Identification of Specific Vacuolar-ATPases by Gene Expression Profiling in renal cells and testis of Nephropathic Cystinosis
Presented by Dr Swastika Sur, Department of Surgery, University of California San Francisco, SF, CA, USA
Authors: Swastika Sur*, James Smith** and Minnie Sarwal*
Lay Summary: The primary cause of renal Fanconi and male infertility in cystinosis patients are poorly understood. There is now a growing population of cystinosis patients, who are treated with Cysteamine yet characterized by progressive end-stage renal disease (ESRD) and reaching young adulthood, without the ability to have children. Understanding the underlying cellular and molecular processes that result in renal Fanconi and male infertility in cystinosis is thus a critical unmet need. Our current study aims to understand the triggers driving both these pathologies in Cystinosis patients.
Objectives: The kidney is the first organ to get affected, and the most severe form of infantile nephropathic cystinosis is characterized by the renal Fanconi syndrome and progressive ESRD despite cysteine depletion. In addition, infertility is also reported as a frequent finding in these male cystinosis patients. Although spermatogenesis has shown to be intact at the testicular level in some patients, no male cystinosis patient is known to have naturally fathered a child. To this date, the etiology of renal Fanconi and azoospermia in cystinosis patients remains unclear, consequently, finding novel therapeutic targets for these patients is challenging. We are focused to study, in-depth, the dysregulation of autophagy in the generation of renal injury and male infertility in nephropathic cystinosis.
Methods: We have conducted genome wide transcriptional profiling of both kidney and testicular tissue types in patients with infantile nephropathic cystinosis to assess if there are unique or overlapping perturbations of specific molecular pathways that may provide a link between kidney and male infertility in cystinosis. For this purpose, we have studied mRNA changes in renal proximal tubular cells isolated from urine samples from normal controls (n=6) and patients with a confirmed diagnosis of nephropathic cystinosis (n=6). In addition, we obtained testicular biopsy samples from normal controls (n=10) and infertile males (n=10), with and without nephropathic cystinosis. We identified significant gene expression specific to a diagnosis of cystinosis and their dysregulated pathways involved in mitochondrial integrity and cellular acidification. We performed immunoblotting and confocal microscopy to confirm the expression of specific proteins, and assayed changes in tissue specific mitochondrial function. To confirm specific gene expression changes in cystinosis, specific primers were utilized to amplify gene expression by Fluidigm multiplex microfluidic qPCR.
Results: We have identified common kidney-specific and male reproductive organ-specific targets and their related pathways, many of which are downregulated in cystinosis, and play a crucial role for maintaining normal renal function and male fertility. The interaction and co-localization of some of these genes of interest with CTNS appear to be essential to maintain normal cellular function.
Conclusions: The common dysregulation of the specific genes, exclusive to the cystinotic kidney and testis, provide the first common link of molecular perturbations that may reflect in the observed functional decline of both the kidney and the male reproductive system in cystinosis. This has the potential to pinpoint a target to screen for therapeutic agents that may provide new treatments for renal Fanconi and male-infertility in cystinosis.
* Department of Surgery, University of California San Francisco, SF, CA, USA
** Department of Urology, University of California San Francisco, SF, CA, USA