Detlef Schlondorff

The glomerular mesangial cell: an expanding role for a specialized pericyte.

The mesangial cell occupies a central position in the renal glomerulus. It has characteristics of a modified smooth muscle cell, but is also capable of a number of other functions. Among these are generation of prostaglandins (PGs) and mediators of inflammation; production and breakdown of basement membrane and other biomatrix material; synthesis of cytokines; and uptake of macromolecules, including immune complexes. In terms of its smooth muscle activity, the mesangial cell contracts or relaxes in response to a number of vasoactive agents. This ability allows the cells to modify glomerular filtration locally. The cellular mechanism of action of many agents influencing mesangial cells involves activation of phospholipase C for phosphatidylinositol 4,5‐bisphosphate. This results in generation of inositol trisphosphate and release of intracellular calcium. Mesangial cell relaxation can be mediated by enhanced cAMP or cGMP generation. Many vasoactive substances also stimulate PG production by mesangial cells. This involves activation of both phospholipase C and A2, the latter being responsible for the release of arachidonic acid. Mesangial cells are also capable of endocytosis of macromolecules, including immune complexes. This is initiated by binding to a specific receptor, resulting in formation of PG, platelet‐activating factor, and reactive oxygen species. Mesangial cells can generate interleukin 1 and platelet‐derived growth factor and respond to these in an autocrine manner. Thus, the mesangial cell not only can control glomerular filtration, but may also be involved in the response to local injury, including cell proliferation and basement membrane remodeling.— Schlondorff, D. The glomerular mesangial cell: an expanding role for a specialized pericyte. FASEB J. 1: 272‐281; 1987.

Cellular Mechanisms of Lipid Injury in the Glomerulus

Hyperlipidemias may play a role in the progression of various renal diseases, including diabetes mellitus. We therefore examined the characteristics of low-density lipoprotein (LDL) binding and uptake in cultured rat mesangial cells. Mesangial cells bound and took up LDL in a manner consistent with specific receptor mediation. Furthermore, exposure of mesangial cells to LDL enhanced intracellular cholesteryl esterification and decreased de novo cholesterol synthesis. Mesangial cells expressed mRNA for LDL receptor and their expression was downregulated after preloading of cells with LDL. These results are consistent with regulation of cholesterol uptake and metabolism by a specific LDL receptor mechanism. During diabetes the apolipoprotein B of LDL undergoes nonenzymatic glycation, which may alter its affinity for the LDL receptor. Glycation of LDL reduced its affinity for binding to the receptor sites and decreased its uptake by mesangial cells. Thus, during diabetes less LDL may be taken up and more remain extracellularly, where it can be trapped in the matrix. Oxidation of LDL bound to extracellular matrix is believed to be a major factor in the pathobiology of hyperlipidemias. Specific scavenger receptors for oxidized LDL have been described and cloned. We therefore examined whether rat mesangial cells bound and took up oxidized LDL. We demonstrated low-affinity but high-capacity binding sites for oxidized LDL on mesangial cells. In contrast to LDL, which supported mesangial cell proliferation, oxidized LDL was cytotoxic for the cells and resulted in stimulation of mesangial cell prostaglandin E2 production. Trapping of LDL in the extracellular matrix is considered an initial event in LDL-induced vascular pathology. We therefore evaluated binding of LDL and modified LDL to extracellular matrix produced by cultured mesangial cells. Mesangial matrix had a high capacity to bind LDL and modified LDL (glycated or oxidized) in a nonsaturable manner. These results obtained with cultured mesangial cells and their matrix allow the formulation of a working hypothesis. Under normal eulipemic conditions mesangial cells handle LDL in a regulated manner. During hyperlipidemia or expansion of extracellular matrix LDL accumulates in the matrix. There LDL would be subject to oxidative modifications, especially under conditions of mesangial cell stress, such as inflammatory, mechanical, or ischemic injury. Part of the oxidized LDL could be taken up by scavenger receptors on mesangial cells and monocyte-macrophages, resulting in foam cell formation. Excess oxidized LDL, and specifically the lipid peroxides and lysolipids of oxidized LDL, would act as cytotoxic agents on mesangial, epithelial, and endothelial cells, thereby contributing to a vicious cycle of cell damage and sclerosis.

