The tetraspanin CD151’s role in the kidney and mapping of genetic modifiers of glomerular disease

Naudin, Crystal

Title: The tetraspanin CD151’s role in the kidney and mapping of genetic modifiers of glomerular disease
Creator: Naudin, Crystal
Relation: University of Newcastle Research Higher Degree Thesis
Resource Type: thesis
Date: 2015
Description: Research Doctorate - Doctor of Philosophy (PhD)
Description: Glomerular diseases represent a major burden for both patients and the community. They are responsible for a significant proportion of chronic kidney disease, which can ultimately progress to end stage renal disease, requiring dialysis or transplantation. Glomerular diseases are associated with leakage of proteins across the glomerular filtration barrier into the primary urine (proteinuria or albuminuria – as albumin is the major protein involved). The glomerular filtration barrier is composed of three interconnected layers: podocyte foot processes, the glomerular basement membrane (GBM) and a fenestrated endothelium. The tetraspanin protein CD151 is a crucial component of the glomerular filtration barrier, where it is known to complex with integrins to strengthen podocyte foot process anchorage to the GBM. In addition, previous findings in our laboratory have shown that in FVB/N Cd151^-/- mice the disruption and abnormal development of the GBM precedes podocyte foot process abnormalities, suggesting that CD151 also plays a role in the maturation and remodelling of the GBM. In other settings CD151 has been shown to regulate the proteolytic activity of matrix metalloproteinases (MMPs), important players in the homeostasis of basement membranes, and thus CD151 has many potential roles in glomerular homeostasis and disease. Similar to human mutation, Cd151 knockout in the FVB/N mouse strain leads to severe early-onset glomerular disease associated with GBM abnormalities, whereas knockout in the C57Bl/6 mouse strain does not lead to glomerular disease with kidneys presenting healthy. This strong influence of genetic background suggests the action of modifier genes, which may have important roles in human kidney disease where the course of glomerular diseases can vary significantly between patients. It is therefore important to identify the genes modulating progression as they could be used as biomarkers to predict the course of these heterogeneous diseases in patients or as potential therapeutic targets. In order to understand the molecular mechanisms contributing to progression and onset of glomerular disease in FVB/N Cd151^-/- mice, whole genome mRNA expression profiles of glomeruli from Cd151^+/+ and Cd151^-/- mice were investigated on both the C57Bl/6 and FVB/N backgrounds. Analysis was conducted at 3 weeks of age, at this stage in FVB/N Cd151^-/- mice changes to the GBM are evident but secondary changes such as glomerulosclerosis are not yet significant or widespread, and therefore allows the identification of genes relevant to early stage disease. The FVB/N Cd151^-/- mouse glomeruli showed 24 highly significant transcript changes compared to FVB/N Cd151^+/+, including changes in transcription factors, inflammatory factors and extracellular matrix regulators. Many of these changes did not occur in the corresponding comparison in the C57Bl/6 strain (Cd151^-/- versus Cd151^+/+) and therefore are likely specific to glomerular disease development. Following on from identified changes in transcript expression of MMPs, it was found that the proteolytic activities of pro-MMP-9, MMP-9 and MMP-2 were reduced in FVB/N Cd151^-/- glomeruli compared to FVB/N Cd151^+/+, C57Bl/6 Cd151^+/+ and C57Bl/6 Cd151^-/- mice. Furthermore the protein expression of MMP-10 was upregulated specifically in FVB/N Cd151^-/- glomeruli. Therefore the GBM defects observed in FVB/N Cd151^-/- mice may be due to reduced turnover of basement membrane proteins by MMPs in FVB/N Cd151^-/- mice. Eleven pathways were enriched specifically in FVB/N Cd151^-/- mice, including robust changes in two cellular signalling gene networks: the T-cell receptor signalling network and the axon guidance network. Firstly, this suggests that the development of glomerular disease in this mouse model may have immune involvement. Secondly this finding supports recent parallels that have been drawn between the signalling molecules involved in elongation and adhesion signalling