Binding of insulin-like growth factor-I by glomerular endothelial and epithelial cells: further evidence for IGF-I action in the renal glomerulus

The renal glomerulus is both a site of action and synthesis of IGF-I. We previously demonstrated the presence of IGF-I receptor and synthesis in glomerular mesangial cells. In this study we investigated the presence of specific IGF-I receptors on mouse glomerular endothelial and epithelial cells in culture. [125I]IGF-I specifically bound to the cell surface of both cell types. Maximum specific binding, 0.141 B/F for endothelial cells and 0.301 B/F for epithelial cells, was obtained at 22 degrees C after 150 min incubation. The estimated Kd values were 2.25 x 10(-9) for endothelial cells and 1.5 x 10(-9) for epithelial cells. Cross-linking studies showed a single band of radioactivity with an estimated mol wt of 145K, consistent with the alpha-subunit of the IGF-I receptor. Radiolabelled IGF-I was not degraded by either cell types. These findings suggest a possible paracrine action of IGF-I in the renal glomerulus.     

Localization and distribution of anionic charges in the glomerular mesangium of normal and nephrotic rats

Sulfated glycosaminoglycans and sialoglycoproteins are thought to play a pivotal role in the glomerular capillary wall barrier to filtration since these anionic charged elements are important in the maintenance of capillary wall integrity and constitute a charge-selective filter. The development of proteinuria in puromycin aminonucleoside (PAN) nephrosis is associated with polyanion loss from the glomerular capillary wall structures. Since in PAN nephrosis the permeability of the mesangial area to plasma proteins and tracer substances has also been shown to be increased, the purpose of this study was to analyse the localization and distribution of anionic charges in the glomerular mesangium in this experimental model. Glycosaminoglycans were labeled by perfusion of the kidneys with ruthenium red solution (RR). Electron microscopic examination revealed the presence of distinct small RR granules ("anionic sites") in the mesangial intercellular matrix substance and in the laminae rarae of the glomerular basement membrane (GBM). The center-to-center spacing of the granules was measured and a frequency distribution of intervals in different interval classes was constructed. In normal glomeruli the anionic sites in the mesangial matrix showed a distribution pattern identical to the GBM with a maximal interval incidence at the 31-40 nm class. In nephrotic rats anionic site distributions in matrix and GBM did not change significantly. Sialoglycoproteins were labeled with colloidal iron (CI). In PAN nephrosis a decrease of CI binding was observed at the epithelial-basement membrane junction of the glomerular capillary wall. However, CI labeling of the mesangial matrix and mesangial cell membranes did not differ from that of normal glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between ricin and Zajdela ascitic hepatoma cells

The binding to and toxicity of ricin on Zajdela hepatoma ascites cells were studied. The kinetic analysis of [125I]-ricin binding to hepatoma cells indicated that maximal specific binding was reached within 30 min. at 4 degrees C and 60 min. at 25 degrees C and that toxin binding to hepatoma cells was saturable. When the binding data were plotted according to the method of Scatchard, curvilinear graphs were obtained suggesting that hepatoma cells have both high and low affinity receptors for ricin. The number of high and low affinity receptors was identical at 4 and 25 degrees C, i.e., 8 x 10(5) and 1.2 x 10(7) sites per cell respectively. However, the capacity of hepatoma cells to bind ricin is stronger at 4 degrees C than at 25 degrees C. The toxic activity of ricin was totally abolished in the presence of lactose suggesting that ricin binding to cells occurs through binding sites containing galactosyl residues.     

Characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal antibody specific for human fibrin fragment D-dimer

