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.     

Prostaglandin E2 and atriopeptin III oppose the contractile effect of angiotensin II in rat kidney mesangial cell cultures

Effects of vasoconstrictory and of dilatory hormones were studied on the contractile activity of cultured rat kidney mesangial cells. By phase contrast microscopy, a rapid contraction was seen of most cells treated with angiotensin II (10(-6) - 10(-10) mol/L), which was sometimes followed by autonomous relaxation after 10 to 20 min. Prostaglandin E2 and atriopeptin III prevented the contractile effect of angiotensin II in a dose-dependent manner. Angiotensin II, but not atriopeptin III, stimulated prostaglandin E2 synthesis in mesangial cell cultures.     

Renal cyclo-oxygenase and lipoxygenase products in health and disease

Renal glomeruli have cyclo-oxygenase and lipoxygenase enzymes which convert arachidonic acid to prostaglandins, thromboxane and 12-hydroxyeicosatetraenoic acid. Glomerular epithelial and mesangial cells, in culture, also synthesize these arachidonate products. Angiotensin and vasopressin contract mesangial cells and stimulate mesangial synthesis of PGE2. PGE2, in the glomerulus, antagonizes the actions of angiotensin on the mesangium and hence reduces angiotensin-mediated glomerular contraction. Glomerular immune injury (nephrotoxic serum nephritis) augments glomerular production of prostaglandins and thromboxane. Thromboxane reduces glomerular function and inhibition of thromboxane synthesis preserves glomerular filtration rate and renal plasma flow in this disease model. Spontaneously hypertensive rats also have enhanced glomerular prostaglandin and thromboxane synthesis. Although acute inhibition of thromboxane synthesis will vasodilate the hypertensive rat kidney, chronic inhibition does not reduce blood pressure or increase renal blood flow.     

Bidirectional action of extracellular ATP on intracapillary volume of isolated rat renal glomeruli.

Receptors for extracellular nucleotides (P2-purinoceptors) are expressed in renal glomerulus; both on mesangial and endothelial cells. In the present study we have evaluated the potential role of ATP in the regulation of glomerular contraction and relaxation. Using [3H]-inulin we measured the Glomerular Inulin Space (GIS), (that reflects mainly glomerular intracapillary volume), in the presence of ATP and its analogues e.g. 2-methylthio-ATP (P2Y-receptor agonist) and beta,gamma-methylene-ATP (P2X-receptor agonist). Incubation of the intact glomeruli with ATP or 2-methylthio-ATP or beta,gamma-methylene-ATP induced a decrease of GIS in similar magnitude as angiotensin II e.g.: about 10% of the basal value. When glomeruli were precontracted with angiotensin II it was observed that both ATP and 2-methylthio-ATP induced an increase of GIS to the basal value, similarly to atrial natriuretic factor. Furthermore, there was no relaxing effect with beta,gamma-methylene-ATP. We suggest that, these bidirectional changes of the intracapillary volume induced by the extracellular ATP may contribute to regulation of glomerular dynamics.     

Prostaglandin E2 and atriopeptin III oppose the contractile effect of angiotensin II in rat kidney mesangial cell cultures

Effects of vasoconstrictory and of dilatory hormones were studied on the contractile activity of cultured rat kidney mesangial cells. By phase contrast microscopy, a rapid contraction was seen of most cells treated with angiotensine II (10⁻⁶ − 10⁻¹⁰ mol/L), which was sometimes followed by autonomous relaxation after 10 to 20 min. Prostaglandin E₂ and antriopeptin III prevented the contractile effect of angiotensin II in a dose-dependent manner. Angiotensin II, but not atriopeptine III, stimulated prostaglandin E₂ synthesis in mesangial cell cultures.     

Effect of age on contractile response to angiotensin II in rat aorta

The effects of aging on contractile response to angiotensin II and tachyphylaxis to it were investigated using aortic strips from rats aged 1.5, 4 and 22 months. Whether the endothelium was present or not, the contractile response to angiotensin II was greater and tachyphylaxis to it was less in 1.5-month-old rats than in 4- and 22-month-old rats. The differences between 4- and 22-month-old rats were not significant. Removal of the endothelium enhanced angiotensin II-induced maximal contraction and depressed the tachyphylaxis, these endothelial effects being greater in 4- rather than in 1.5-month-old rats. When the contractile force of angiotensin II was adjusted to a similar level for 1.5- and 4-month-old rats, the endothelial effect on the tachyphylaxis was greater in the 4-month-old rats, but no significant difference was noted in the endothelial effect on the contractile force. These results suggest that during growth, the contractile response of rat aorta to angiotensin II decreases while the endothelial effect on it increases.     

