Date of Award

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Regenerative Medicine and Cell Biology

College

College of Graduate Studies

First Advisor

W. Scott Argraves

Second Advisor

Kelley M. Argraves

Third Advisor

Phillip Darwin Bell

Fourth Advisor

Lina M. Obeid

Fifth Advisor

Bryan P. Toole

Abstract

Cubilin is an endocytic receptor capable of mediating the endocytosis of multiple ligands including High Density Lipoprotein apolipoprotein A-I (apoA-1) and albumin. It is highly expressed on the epithelium of intestinal villi and renal proximal tubules. In the kidney, endocytosis mediated by cubilin and its co-receptor megalin is the principal mechanism for proximal tubule reabsorption of filtered proteins, such as albumin and apoA-1. Yet, the consequences of cubilin deficiency on the blood levels of albumin and apoA-1 have not been studied. Furthermore, cubilin is downregulated in chronic diseases such as diabetes. However, little is known about the regulation of cubilin expression. In this study, we show that cubilin expression is under epigenetic regulation by at least two processes. The first process involves inactivation of expression of one of the cubilin alleles. This monoallelic expression state could not be transformed to biallelic by inhibiting DNA methylation or histone deacetylation. The second process involves transcriptional regulation of cubilin by peroxisome proliferator-activated receptor (PPAR) transcription factors that are themselves regulated by DNA methylation and histone deacetylation. This is supported by findings that inhibitors of DNA methylation and histone deacetylation, 5Aza and TSA, increase cubilin mRNA and protein in renal and intestinal cell lines. Not only was the expression of PPARα and γ inducible by 5Aza and TSA, but also the positive effects of TSA and 5Aza on cubilin expression were dependent on both increased PPAR transcription and activation. Additionally, 5Aza and TSA had similar effects on the expression of the cubilin co-receptor, megalin. Together, these findings reveal that cubilin and megalin expression is under epigenetic control and thus point to new avenues for overcoming pathological suppression of these genes through targeting of epigenetic regulatory processes. We also demonstrate that cubilin is necessary for maintaining the blood levels of albumin and HDL. In a heterozygous mouse model of targeted cubilin gene deletion (cubilin HT), cubilin haploinsufficiency led to a significant decrease in blood levels of albumin and apoA-I as well as HDL and HDL-cholesterol. Cubilin HT mice displayed reduced renal proximal tubule uptake of albumin and apoA-I, and significantly increased urinary loss of albumin and apoA-1. Urinary levels of apoA-I inversely correlated with plasma apoA-1 levels. Furthermore, the fractional clearance of smaller HDL particles (d>l.13 g/mL) from the blood was significantly increased in cubilin HT mice, indicating a decreased half-life for HDL particles that are filtered through the glomerulus. In contrast, there was no significant change in the fractional lclearan9e of larger HDL2 particles, which are not filtered through the glomerulus. Furthermore, no significant change was observed in the levels of albumin and apoA-I protein or mRNA extracted from liver, kidney or intestine of cubilin HT mice as compared to wildtype. Therefore, cubilin deficiency leads to reduced albumin and apoA-I renal proximal tubule salvage back to blood, which results in reduced albumin and apoA-I/HDL blood levels. These findings are consistent with previous studies demonstrating renal proximal tubule transcytosis of albumin and the necessity of this process in maintaining blood albumin levels. In addition, clinical data indicate that proteinuria in patients with intact glomerular function leads to reduced blood levels of HDL. Therefore, our study is relevant to pathological proteinuria where patients might benefit from increased cubilin expression to reduce proteinuria and raise albumin and HDL levels.

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