A recombinant single-chain molecule, scFv-K12G0, containing the variable domains of the monoclonal antibody MA-15C5, specific for fragment D-dimer of human cross-linked fibrin, was constructed and expressed in Spodoptera frugiperda, Sf9, insect cells. The Arg108 carboxyl-terminal amino acid of the variable domain of the light-chain of the antibody was connected through a synthetic Ala-Gly-Gln-Gly-Ser-Ser-Val peptide linker with the Gln1 amino-terminal amino acid of the variable domain of its heavy chain. scFv-K12G0 was secreted by the infected Sf9 cells at a rate of 10 micrograms/10(6) cells within 48 h, resulting in conditioned medium with a maximal concentration of 15 mg of scFv-K12G0/liter. The molecule, purified to homogeneity by ion exchange chromatography and gel filtration, migrated as a single Mr band on reduced sodium dodecyl sulfate-gel electrophoresis. It bound to immobilized fragment D-dimer with an affinity constant of 4.0 x 10(9) M-1 (2.0 x 10(10) M-1 for intact MA-15C5). Clearing of scFv-K12G0 from the circulation in rabbits occurred with an initial half-life (t1/2 alpha) of 10 min and a clearance of 5.1 ml min-1, as compared to 90 min and 210 ml min-1 for intact MA-15C5. Nephrectomy resulted in a prolongation of t1/2 alpha to 110 min, suggesting that the rapid clearance of scFv-K12G0 occurs primarily via the kidney, presumably by glomerular filtration. The results indicate that the single-chain recombinant molecule scFv-K12G0 is secreted in functionally intact form and suggest that it may be useful for targeting of radioisotopes or plasminogen activators to blood clots in vivo.     

Dual effect of vasoactive intestinal peptide on the mitogenic response of rabbit spleen lymphocytes

The effects of vasoactive intestinal peptide (VIP) have been investigated on the mitogenic response of rabbit spleen cells. Specific binding of 125I-VIP to these mononuclear cells is rapid and saturable. Analysis of binding reveals two classes of binding sites, a class of high-affinity binding sites with KD = 0.93 +/- 0.11 nM and maximal binding capacity of 2000 +/- 560 sites/cell, and a class of low-affinity binding sites with KD = 225 +/- 58 nM and maximal binding capacity of 280,000 +/- 60,000 sites/cell. The VIP regulatory effect on mitogen-stimulated rabbit spleen cell proliferation appears to be time dependent and bimodal. When VIP was added simultaneously with mitogens, it induced an inhibition of the proliferative response. With concanavalin A (Con A) or pokeweed mitogen (PWM), addition of 10(-8) M VIP resulted in a maximal 30% inhibition of [3H]thymidine incorporation after 96 h of culture. This inhibitory effect was significant at concentrations from 10(-8)-10(-6) M and half-maximal inhibition was obtained with 1.2 x 10(-9) M VIP. By contrast, when rabbit spleen cells were preincubated for 18 h with VIP alone, the lymphocyte proliferative response to Con A was increased. However, this increase was mitogen-selective, since it was only observed when the T-cell mitogen Con A was used. The maximal response was obtained after 96 h of culture in the presence of Con A. The VIP stimulatory effect was dose-dependent with a maximal effect at 10(-7) M and a half-maximal effect at 1.7 x 10(-9) M VIP. The effect of VIP was also time-dependent, since a 6 h preincubation was sufficient to induce a significant increase in the proliferative response which was maximal after an 18 h preincubation.     

Receptor-mediated induction of aminopeptidase A (APA) of human glomerular epithelial cells (HGEC) by glucocorticoids.

Membrane-bound peptidases are critical regulators of peptide hormones. We therefore characterized aminopeptidase A (APA) activity in human glomerular epithelial cells (HGEC) and studied the control of its expression. APA, which splits off the N-terminal Asp from angiotensin II (AII), was present at the surface of HGECs (55% of the total enzyme). APA activity was calcium-dependent and was inhibited by amastatin. Treatment of HGECs by dexamethasone (DEX) increased ecto-APA activity in a dose- and time-dependent manner. Maximal increase of APA activity (x 2) occurred after treatment with 0.5 microM DEX for 5 days. HIgher concentrations (1-10 microM) of aldosterone (ALD) stimulated APA activity to a lesser extent (x 1.25). Actinomycin D and cycloheximide prevented and RU 38486, a glucocorticoid receptor antagonist, suppressed the DEX-induced increase in APA activity. These results indicate that AII availability at glomerular receptor sites may be reduced by DEX and suggest a role for glucocorticoids in AII-dependent changes of glomerular filtration rate.     