Renal prostaglandin synthesis: Sites of production and specific actions of prostaglandins

Prostaglandins are substances that exert their effects at the site of their production. Therefore, the synthesis and effects of prostaglandins have to be considered separately for each nephron segment. In the cortex, major sites of prostaglandin synthesis include arteries and arterioles as well as the glomerulus. At these sites, prostaglandins are important in maintaining blood flow and glomerular filtration, especially during conditions of enhanced vasoconstrictor activity. Vasoconstrictors such as angiotensin II, norepinephrine, and vasopressin increase production of the vasodilator prostaglandins, thereby preventing an overshoot of their action. The role of arteriolar-glomerular prostaglandins in maintaining blood flow and filtration may be even more prominent during renal diseases. The proximal tubule and the loop of Henle show little ability to produce prostaglandins, but may generate considerable amounts of epoxygenase products of arachidonic acid. These epoxygenase products may play a prostaglandin-independent role in water and electrolyte transport in the thick ascending loop of Henle and the collecting tubule. Both the cortical and the medullary collecting tubules produce large amounts of prostaglandins, predominantly prostaglandin E2 (PGE2). In these segments, synthesis of PGE2 is stimulated by bradykinin and to a somewhat more variable degree by vasopressin. The PGE2 generated antagonizes the hydroosomotic effect of vasopressin both in vivo and in vitro, and may influence electrolyte excretion. Thus, the overall role of PGE2--and possibly of epoxygenase products of arachidonic acid--in tubular functions seems to be one of local modulation of water and electrolyte transport. Finally, interstitial cells are a major site of medullary prostaglandin production. Prostaglandins generated by the interstitial cells may play a role in maintaining blood flow to this poorly oxygenated and hypertonic region of the kidney.

Prostaglandin synthesis in isolated glomeruli

Prostaglandins are thought to play an important role in the local regulation of glomerular blood flow and in the release of renin from the juxtaglomerular apparatus. We therefore examined prostaglandin synthesis by isolated rat glomeruli. Isolated glomeruli were either prelabeled with [14C] arachidonic acid or were incubated with [14C] arachidonic acid for the entire experimental incubation in Krebs buffer. Prostaglandin synthesis was determined by thin layer radio-chromatography of acid extracts of the supernatant solutions. Indomethacin inhibitable synthesis of small amounts of 6-keto-PGF1 alpha, the metabolite of prostacyclin (PGI2,) and larger amounts of PGF2 alpha, and PGE2, and possibly thromboxane B2 (TXB2) by isolated glomeruli could be demonstrated with either prelabeling or direct incubation. These findings support the hypothesis that prostaglandins are produced within the glomerulus where they may affect local glomerular blood flow and function.

Mouse mesangial cells produce colony-stimulating factor-1 (CSF-1) and express the CSF-1 receptor.

CSF-1 stimulates the survival, proliferation, and differentiation of mononuclear phagocytes and may also play a role in placental development. The expression of CSF-1 and the CSF-1 receptor (CSF-1R) and their regulation were examined in cultures of mouse mesangial cells (MC). The concentration of CSF-1 in the medium of cultured MC increased linearly with time over 24 h. IFN-gamma stimulated and dibutyryl cyclic AMP inhibited CSF-1 production in a dose-dependent manner. MC expression of CSF-1 mRNA was shown by Northern blot analysis, and CSF-1 mRNA levels were increased within 4 h of IFN-gamma addition and inhibited within 4 h of dibutyryl cyclic AMP addition. Indirect immunofluorescence indicated that 90% of the untreated cultured MC expressed CSF-1. In addition, CSF-1R expression by MC was demonstrated by immunofluorescence with anti-receptor antibody, specific binding of [125I] CSF-1, and expression of the CSF-1R mRNA by Northern blot analysis. Thus, mouse MC, specialized pericytes of non-bone marrow origin, not only produce CSF-1 but also express receptors for CSF-1. The effects of CSF-1 on MC may be important in the control of immune function in the glomerulus.

Dopamine attenuates the contractile response to angiotensin II in isolated rat glomeruli and cultured mesangial cells.

Recent evidence suggests that dopamine may alter kidney function by actions not only in the renal vasculature but also at the glomerular-mesangial level. We studied this phenomenon by examining the ability of dopamine to antagonize the contractile properties of angiotensin II hi isolated rat glomeruli and cultured mesangial cells. In isolated rat glomeruli angiotensin II caused a decrease in the planar surface area, indicating glomerular contraction, an effect that was abolished by coincubation with dopamine. Angiotensin II also mediated shape changes in cultured mesangial cells, which resulted in a decline in their planar areas. Simultaneous addition of dopamine prevented these decreases in cell size. In mesangial cells grown on a flexible silicone rubber support, angiotensin II addition enhanced wrinkling of the mobile surface. This indicated that the angiotensin-II-induced decrease in cell size observed in cells grown on conventional substrata represented contraction. Conversely, dopamine caused a rapid reduction in wrinkling of the surfaces from control cells as well as those previously treated with angiotensin II, actions consistent with cell relaxation. The prostaglandin inhibitor indomethacin did not alter the ability of dopamine to attenuate angiotensin-II-associated reductions in mesangial cell surface area. Direct determination of mesangial cell prostaglandin-E2 production showed that dopamine did not change either basal synthesis or angiotensin-II-stimulated synthesis of prostaglandin. The results demonstrate that dopamine antagonizes the constrictor effect of angiotensin II at the glomerular-mesangial level. This action of dopamine is prostaglandin independent. These findings support a role for dopamine in the regulation of glomerular filtration and may provide a rationale for its use during states of renal, vasoconstriction.

