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Genetic factors that determine the degree of susceptibility to atherosclerosis may also influence the effects of estrogens and progestins in arterial vessel disease. We examined and compared estrogen receptor (ER) and progesterone receptor (PR) expression and the effects of 17beta-estradiol (E2) and progesterone (P) on collagen synthesis and matrix metalloproteinase (MMP) activities in the aortic arch and in cultured aortic smooth muscle cells (ASMC) of atherosclerosis-susceptible (C57Bl6/J, B6) or -resistant (C3H/HeJ, C3H) mice. ERalpha, ERbeta, and PR levels were higher in the aorta and ASMC of atherosclerosis-susceptible B6 mice. In transfection studies using an estrogen response element-driven reporter plasmid, E2 elicited a >2-fold increase in luciferase activity in ASMC of B6 (B6-ASMC), which demonstrated the transcriptional activity of ER in atherosclerosis-susceptible cells. Importantly, the response of endogenous target genes to E2 and P was different in B6-ASMC and C3H-ASMC. E2 decreased collagen synthesis but had no effect on MMP activities in B6-ASMC. P decreased MMP-2 and MMP-9 activity in B6-ASMC. In contrast, E2 increased MMP-2 and decreased MMP-9 activity but had no effect on collagen synthesis in C3H-ASMC. P had no effect on collagen synthesis and MMP activity in C3H-ASMC. These differences in response to sex hormones may have important implications for women who receive hormone replacement therapy.

We found that peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA was reduced by 77% in glomeruli of diabetic mice. Because mesangial cells play an important role in diabetic nephropathy, we examined regulation of type I collagen expression by PPARgamma and transforming growth factor-beta(1) (TGF-beta(1)) in mouse mesangial cells in the presence of 6 and 25 mM glucose. Mesangial cells contained functionally active PPARgamma. Exposure to 25 mM glucose resulted in reduced PPARgamma expression and transcriptional activity, accompanied by increased type I collagen expression. Restoration of PPARgamma activity to normal levels in cells cultured in 25 mM glucose, by transfection with a PPARgamma expression construct and treatment with the PPARgamma agonist troglitazone, returned type I collagen levels toward normal values. Activation of PPARgamma by troglitazone also decreased type I collagen mRNA and blocked TGF-beta(1)-mediated upregulation of type I collagen mRNA and protein. Moreover, PPARgamma activation suppressed basal and activated TGF-beta(1) responses in mesangial cells. This action was blocked by transfection of cells with a dominant-negative PPARgamma construct. In summary, PPARgamma suppresses the increased type I collagen mRNA and protein expression mediated by TGF-beta(1) in mesangial cells.

The macrophage mannose receptor, a carbohydrate-binding membrane protein, mediates endocytosis and phagocytosis. This study was undertaken to determine whether mannose receptors were expressed in resting glomerular mesangial and endothelial cells and whether their level was affected by cytokines. Neither mannose receptor mRNA nor proteins were found in resting mesangial or endothelial cells. Mannose receptor mRNA was induced in a dose- and time-dependent manner in mesangial cells by interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) but not by platelet-derived growth factor-B or IL-6. Cell surface receptors were found by fluorescence-activated cell sorter analysis. Binding to stimulated mesangial cells was saturable and inhibited by excess mannose-bovine serum albumin (BSA) but not by galactose-BSA. TNF-alpha and IL-1 alpha also induced apoptosis in mesangial cells. Mannose receptor expression was not restricted to apoptotic stimulated mesangial cells. Neither agonist induced mannose receptor expression or apoptosis in endothelial cells. Because immunoglobulin A, M, and G contain mannose residues, immune aggregates may be removed from the mesangium through cytokine-induced mannose receptors.

Changes in the composition of the mesangial extracellular matrix (ECM) and cell turnover are present in glomerular disease. To determine if ECM changes play a role in perpetuating mesangial cell dysfunction, we examined a line of mouse mesangial cells cultured on films or gels of several ECM components and also on methyl cellulose, an inert substrate that prevents attachment. Cells on films of fibronectin or type IV or I collagen had persistently high growth rates and high levels of alpha 1-I and alpha 1-IV collagen mRNAs. In contrast, on gels of type IV or I collagen or matrigel, the growth rate was low. The alpha 1-IV collagen mRNA levels were low on type IV collagen gel or matrigel, whereas the alpha 1-I collagen mRNA levels remained high. In contrast, the alpha 1-I collagen mRNA levels were low on type I collagen gel, and the alpha 1-IV collagen mRNA levels were high. Cells on methyl cellulose formed floating aggregates, did not proliferate, and had a 5- to 10-fold decrease in both alpha 1-I and alpha 1-IV collagen mRNA levels. These phenotypic changes were largely reversible. Finally, when matrigel was layered over cells on fibronectin films, alpha 1-IV collagen mRNA levels decreased, but alpha 1-I collagen mRNA levels and proliferation remained high. Thus proliferation and alpha 1-I and alpha 1-IV collagen mRNA levels in mesangial cells were independently regulated and depended on attachment and the nature of the adjacent matrix.

