Age-related changes in α₁- and α₂-chain type IV collagen mRNAs in adult mouse glomeruli: Competitive PCR

Studies of age-related changes in glomerular extracellular matrix (ECM) synthesis in normal mice have been hampered by the difficulty of isolating sufficient numbers of intact glomeruli and by the inability to quantify different mRNA species. The purpose of this study was to identify and quantitate the individual mRNAs coding for α₁- and α₂-chains of type IV collagen in isolated, single glomeruli of normal mice at different ages. These data on normal ECM synthesis were necessary for the understanding of glomerulosclerosis, a condition characterized by excess deposition of collagen. Pools of freshly microdissected adult mouse glomeruli were reverse transcribed in situ, and α₁-IV and α₂-IV collagen mRNAs were individually amplified by means of specific primers and the polymerase chain reaction (PCR), according to a previously published method. A competitive PCR assay, based on utilization of mutated cDNAs, allowed the reproducible, quantitative, and separate determination of the absolute amounts of both α₁-IV and α₂-IV mRNAs measured, as their respective cDNAs, in one-tenth of one glomerulus. The levels of α₁-IV and α₂-IV collagen mRNA were 208 ± 36.0 × 10^-4 and 161.2 ± 18.6 × 10^-4 amol/glomerulus in 5-wk-old mice. There were no significant age-related differences at 8, 12, and 24 wk. The mean levels over this period were 60.2 ± 4.9 × 10^-4 for α₁-IV collagen mRNA and 63.9 ± 5.8 × 10^-4 amol/glomerulus for α₂-IV collagen mRNA. Two of three 24-wk-old mice had mild glomerulosclerosis. The glomerular levels of both mRNAs were elevated 1.8-fold in these mice, compared with normal 8- to 24-wk-old animals.