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published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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Chronic kidney disease (CKD) is a major cause of morbidity and mortality, affecting more than 10% of the world’s population. CKD is associated with excessive renal fibrosis, which leads to declining kidney function and eventual kidney failure. In CKD, glomerular mesangial cells (MC), resident fibroblasts and tubular epithelial cells undergo phenotypic activation and transition in response to profibrotic and proinflammatory cytokines such as transforming growth factor β1 (TGFβ1). These activated renal cells excessively produce extracellular matrix (ECM) proteins that replace functional renal tissue and lead to renal fibrosis. Caveolae are small omega-shaped invaginations of the plasma membrane that mediate signaling transduction events. Formation of caveolae require the protein caveolin-1 (cav-1). We have previously shown that the ability of MC to produce matrix proteins is dependent on cav-1 expression. Unfortunately, clinically targeting cav-1 within the kidneys, specifically within MC, is technically challenging and as of yet unfeasible. Thus, to better understand how cav-1 deletion is protective, we carried out a microarray screen comparing cav-1 wild-type (WT) and knockout (KO) MC. Here, we discovered significant up-regulation of a TGFβ superfamily inhibitory protein, follistatin (FST). FST specifically targets and neutralizes activin A (ActA) but not TGFβ1. TGFβ1 and ActA both belong to the TGFβ superfamily of cytokines and growth factors. While TGFβ1 itself is a known key mediator of renal fibrosis, therapies aimed at directly inhibiting TGFβ1 in kidney diseases have not been successful due to opposing profibrotic and anti-inflammatory effects. ActA has been shown to act as a strong profibrotic and proinflammatory agent in various organs, including the lungs and liver. We along with others have observed elevated levels of ActA within the kidneys and serum of mice and humans with CKD. Functionally, ActA has been shown to contribute to ECM production in the kidneys. Hence, we hypothesized that ActA inhibition through FST could prove beneficial in CKD. In this thesis, our first study elucidated a novel molecular pathway by which cav-1 regulates expression of the FST in MC. Our results indicate that FST is negatively regulated by cav-1 through a PI3K/PKC zeta/Sp1 transcriptional pathway. Our second study expands on these findings and tests whether exogenous FST administration protects against the progression of CKD in a surgical mouse model of CKD. Here, we discovered that FST acts as a reactive oxygen species (ROS) scavenger and that exogenous administration of FST protects against the development of CKD through the inhibition of renal fibrosis and oxidative stress. Lastly, our third study determined whether microRNAs (miRNAs) are implicated in post-transcriptionally regulating FST through cav-1 and whether these FST-targeting miRNAs can be utilized therapeutically to protect against the development and progression of CKD. Here, we determined that a FST-targeting miRNA, microRNA299a-5p, is significantly downregulated in cav-1 deficient MC, upregulated in vivo in a mouse model of CKD and that its inhibition, in vitro and in vivo protects against the accumulation of ECM proteins and renal fibrosis. These studies collectively suggest that FST is an effective therapeutic option for the management of CKD. / Thesis / Doctor of Philosophy (PhD) / Chronic kidney disease results from excessive fibrosis (scarring) within the kidneys. The goal of this thesis is to understand the molecular mechanisms involving the regulation of an antifibrotic protein, follistatin, in glomerular mesangial cells and to identify its therapeutic potential in chronic kidney disease. This thesis has identified that follistatin, an endogenous inhibitor of the profibrotic cytokine activin A, is regulated transcriptionally by Sp1 and post-transcriptionally by microRNA299a-5p. Furthermore, this thesis has demonstrated that exogenous recombinant follistatin administration protects against the progression of chronic kidney disease and that microRNA299a-5p targeting may be an alternative approach to block renal fibrosis. These studies collectively show that follistatin is an effective treatment for the management of chronic kidney disease.

Wang Yajun. / "April 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 212-248). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 103-116).