Oligosyndactyly mice (ROP Os/+) are a radiation-induced mutant strain with reduced glomerular number and increased glomerular size. We found that they develop glomerulosclerosis. At 3 mo, ROP Os/+ mice had diffuse mesangial expansion by light microscopy, whereas their +/+ littermates did not. Electron microscopic morphometry revealed a twofold increase in mesangial areas but no changes in the thickness of glomerular basal laminae. Mean glomerular volume was increased 1.8-fold. Cell number and thymidine labeling index were increased 1.3- and 2.4-fold, respectively. The amount of glomerular type IV collagen and tenascin but not laminin was increased by immunofluorescence microscopy. mRNA levels in microdissected glomeruli were measured by competetive reverse transcription-polymerase chain reaction and corrected for cell number. alpha 1-Chain type IV collagen and tenascin mRNAs were increased 3.2-fold and 1.8-fold, whereas laminin B1 mRNA levels were not. The levels of 72-kDa collagenase mRNA were increased 1.6-fold. Transforming growth factor-beta 1 mRNA levels were elevated 1.8-fold, but platelet-derived growth factor-B mRNA levels remained normal. This is the first analysis of glomerular molecular and cellular changes in a model of congenital nephron reduction.

Endothelial cells isolated from a variety of vascular beds bind and transport insulin but exhibit relatively low insulin degrading activity. Because endothelial cells exhibit heterogeneity and since kidney is a major site of insulin degradation, we studied the processing of insulin by glomerular endothelial cells (GEC). When exposed to 2 x 10(-10) M 125I-labeled insulin, GEC associated with the hormone in a specific manner. This interaction was inhibited by insulin but not by a number of unrelated peptide hormones. Over a 90-min period, GEC degraded 42 +/- 3% of the 125I-insulin, as measured by solubility in trichloroacetic acid (TCA). Degradation was inhibited 90% by an excess of insulin or adrenocorticotropic hormone (10(-6) M) and 57% by glucagon, whereas growth hormone and calcitonin were without effect. Separation of plasma membrane bound from internalized insulin was achieved by decreasing extracellular pH. In the steady state, 43% of cell-associated insulin was membrane bound and 57% internalized. The fate of the internalized 125I-insulin was examined by incubating acid-washed cells at 37 degrees C for 60 min. Over this time 18% of the radioactivity was released as TCA insoluble- and 72% as TCA-soluble radioactivity. Release was increased by insulin (10(-6) M) but not by unrelated peptide hormones. In the presence of chloroquine, 125I-insulin release increased by one third while degradation fell. High-performance liquid chromatography revealed that GEC released both intact insulin and large intermediates and that chloroquine inhibited intermediate formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the balance between synthesis and degradation of extracellular matrix may result in glomerulosclerosis. The interaction between metalloproteinases and their inhibitors presumably modulates the rate of glomerular matrix degradation. We examined the gene expression of tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in human glomeruli and TIMP-2 protein in tissue sections. Kidney tissue was obtained from adults undergoing nephrectomy for renal tumor (n = 9) or biopsy for nephrosis and renal failure (n = 1). Glomeruli were microdissected and subjected to reverse transcription. TIMP cDNAs were quantitated by competitive polymerase chain reaction assays. Five nephrectomy specimens had normal glomeruli and four had diffuse glomerulosclerosis. TIMP-1 and TIMP-2 cDNA levels, detected in glomeruli from all patients, were increased fourfold and threefold, respectively, in patients with glomerulosclerosis. The elevated TIMP cDNA levels could not be attributed to an increased number of glomerular cells. TIMP-2 protein was detected within normal and sclerotic glomeruli. In conclusion, both TIMP genes were expressed in normal glomeruli, and their level of expression was increased in glomerulosclerosis associated with renal carcinoma, suggesting that expression of these inhibitors may correlate with the development of sclerosis.

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 alpha 1- and alpha 2-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 alpha 1-IV and alpha 2-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 alpha 1-IV and alpha 2-IV mRNAs measured, as their respective cDNAs, in one-tenth of one glomerulus. The levels of alpha 1-IV and alpha 2-IV collagen mRNA were 208 +/- 36.0 x 10(-4) and 161.2 +/- 18.6 x 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 x 10(-4) for alpha 1-IV collagen mRNA and 63.9 +/- 5.8 x 10(-4) amol/glomerulus for alpha 2-IV collagen mRNA. Two of three 24-wk-old mice had mild glomerulosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Mesangial cell proliferation is a common hallmark of many glomerular diseases. The exact mechanisms inducing cell proliferation in glomerulosclerosis are not completely understood, and it remains to be determined whether growth factors play a role in this process. Insulinlike growth factor I (IGF I) has been shown to be synthesized in the kidney, and glomerular mesangial cells have receptors for and exhibit mitogenic response to IGF I. We found that mouse glomerular mesangial cells in culture synthesized and released into the culture medium a molecule with immunological and biological features of IGF I. This molecule specifically bound to mesangial cell IGF I receptors; high-pressure liquid chromatographic analysis provided further evidence of its similarity to human recombinant IGF I. Mesangial cells released into the culture medium 6 ng/10(6) cells of IGF I-like material per 24 h in a time-dependent and actinomycin-D inhibitable fashion. These data suggest that IGF I might be locally released by mesangial cells in the glomerulus and act in an autocrine and paracrine fashion.