chemokine (C-X-C motif) ligand-5 (Cxcl5), interferon-gamma (Ifn-γ), interleukin-6 (Il6), interleukin-7 (Il7), interleukin-9 (Il9), toll-like receptor-2 (Tlr2), toll-like receptor-3 (Tlr3), and toll-like receptor-6 (Tlr6) cytokines and chemokines were either up- or down-regulated by both antibiotic treatment and ALA supplement in homozygote Slc7a9G105R mice. Our findings prove that developing personalised translational models of monogenic kidney stone diseases is feasible. Spironolactone and microbiota do not impact cystine stone formation in Slc7a9G105R mice; however, it is too early to conclude their effect on cystine stones, and further studies could be required to test with immunohistochemistry, qPCR, flowcytometry and RNA-sequencing. Nevertheless, ALA is effective in primary or secondary prevention of type B cystinuria. The crucial next step is to take ALA into broader clinical trials, including in paediatrics.
- Subject
- cystinuria; cystine stones; nephrolithiasis; kidney stones; intervention
- Identifier
- http://hdl.handle.net/1959.13/1510902
- Identifier
- uon:56456
- Rights
- Copyright 2024 Nirmal Prasad Bhatt, This thesis is under embargo and will be made available 23.01.2025.
- Language
- eng
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