Cadmium uptake by cells of renal origin

We compared the ability of rat glomerular mesangial cells and LLC-PK1 cells to take up Cd2+ from solution. The former are smooth muscle-like cells of mesenchymal origin, the latter an established line of proximal tubular epithelium. Both cells, as well as primary glomerular epithelia, accumulated Cd2+ against a concentration gradient in a time-dependent manner. Uptake by mesangial cells obeyed a Michaelis model with an apparent Km of 19 microM and could be described by an initial rapid step of surface binding followed by rate-limiting internalization. In contrast, uptake by LLC-PK1 cells was non-saturable under accessible concentrations of Cd2+ and internalization was not a necessary consequence of association with the cell surface. In several other cell types, Cd2+ uptake has been shown to be inhibited by blockage of cell-surface sulfhydryl groups. In contrast, uptake by neither mesangial nor LLC-PK1 cells was depressed by N-ethylmaleimide, which actually enhanced the surface binding and to a lesser extent the uptake by the LLC-PK1 cell line. Neither depended on metabolic energy for uptake or utilized Ca2+ channels. The internalization process was temperature dependent and was obliterated at 2 degrees C. In mesangial cells, this allowed direct observation of the internalization event from a presaturated surface pool. The rate of this process was consistent with the Vmax calculated from the Michaelis model. Surface binding and uptake were decreased by binding of Cd2+ to serum proteins and albumin and were much less dependent on the presence of low molecular weight components of serum. Therefore, these cells may be especially sensitive to Cd2+ at concentrations encountered in vivo because of the low protein content of the plasma ultrafiltrate. Surface binding of Cd2+ to mesangial cells was suppressed by competing divalent ions following the order of the Irving-Williams series (Mn less than Co less than Ni less than Cu greater than Zn), although Zn2+ showed the greatest effect on internalization. In LLC-PK1 cells, Zn2+ and Cu2+ were both effective in decreasing Cd2+ uptake. We conclude that Cd2+ uptake by the tubular epithelial cells is rapid and independent of specific cell surface interactions, whereas uptake by rat mesangial cells follows binding to a specific surface ligand saturating at about 1.5 x 10(7) copies/cell. In both types of cells the uptake appears quite specific for Cd2+ and shows some cross-reactivity with other metal cations explicable by competitive ligand binding.     

Freeze-fracture cytochemistry of rat glomerular capillary tuft. Determination of wheat germ agglutinin binding sites and localization of anionic charges

We propose here the use of freeze-fracture to gain access and to label in vitro glomerular components and locate WGA receptors and anionic sites. Tissues are frozen, fractured under liquid nitrogen, and thawed. Freeze-fracture rendered all glomerular structures directly accessible to the reagents. This made possible study of the nature and topology of cationized ferritin and WGA binding sites. WGA-gold complexes were observed over plasma membranes of podocytes and of endothelial and mesangial cells. Labeling of podocytes and endothelial cells was similar in the mesangial area and in the peripheral part of the capillary loop. Cross-fractures of extracellular matrices showed that WGA bound uniformly to the glomerular basement membrane (GBM) as well as to mesangial matrix. In fractured specimens treated with neuraminidase, WGA was no longer observed over podocytes but it consistently labeled the surface of endothelial and mesangial cells. Whereas in GBM cross-sections WGA binding was greatly reduced or even abolished, it remained unmodified in the mesangium. This shows that only NeuNAc (sialic acid) might account for the binding of WGA to podocytes, whereas GlcNAcs appear to be the main WGA binding sites on endothelial and mesangial cells and in the mesangial matrix. Both NeuNAc and GLcNAc residues are probably associated in GBM. With cationized ferritin (pI 8.3) at pH 7.4, intense, continuous labeling was seen all over the different plasma membranes, denser in podocytes than in endothelial cells. CF was also observed in cross-fractured profiles of extracellular matrices and never appeared agglutinated in discrete sites.     

The plasma membrane of epididymal epithelial cells has a specific receptor which binds to androgen-binding protein and sex steroid-binding protein.

The binding of [3H] delta 6-testosterone photoaffinity-labelled rat androgen-binding protein (rABP) has been studied in an enriched fraction of plasma membranes of epithelial epididymal cells in immature (15 days) and adult rats (40 days). The binding was maximal in less than 30 min and more rapid at 4 degrees C than at 34 degrees C. It was calcium and pH dependent. Scatchard plots of the binding data gave curvilinear plots with two types of binding sites corresponding to a K(ass1) of 18.2 nM-1 and K(ass2) of 1.6 nM-1 (2.2 x 10(11) sites/mg protein and 5.4 x 10(11) sites/mg protein, respectively). In adult rats, only one type of binding site was found, with a K(ass) of 3.7 nM-1 (4.5 x 10(11) sites/mg protein). The number of receptors was 5-fold lower in the cauda than in the caput of the epididymis. The pretreatment of the isolated intact cells with streptozotocin induced a 45% reduction of the binding. Only unlabelled rABP and hSBP (human sex steroid-binding protein) but not other proteins (lactotransferrin, serotransferrin, asialofetuine, fetuine and bovine serum albumin) competed with the labelled ligand to bind plasma membranes. The membrane fraction was solubilized by triton X-100. Its incubation with labelled rABP and hSBP provoked the elution of the tracer as an aggregate into the void volume fraction of superose 6B mini-gel filtration columns. Structural homology between hSBP and rABP could be responsible for the common behaviour of the steroid-carrier molecules for the ABP receptor of rat epididymal epithelial cells.     

