Glomerular deposition of homotrimeric type I collagen in the COL1A2 deficient mouse

Title from title screen of research.pdf file (viewed on December 22, 2006).The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file."May 2006"Vita.Thesis (Ph. D.) University of Missouri-Columbia 2006.Type I collagen is the most abundant structural protein in the body. Type I collagen generally exists as a heterotrimeric protein; however, a homotrimeric isotype of type I collagen has been identified. Our lab currently studies the COL1A2 deficient mouse model which is homozygous for a mutation that causes the mice to synthesize homotrimeric type I collagen exclusively. We recently identified deposition of type I collagen in the kidneys of the COL1A2 deficient mouse model and under normal physiologic conditions such accumulation is pathologic. The primary goal of this research is to investigate the molecular mechanisms involved in the regulation of type I collagen within the kidney by evaluating synthetic and degradative pathways. As presented here, the accumulation of homotrimeric type I collagen in the kidneys of the COL1A2 deficient mice occurs postnatally and appears to be due to an increase in the synthetic pathway, as well as an alteration in the degradative pathway. Gaining a further understanding of the mechanisms behind the deposition of collagen within the kidney will hopefully lead to a greater understanding of secondary renal injury seen in the progression to end-stage renal disease, a complication of a variety diseases, including diabetic and IgA nephropathy. Further, characterization of the role of homotrimeric type I collagen will also lead to a greater understanding of its role in developmental and pathological events.Includes bibliographical reference

Investigation of type I collagen deposition in the glomeruli of COL1A2 deficient mice

Abstract only availableType I collagen is the most abundant structural protein in the body, playing a major role in the strength and integrity of connective tissues. Alterations in the synthesis and structure of type I collagen result in a number of connective tissue disorders, such as osteogenesis imperfecta and Ehlers-Danlos syndrome. Type I collagen is normally a heterotrimeric type I collagen molecule composed of three pro 1(I) collagen chains and one pro2(I) collagen chain. The COL1A2 deficient mouse produces only homotrimeric molecules, composed of three pro1(I) collagen chains resulting from a functional null mutation in the COL1A2 gene. Recently our lab discovered a novel type I collagen glomerulopathy in the COL1A2 mouse. The novel glomerulopathy demonstrated increased collagen deposition in the renal glomerular mesangium. The collagen accumulation is similar to what is observed in the secondary progression of renal damage, caused by a variety of kidney disorders. This project entails the measurement of COL1A2 and COL1A1 mRNA levels discerning whether increased collagen deposition in the glomeruli mesangium in COL1A2 deficient mice is related to increased type I collagen mRNA expression (pretranslational mechanism). These studies required harvesting kidneys from the COL1A2 deficient, heterozygous, and wildtype mice at one week, two weeks, and one month of age. Total RNA was isolated from harvested kidneys using the Qiagen RNAeasy RNA isolation kit, post mortar and pestle homogenization. The isolated total RNA was then analyzed for amount and quality via spectrophotometer at 260nm and 280nm, and 80ng of total RNA was used for cDNA generation via reverse transcriptase polymerase chain reaction using Promega ImpromII reagents. PCR products were generated for mouse COL1A2 transcripts using primers optimized for Real Time PCR analysis (RT-PCR). Quantitation of the COL1A1 and COL1A2 transcripts was obtained on the Roche LightCycler, with SYBR green monitoring after each amplification cycle. Previous in situ hybridization data suggests that there may not be an increase in mRNA. The study will attempt to gain a greater understanding of the molecular mechanism leading to type I collagen accumulation in the renal mesangium, with future application to understanding the role of extracellular matrix deposition in renal pathology and disease.Life Sciences Undergraduate Research Opportunity Progra

Relations between matrix metalloproteinase (MMP) activity, type I collagen accumulation, and glomerular sclerosis in the oim mouse [abstract]

Abstract only availableFaculty Mentor: Dr. Charlotte Phillips, BiochemistryA novel type I collagen glomerulopathy was identified in oim mice [pro2(I) collagen (COL1A2 deficient)], which synthesize exclusively homotrimeric type I collagen, [1(I)3]. Type I collagen exists predominantly as a heterotrimer [1(I)2 2(I)], although the homotrimeric form is present in small amounts in skin, embryologic tissues and would healing. However, the functional role of this homotrimer is unknown. In the oim mouse kidney the homotrimer accumulates in the glomeruli. Under normal physiologic conditions type I collagen is not present in the glomeruli; its accumulation is pathologic. In the following study we use an innovative perfusion technique that utilizes magnetic beads to isolate the glomeruli from surrounding tissue to investigate whether the accumulation of homotrimer is a result of either increased synthesis or a decreased degradative function of glomerular MMPs. We demonstrate through RT-PCR amplification that wild type and oim glomerular cells do, in fact, produce transcripts for MMP-1, MMP-2, MMP-9, and MMP-13. Furthermore, preliminary studies from collagen gel zymography suggest that glomerular MMPs cleave heterotrimeric type I collagen preferentially