of podocyte processes and axonal dendrites. Overall the loss of CD151 significantly affects the expression of molecules likely to influence inflammatory signalling in the glomerulus, and cytoskeletal organisation within podocyte foot processes. Mindin, an inflammatory mediator, was significantly and specifically induced in the GBM of FVB/N Cd151^-/- mice, as detected by immunofluorescence and was also observed in the urine of these mice by immunoblotting. The functions of mindin have not been investigated in the kidney; however, as it is known to be pro-inflammatory, the potential for mindin to be pathologically contributing to disease progression was investigated. Inflammatory infiltrates including lymphocytes, neutrophils, macrophages and eosinophils, were observed as early as 3 weeks of age in FVB/N Cd151^-/- but not in FVB/N Cd151^+/+, C57Bl/6 Cd151^+/+ and C57Bl/6 Cd151^-/- mice. This infiltration was progressive and more pronounced in 12 week old FVB/N Cd151^-/- mice, which had developed extensive inflammatory lesions. It can be speculated therefore that mindin is involved in recruiting inflammatory cells into kidneys of FVB/N Cd151^-/- mice early in disease, which may then contribute to disease progression. As FVB/N Cd151^-/- mice show severe early onset glomerular disease compared to C57Bl/6 Cd151^-/- mice, which show a healthy kidney phenotype, this model lends itself to the identification of quantitative trait loci (QTL) influencing glomerular disease development. Therefore, a backcross of the resistant line (C57Bl/6) for two generations onto the permissive line (FVB/N) was carried out. F1 Cd151^-/- mice (FVB/N × C57Bl/6) show complete absence of glomerular disease, suggesting that the protective alleles from the C57Bl/6 background are dominant. N2 (F1 x FVB/N) Cd151^-/- mice were found to have a highly variable kidney phenotype, with 68% developing albuminuria. Analysis of age at onset of albuminuria in N2 mice showed that there were three statistically distinct groups: no onset (followed up to 12 months of age), early onset (<2 months of age) and late onset (3-4 months of age). Linkage analysis identified 2 regions that account for >50% of the variability in inheritance of the trait. Specifically inheritance of an FVB/N homozygous genotype at a chromosome 14 QTL (45.34cM - 46.34cM) was strongly associated with early onset albuminuria. This region includes only 1 gene, protocadherin 9, which is known to be expressed in human foetal kidney tissue, suggesting a role in kidney development. Taken together, the data suggests that protocadherin 9 represents a modifier gene influencing glomerular phenotype in the absence of CD151. A second linked region on chromosome 1 (40.859cM-44.046cM) also strongly influenced the development of glomerular disease, with susceptibility associating with heterozygosity. Within this region 22 genes, including GBM proteins collagen IV chains α3 and α4 are located. The genetic modifiers responsible for influencing glomerular phenotype in the absence of CD151 in the two QTL regions remain undefined; however, protocadherin 9 and the collagen IV chains α3 and α4 represent likely candidate modifier genes, and require further investigation. To further assess the relationship between genetic background and disease severity and progression, a multiple-trait analysis was performed using albuminuria, GBM defects, creatinine clearance and serum urea as quantitative traits. Using this approach a further 13 QTLs were identified which relate to Cd151^-/- glomerular disease progression and severity. Several of the mapped QTLs demonstrated concordance with previously identified QTLs for rodent models of glomerular disease, and concordance with a human locus influencing diabetic nephropathy. In conclusion this is the first reported comprehensive analysis of gene expression changes as well as QTLs in the Cd151^-/- model of glomerular disease. The study has identified a number of genes and proteins that likely contribute to disease onset or progression which now require further investigation to determine their function in human kidney diseases.
Subject: glomerular disease; tetraspanin; CD151; quantitative trait loci; gene expression; matrix metalloproteinases; inflammation
Identifier: http://hdl.handle.net/1959.13/1305765
Identifier: uon:21095
Language: eng
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