Villous adenoma depletion syndrome: evidence for a cyclic nucleotide-mediated diarrhea

Massive secretory diarrhea is associated with some villous adenomas. The mechanism of this secretion is unknown but the character of the diarrhea resembles that of cyclic nucleotide-mediated diarrheas. We have compared the cyclic nucleotide metabolism of a large secretory villous adenoma with a nonsecretory villous adenoma, a solid, carcinoma and their normal mucosae. The adenylate cyclase, cyclic AMP content, and a cyclic AMP-dependent protein kinase ratios in the secretory tumor were increased as compared to these values in the nonsecretory tumors and normal mucosae, a situation similar to that seen with cholera toxin-induced diarrhea. Our data suggest that the massive diarrhea in our patient with a secretory villous adenoma may be related to increased adenylate cyclase activity.

Effect of vasopressin on cyclic AMP-dependent protein kinase in toad urinary bladder

The effect of vasopressin on the toad urinary bladder has been shown to be mediated by cyclic AMP. It has been assumed that, as demonstrated for other systems, this involves activation of cyclic AMP-dependent protein kinase. In order to test this hypothesis we investigated the effect of vasopressin on cyclic AMP-dependent protein kinases in epithelial cells of toad bladders. About 80% of protein kinase activity and cyclic AMP-binding capacity was found to be in the cytosol. DEAE-cellulose chromatography showed a pattern of 15--20% type I and 80--85% type II cyclic AMP-dependent protein kinase. Cytosolic kinase was activated 3--4-fold by cyclic AMP with half-maximal activation at 5 . 10(-8) M. Similarly, half-maximal binding of cyclic AMP occurred at 7 . 10(-8) M. Incubation of toad bladders in Ringers solution containing 0.1 mM 3-isobutyl-1-methylxanthine, prior to homogenization and assay, showed stable cyclic AMP-binding capacity and protein kinase ratio --cyclic AMP/+cyclic AMP. Exposure of bladders to 10 mU/ml of vasopressin for 10 min caused intracellular activation of protein kinase and decrease in cyclic AMP-binding capacity that were maintained for at least 30 min. Incubation of bladders with increasing concentrations of vasopressin (0.5--100 mU/ml) resulted in a discrepancy between a progressive increase in cyclic AMP levels and a levelling off at 10 mU/ml of vasopressin for the changes in protein kinase ratio and cyclic AMP-binding capacity. The increase in kinase ratio was due to higher activity in the absence of exogenous cyclic AMP and was fully inhibitable by a specific protein kinase inhibitor. Using Sephadex G-25-CM50 column chromatography for separation of holoenzyme and free catalytic subunit we demonstrated that the activation of protein kinase in the vasopressin-treated bladders is due to intracellular dissociation of the kinase. These results show that the effect of vasopressin on the toad bladder involves activation of a cytosolic cyclic AMP-dependent protein kinase. The time course and the dose-response curve of the kinase activation closely parallel vasopressins effect on osmotic water flow.

Angiotensin II causes formation of platelet activating factor in cultured rat mesangial cells.

Angiotensin II may contribute to the progression of renal glomerular diseases. Beneficial effects of converting enzyme inhibition in models of renal disease are, however, not always explicable by hemodynamic consequences of angiotensin II inhibition. Angiotensin increases intracellular calcium in glomendar mesangial cells and activates phospholipase A2, factors required for the formation of the lipid mediator of Inflammation platelet activating factor (PAT). We therefore examined whether angiotensin II could stimulate PAF production in cultured rat mesangial cells. During a 15-minute incubation angiotensin II caused formation of PAF in a dose- dependent manner with a threshold around 10-9 M. In four experiments PAF formation in response to angiotensin II (10-8 M) occurred within 5 minutes and was 29±8 pmol PAF/mg protein. The amount of PAF detected then declined to 9±2 and 13±3 pmol after 15 and 30 minutes of incubation with angiotensin II. More than 90percent; of the PAF remained cell-associated. The PAF formation was confirmed by negative ion chemical ionization mode of mass spectrometry. A single species of PAF was detected and identified as hexadecyl PAF. We speculate that part of the detrimental effects of angiotensin II in progressive renal disease may relate to PAF formation. The PAF generated may in turn influence glomendar function, platelets, and eicosanoid synthesis, all factors implicated in renal disease. Furthermore, we speculate that angiotensin II-induced PAF formation may contribute to microvasculature pathology in general.

Hyperosmolal State Associated with Rhabdomyolysis

We report a case of nonketotic hyperosmolal state associated with rhabdomyolysis. None of the known predisposing factors for rhabdomyolysis, e.g. coma, potassium or phosphate depletion, were present in this patient. We propose that severe hyperosmolality per se may represent another predisposing factor for nontraumatic rhabdomyolysis.