Atrial natriuretic factor (ANF) binding sites in frog kidney and adrenal.

Atrial natriuretic factor (ANF) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. [125I]-rat ANF(99-126) binding was present in kidney glomeruli and in the outer layer of interrenal tissue in the adrenal gland. ANF binding exhibited positive cooperativity with a half-maximal binding concentration (EC50) of 102 +/- 16 pM in glomeruli and 93 +/- 19 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 1.33 +/- 0.16 and 1.21 +/- 0.36 fmol/mm2. [125I]-Rat ANF(99-126) binding was competitively displaced by unlabeled ANF analogues with an intact disulfide bridge showing a lower affinity than the iodinated ligand. The presence of ANF binding in glomeruli and steroidogenic interrenal cells suggests physiological functions of ANF for the osmomineral regulation in the frog by influencing glomerular filtration rate and adrenal steroid secretion.     

Time-dependent biphasic regulation of Na+/K+/Cl- cotransport in rat glomerular mesangial cells

Time-dependent regulation of loop diuretic-sensitive Na+/K+/Cl- cotransport and [3H]bumetanide binding was investigated in cultured rat glomerular mesangial cells. Angiotensin II or epidermal growth factor induced stimulation of Na+/K+/Cl- cotransport within 5 min, with a return to the control values by 30 min. Treatment of cells with phorbol 12-myristate 13-acetate (0.1 microM) (PMA), the calcium ionophore A23187 (1 microM), or the combination of 5 mM NaF and 10 microM AlCl3 produced a transient stimulation of Na+/K+/Cl- cotransport in 5-10 min to 148, 135, and 163% of control, respectively, which was followed by a progressive decrease to 34, 64, and 20% of the base-line activity, respectively, by 60 min. Exposure to cyclic 8-bromo-AMP (0.1 mM) or to forskolin (1 microM) and isobutylmethylxanthine (0.1 mM) caused a maximal inhibition of the cotransport in 5 min to 79 and 60% of control, respectively, with a subsequent gradual increase to 137 and 164% of the base-line activity, respectively, by 60 min. The effects of PMA, forskolin, and cyclic 8-bromo-AMP were concentration-dependent. In order to characterize further the alterations in the cotransport activity, binding of [3H]bumetanide was determined. Saturation binding analyses showed that the late inhibition of the cotransport by PMA and stimulation by forskolin were associated with a significant decrease and increase, respectively, in Bmax, with no significant changes in binding affinity. Correlations between changes in the cotransport activity and [3H]bumetanide binding were also observed in cells treated with cyclic 8-bromo-AMP or with NaF and AlCl3. Incubation of cells in Cl- or Na+ free solution greater than or equal to 60 min resulted in an increase in both the cotransport activity and [3H]bumetanide binding. These observations indicate that, in glomerular mesangial cells, persistent stimulation of second messengers that regulate the cotransporter induces a time-dependent, biphasic regulation of Na+/K+/Cl- cotransport and that the regulation occurring after greater than or equal to 60 min of treatment is primarily due to changes in the number of the active cotransport sites. Because long term removal of the transported ions also increases the number of active cotransport sites, these results suggest that alterations in intracellular ionic homeostasis may also mediate cotransport activity.     

