Publications

To examine the capability of glomerular mesangial cells (MCs) to produce extracellular matrix, the authors studied MCs in culture by light and electron microscopy as well as immunocytochemistry. MCs were obtained from isolated rat glomeruli and maintained up to 12 weeks in medium containing 20% fetal calf serum. MC outgrowth of primary culture and of up to three subcultures showed characteristic organization consisting of bands of elongated or stellate intertwined cells. After confluency at 10-16 days, MCs continued to grow in irregular multilayers. MCs produced extracellular matrix material within 2-4 days after plating, and large amounts of matrix accumulated with time. By 2-3 weeks, foci of exaggerated MC proliferation, matrix secretion, and necrotic cell debris formed nodular protrusions, which gradually produced large hillocks. Immunocytochemical studies of MC outgrowths were performed on culture plates or on sectioned material with the use of specific rabbit polyclonal antibodies to isolated matrix proteins and FITC-conjugated, affinity-purified second antibodies. Within 3 days of culture, MCs elaborated fibronectin and collagen Types I, III, IV, and V. With time, strands of matrix, notably in the central mass of hillocks, stained extensively for these constituents. Staining for laminin was less pronounced. Smooth muscle cell myosin was regularly found on distinct intracellular fibrils and in the extracellular material of hillocks. Electron microscopy revealed the hillocks to be composed of elongated cells on the surface and stellate cells intermingled with matrix and necrotic cell debris in the core. The results show that proliferating MCs can be maintained in homogeneous culture for a prolonged time period. MCs produce large amounts of the extracellular matrix proteins (Type IV and V collagen, fibronectin, laminin), which are found in normal glomeruli. Cultured MCs also produce interstitial collagen Types I and III. MC hillocks show the nodular accumulation of matrix similar to that seen in the mesangium of diseased glomeruli. It is concluded that the in vitro model of prolonged MC outgrowth may facilitate the investigation of factors that govern mesangial matrix production. Such a model could be used in examining the response of the mesangium to defined inflammatory or metabolic stimuli.

Murine interleukin 1 (IL 1) inhibited concentration dependently the proliferation of murine T cell lymphomas and the human leukemic cell line K 562. The cytostatic action of IL 1 was not associated with cytotoxicity and appeared to be irreversible. Changes in the expression of surface antigens, like a rapid decrease of transferrin receptors or, more delayed, an increase in HLA-A, B, C antigen density suggested that a differentiation step was induced by IL 1. This effect of IL 1 was a direct one and most likely mediated by a specific receptor molecule. In order to characterize the receptor for IL 1, highly purified plasma membranes from K 562 were incubated with murine IL 1, and the phosphorylation pattern of plasma membrane proteins was investigated by the addition of radiolabeled ATP. At 0 degree C, IL 1 induced the specific phosphorylation of a 41 kDa membrane protein in a time- and concentration-dependent manner. Analysis of the phosphoamino acid composition revealed that IL 1 induced specifically the phosphorylation of tyrosine residues of the 41 kDa protein. Crosslinking experiments proved that the 41 kDa protein had an IL 1 binding site, strongly suggesting that the 41 kDa protein was the receptor for IL 1 itself. Affinity labeling with an ATP-analogue showed that this protein possessed an ATP binding and cleaving site. We conclude from this that the receptor for IL 1 in the plasma membranes of K 562 is a transmembranous protein of 41 kDa, which possesses a tyrosine specific protein kinase activity with an autophosphorylating capacity.

In this paper, we have attempted to provide an overview of the methods and findings of a large number of investigators who have dealt with an analysis of the glomerular inflammatory response using tissue culture techniques. These observations represent only a beginning. With the growing interest in this aspect of kidney disease, it is to anticipated that many further advancements in the understanding of the cell biology of the glomerulus are forthcoming. The translation of this fundamental information into new diagnostic and therapeutic modalities is an exciting challenge to investigative nephrology.