To investigate the physiological action of follistatin in the swine ovary a recombinant porcine follistatin (rpFS) with apparent molecular weights of 39, 46, 48, and 50 kDa was expressed and characterized. The rpFS crossreacted with antibodies against native porcine follistatin and with activin A. One rpFS isotype was purified by monoclonal antibody affinity chromatography (rp-305 a.a. FS). In porcine granulosa cell cultures, rp-305 a.a. FS suppressed estradiol-17<math> <f> <g>b</g></f> </math> accumulation (ED50 = 0.9 <math> <f> <g>m</g></f> </math>g/ml) independent of activin. Gilts were vaccinated against rpFS to determine the effect of follistatin immunoneutralization on litter size or ovulation rate. In Experiment 1, forty-seven gilts were vaccinated four times with rpFS (FS n = 23) or with a control vaccine (CTL n = 24). The naturally matured, cycling gilts were bred and piglet numbers were recorded at farrowing. FS vaccination did not increase total litter size. However, grouping by low (<math> <f> <=</f> </math>1:400 n = 16) or high anti-follistatin antibody titer (>1:400 n = 7) responses showed an increased litter size in gilts with a high titer (total piglets: FS high titer = 13.0 ± 0.8; FS low titer = 10.8 ± 0.6; CTL 11.4 ± 0.5; p = 0.08). In Experiment 2, sixty-nine gilts were vaccinated three times with rpFS (n = 35) or CTL (n = 34) vaccines. The gilts were induced and synchronized into estrus using PG600, <math> <f> <rm>PGF<inf>2<g>a</g></inf></rm></f> </math> and boar exposure; cycling gilts received a fourth vaccination. Reproductive tracts were collected two weeks after the second observed estrus (FS n = 14, CTL n = 15). FS vaccination did not increase the number of corpora lutea (FS = 13.2 ± 0.5, CTL = 14.5 ± 0.7) or corpora albicantia (FS = 12.1 ± 1.9, CTL = 12.3 ± 2.0), but appeared to effect normal ovarian morphology increasing the number of blood-filled follicles and corpora lutea. In conclusion, rp-305 a.a. FS appears to negatively affect estradiol accumulation 'in vitro'. Vaccination against follistatin in naturally cycling gilts enhanced litter size. Follistatin vaccination of pharmacologically induced gilts did not increase ovulation rate, but biological effects were apparent suggesting that pharmacological induction of estrus may have confounded the follistatin vaccination treatment.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains ix, 109 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 88-104).

The post-natal regenerative capacity of skeletal muscle is attributed to myogenic satellite cells, which function as lineage-committed precursors to replace terminally differentiated muscle. The development and differentiation of the satellite cell lineage is regulated by Pax7 and the myogenic regulatory factors. While the expression of Pax7 is vital to the function of the satellite cell compartment, the paired domain alternative splicing events that regulate its DNA binding potential remain elusive. Interestingly, the generation of Pax7 splice variants differentially regulate Myf5 expression. We performed a global analysis of two Pax7 isoforms, which differ by a glycine-leucine dipeptide, to determine how paired domain splicing events modify the ability of Pax7 to regulate target genes. To this end, we observe that although the homeodomain is important for Pax7 binding, these isoform differences in the paired domain can regulate Pax7 targets during myogenesis. In addition to further examining the role of Pax7 during satellite cell proliferation and maintenance, it remains important to understand their downstream differentiation potential. Since activation of the canonical Wnt signalling pathway results in reduced regenerative efficiency in vivo, we undertook a global analysis of satellite cell derived myoblasts to examine their ability to respond to canonical Wnt signalling. We demonstrate that Wnt/β-Catenin signalling drives myogenic differentiation, via the myogenin-dependent control of follistatin expression, further fine-tuning the myogenic differentiation process. The effects of canonical Wnt signalling on myogenic differentiation complement our observations regarding Pax7 alternative splicing during myoblast proliferation and provide a greater comprehensive understanding of the molecular regulation of satellite cell development and differentiation during adult myogenesis.

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