Arachidonate metabolites and the control of glomerular function

The glomerulus is a dynamic structure capable of regulating the glomerular filtration rate (GFR) by mesangial contraction, thereby decreasing Kf. The mesangium contracts in response to angiotensin II (AII) and arginine vasopressin (AVP), both of which are potent stimuli of vasodilatory prostaglandin (PG) production. We studied interactions among these opposing factors in glomeruli. Normal rat glomeruli synthesized PGF2 alpha greater than PGE2 greater than 6-keto-PGF1 alpha = thromboxane (Tx) B2. Rat glomerular epithelial and mesangial cells, although capable of producing these four cyclooxy-genase end products, responded to AVP and AII stimulation with a preferential increase of PGE2, which suggests an intraglomerular feedback system between constrictor and dilator factors. Whole glomeruli, when incubated in AII, decreased in size, with a maximum decrement of surface area at 10(-10) M AII. In these glomerular contraction studies, preincubation with either arachidonate or PGE2 decreased the contractile response to AII, whereas PG inhibition enhanced the glomerular contractile response. Stable endoperoxide analogs also contracted glomeruli. In the acute phase of nephrotoxic serum nephritis (NSN) there were marked increments in glomerular production of TxA2, which correlated temporally with decrements of GFR and filtration fraction. Inhibition of TxA2 synthesis normalized GFR and filtration fraction 1-3 h after induction of NSN. These studies suggest not only an important physiological feedback role of vasodilatory PGs, as modulators of AII-induced glomerular contraction, but also a direct mesangial contractile effect of the arachidonate metabolite TxA2.     

Urinary selenium excretion in selenite-loaded sheep and subsequent Se dynamics in blood constituents

Renal selenium excretion in sheep was measured during intravenous infusion of sodium selenite, and the post-infusion dynamics of Se levels in whole blood, plasma and red blood cells (RBC) were investigated for the next 5 days. The plasma Se level increased almost twenty fold with the infusion of Na2SeO3 (from 0.39 +/- 0.02 to 7.83 +/- 0.33 micromol x L(-1), P < 0.001) compared with the baseline value. The selenium concentration in urine (0.07 +/- 0.02 vs. 18.53 +/- 2.56 micromol x L(-1), P < 0.001), the amount of Se excreted (0.14 +/- 0.07 vs. 21.40 +/- 2.31 nmol x min(-1), P < 0.001) and the renal clearance of Se (0.1 9 +/- 0.03 vs. 3.01 +/- 0.34 mL x min(-1), P < 0.001) were found to be highly significantly elevated during selenite loading. The clearance measurements showed no changes in the urinary flow rate or in the glomerular filtration rate. During and at the end of infusion the highest Se level was attained in plasma, followed by whole blood and RBC. The plasma Se level fell rapidly within 10 min after the end of infusion, but the concentration of Se in RBC was stable up to the fourth hour, when it started to decrease too. On day 5 the Se concentrations in plasma, RBC and whole blood were found to be only slightly but still significantly higher than before the selenite infusion. The large disproportion between the infusion rate of Se (8.76 microg x min(-1)) and its renal excretion rate (1.69 microg x min(-1)) found in clearance measurements suggests low glomerular filtration of infused selenium, which might primarily be caused by the binding of selenite metabolites to blood constituents. The presented results confirm the low bioavailability to ruminants of Se from sodium selenite.     

Modulation of interleukin 2 high-affinity binding by lymphocyte-derived tetrahydrobiopterin: pterins as potential participants in the control of interleukin 2 receptor assembly

In this report, we have examined whether (6R)-tetrahydrobiopterin (H4biopterin) modulates the binding of interleukin 2 to high-affinity sites of the cloned mouse cytotoxic T-lymphocyte clone CTLL-2. Scatchard plot analysis of the equilibrium binding data reveals increased affinity when the cells are exposed simultaneously to interleukin 2 and to the pterin. The Kd values are statistically significantly reduced from 1.4 x 10(-11) M to 0.78 x 10(-11) M interleukin 2. The dissociation kinetics of the ligand were followed at 4 degrees C after equilibrium binding under high-affinity conditions (1.2 x 10(-10) M interleukin 2). In the presence of H4 biopterin, the dissociation rate constant (k-1) decreases from 6.2 x 10(-3) min-1 to 3.0 x 10(-3) min-1 and the half-time for dissociation increases from 106.8 min to 218.0 min. As a third approach interleukin 2 was bound to the surface of cells under high-affinity conditions by incubation in the cold and the internalization kinetics upon warming were determined. Sigmoidal-shaped kinetics of endocytosis in control cells indicate that the internalization rates increase only gradually. The presence of H4 biopterin causes an apparent immediate transition from higher-order kinetics to a linear response so that maximum internalization rates are reached immediately upon warming. The data show that lymphocyte-derived H4 biopterin in vitro at concentrations ranging from 2-8 x 10(-7) M modulates interleukin 2 high-affinity binding and that H4 biopterin potentially participates in the control of interleukin 2 receptor assembly.     

Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells

Specific binding sites for synthetic porcine endothelin (pET), a novel potent vasoconstrictor peptide isolated from the supernatant of cultured porcine endothelial cells, and its effects on cytosolic free Ca2+ concentrations ([Ca2+]i) and phosphatidylinositol (PI) response were studied in cultured rat aortic vascular smooth muscle cells (VSMC). Binding of 125I-labeled-pET to rat VSMC was time- and temperature-dependent and the cell-bound 125I-labeled-pET was resistant to dissociate. Scatchard analysis of binding studies indicated the presence of a single class of high-affinity binding sites: the apparent Kd was 2-4 X 10(-10) M and the maximal binding capacity was 11,000-13,000 sites/cell. The binding was highly specific for pET because neither well-recognized vasoconstrictors, peptide neurotoxins, nor Ca2+-channel blockers affected the binding. pET dose-dependently (10(-9)-10(-7) M) induced a transient and sustained increase in [Ca2+]i in fura-2-loaded cells of which effect was largely dependent on extracellular Ca2+, whereas it had no significant effect on PI response in 3H-myoinositol-prelabeled cells. The present data clearly demonstrates the presence of specific receptors for pET distinct from those of the well-recognized vasoconstrictors and voltage-dependent Ca2+-channels in cultured rat VSMC, and suggest that pET-induced increase in [Ca2+]i is involved in the mechanism of its vasoconstriction.     

Porcine brain natriuretic peptide, another modulator of bovine adrenocortical steroidogenesis

Porcine brain natriuretic peptide (pBNP) significantly inhibited aldosterone production stimulated by an angiotensin II analog and ACTH-stimulated cortisol secretion, together with simultaneously increasing the formation of cGMP in dispersed bovine adrenocortical cells. Receptors for pBNP were identified in bovine adrenal gland using an in vitro receptor autoradiographic technique and studies of 125I-pBNP binding. In vitro receptor autoradiography demonstrated specific binding sites for 125I-pBNP in bovine adrenal cortex. Complete displacement of 125I-pBNP by unlabeled pBNP or human atrial natriuretic peptide (hANP) can take place at these sites. Analysis of 125I-pBNP binding to bovine adrenocortical membrane fractions showed that the adrenal cortex had high-affinity, low-capacity pBNP-binding sites, with a dissociation constant (Kd) of 2.32 +/- 0.33 x 10(-10) M (mean +/- SE) and a maximal binding capacity (Bmax) of 36.7 +/- 1.6 fmol/mg protein. Moreover, the specific binding sites for 125I-pBNP were completely displaced not only by unlabeled pBNP but also by unlabeled hANP. The hANP dose required for 50% inhibition of specific 125I-pBNP binding was almost identical to that for pBNP (IC50 values for hANP and pBNP: 8.5 x 10(-10) and 6.5 x 10(-10) M, respectively). These results suggest that pBNP exerts a suppressive effect on bovine adrenocortical steroidogenesis via a receptor which may be shared with ANP.     

Immortalization of rat kidney glomerular mesangial cell and its coculture with glomerular epithelial cell

Mesangial cell has several key roles in the control of glomerular function: it participates in the regulation of glomerular filtration rate, macromolecular clearance, and as both a source and target of numerous hormones and autocrines. Many of these insights into mesangial cell function have been obtained by studying mesangial cells in culture. However, no suitable cell lines have been established yet. We here reported the immortalization of rat kidney glomerular mesangial cell by transfection of E6 and E7 genes of human papillomavirus type 16 (HPV-16) via electroporation and lipofection. The results showed that only electroporation could transfect the genes to mesangial cells and the transfected cells maintained the viability for longer than 6 months. Fluorescence microscopic observation showed that cellular contractility and phagocytosis, which are the two main phenotypes of mesangial cells, are well maintained after transfection. The coculture of transfected mesangial cells with rat glomerular epithelial cells showed that the growth of mesangial cells was suppressed by epithelial cell, but the growth of epithelial cells was enhanced by mesangial cells. Moreover, an enhancing effect on the phagocytosis of mesangial cell was also observed in coculture. Such results may imply that the glomerular cell-cell interaction plays an important role in the regulation of cell proliferation and differentiation.     

Glomerular mesangium as an effector locus for the tubuloglomerular feedback system and renal sympathetic innervation

To define the effector loci for the tubuloglomerular feedback system, the determinants of the single-nephron glomerular filtration rate (SNGFR) were assessed in Munich-Wistar rats by direct glomerular puncture during perfusion of Henle's loop with isotonic Ringer's solution at rates of 0 and 40 nl/min. At the higher flow rate, SNGFR averaged only approximately 65% that measured during the lower flow rate. Whereas mean glomerular capillary hydraulic pressure was unaffected, both glomerular plasma flow rate and ultrafiltration coefficient Kf were found to decrease significantly in response to increase in loop perfusion rate, thereby accounting for the measured reduction in SNGFR. These changes were associated with increased afferent (RA) and efferent (RE) arteriolar resistances. Based on the close anatomic contact between mesangial cells and these arterioles, a single effector mechanism channeled through mesangial contractility is suggested to account for the observed reduction in Kf and increase in RA and RE. Mesangial contractility appears to be under sympathetic nerve control. In our recent micropuncture study with Munich-Wistar rats, a marked reduction in SNGFR was observed during high-frequency stimulation (5 Hz) of the renal nerve. This reduction in SNGFR was accompanied by a marked fall in Kf and increase in RA and RE. When kidneys were perfusion-fixed during high-frequency stimulation, a marked reduction in the number of open channels was demonstrated together with marked narrowing of afferent and efferent arterioles. These observations are consistent with the view that sympathetic innervation of mesangium may modulate GFR through its ability to regulate mesangial contractility.     

Cystatin C secretion by rat glomerular mesangial cells: autocrine loop for in vitro growth-promoting activity.

Cystatin C, the major inhibitor of the cysteine proteinases found in human and rat body fluids, is particularly abundant in seminal plasma and cerebrospinal fluid. In a precedent report, we have evidenced noteworthy levels of cystatin C in rat kidney cortex. In the present study, we show that rat mesangial glomerular cells produce cystatin C. Immunoprecipitation of extracts of metabolically labeled cells and culture media showed that the synthesized cystatin C is a 15.5 +/- 0.5 kDa protein. The protein was released into the culture supernatant (1.6 +/- 0.26 micrograms/10(6) cells/24 h). Urinary rat cystatin C and PPPR synthetic peptide (5-8 N-terminal sequence of rat cystatin C) increased mesangial cell proliferation. Affinity chromatography on Ultrogel-avidin-biotin-PPPR of extracts of metabolically labeled cells indicate the existence of a PPPR binding protein of 46 kDa. The results described in this work suggest, for glomerular rat mesangial cells in vitro, an autocrine regulation of proliferation by cystatin C.     

Autoradiographic localization of vasoactive intestinal peptide (VIP) binding sites in the human term placenta. Relationship with activation of adenylate cyclase

The presence of vasoactive intestinal peptide (VIP) binding sites and the adenylate cyclase activity in response to VIP were examined in the human term placenta. Slices were used in order to preserve the physicochemical environment and the structural integrity of this heterogeneous organ. 125I-VIP binding to placental slices was saturable. The steady state was reached after 90 min at 37 degrees C and was maintained up to 3 h. Unlabeled VIP was able to compete in a dose-dependent manner with an IC50 value of 5.2 +/- 1.3 x 10(-10) M. Autoradiography and histological analysis showed that VIP binding sites were essentially located on fetal vascularization, especially arteries of stem villi. VIP produced a stimulatory effect on cAMP synthesis at a concentration as low as 10(-10) M. The dose-response curve was monophasic with an ED50 value of 2.9 +/- 1.6 x 10(-9) M. The specificity of the VIP effect was tested with peptides structurally related to VIP such as glucagon, secretin, gastric inhibitory polypeptide and human growth-hormone releasing factor. Only secretin at high concentrations (greater than 10(-6) M) increased cAMP production. Leu-enkephalin or insulin were ineffective. The presence of both VIP binding sites on fetal vascularization and VIP-induced adenylate cyclase activation would seem to suggest a regulatory role of the peptide on fetoplacental blood flow.