Complement dependence of antibody-induced mesangial cell injury in the rat

Intravenous administration of rabbit anti-rat thymocyte serum (ATS) reactive with Thy-1-like antigens present on rat mesangial cells induces almost immediate (1-hr) mesangial cell injury in rats followed by sequential mesangiolytic and mesangial-proliferative/infiltrative lesions. To determine the role of complement in these ATS-induced glomerular lesions, ATS was given to Lewis rats that had been depleted of C3 by cobra venom factor (CVF). CVF treatment prevented the degenerative changes in mesangial cells and accumulation of even the few polymorphonuclear leukocytes (PMN) seen in the glomeruli (2.67 PMN/glomerulus) 1 hr after ATS-treatment in rats not given CVF. In addition, CVF prevented the mesangiolysis and mesangial hypercellularity seen at day 4. Rat C3 and late complement components identified in the mesangial of ATS-treated rats in close association with the deposition of rabbit immunoglobulin G was also absent as a result of CVF treatment. CVF treatment did not affect binding of ATS to glomeruli as studied by immunofluorescence or paired label radioisotope techniques. The depletion of leukocytes and/or PMN by irradiation or treatment with anti-I-MN serum had no effect on the induction of the acute mesangial cell damage or the mesangiolytic lesion. Irradiation did diminish the 4-day proliferative/infiltrative lesion. Complement depletion normalized the ATS-induced increase in mesangial uptake of heat-aggregated human gamma-globulin (655.0 +/- 35.2 micrograms in ATS-treated vs 20.3 +/- 2.9 micrograms/5 X 10(4) glomeruli in ATS plus CVF-treated rats; mean +/- SEM). Small immune deposits present in the mesangial areas of kidneys 4 to 5 days after CVF treatment represented CVF-anti-CVF antibody-C3 complexes. The model of mesangial cell damage induced by ATS in the rat is complement-dependent and may relate, at least in part, to complement-mediated mesangial cell lysis.     

Responsiveness of renal glomeruli to adenosine in streptozotocin-induced diabetic rats dependent on hyperglycaemia level.

Glomerular filtration rate (GFR) in response to adenosine precursor, NAD, and glomeruli contractility in response to adenosine were evaluated in streptozotocin-induced diabetic rats with severe (blood glucose 27.8 +/- 1.2 mmol/L) and moderate hyperglycaemia (18.2 +/- 0.9 mmol/L) compared with nondiabetic (ND)-rats. In anaesthetised rats, basal GFR was greater in moderately diabetic rats compared with severely diabetic rats (p < 0.05) and ND-rats (p < 0.02). Intravenous infusion of 5 nmol x min(-1) x kg(-1) NAD reduced GFR and renal plasma flow (RPF) in diabetic rats but had no effect on these parameters in ND-rats. Moreover, NAD-induced reduction of GFR and RPF was greater in rats with severe diabetes (41% and 30%, respectively) than in with moderate diabetes (25% and 26%, respectively). Theophylline (0.2 micromol x min(-1) x kg(-1) ) abolished renal response to NAD. Isolated glomeruli contraction in response to adenosine, assessed by glomerular 3H-inulin space reduction, was lowered in moderately diabetic-group and enhanced in severely diabetic-group. compared with ND-group (p < 0.05). Adenosine A1-receptor antagonist DPCPX inhibited adenosine-induced glomeruli contraction. This differential response of diabetic renal glomeruli to adenosine suggests that impaired glomerular contractility in response to adenosine could be responsible for hyperfiltration in moderate diabets, whereas, the increased adenosine-dependent contractility of glomeruli in severe diabetes may increase the risk of acute renal failure in this condition.     

In vitro prevention of cyclosporin-induced cell contraction by mycophenolic acid

Nephrotoxicity is a major side-effect of cyclosporin A (CsA), which induces a vasoconstrictive response in vascular smooth muscle and mesangial cells. Mycophenolic acid (MPA) is used in combination with low-dose CsA to reduce nephrotoxicity. We previously demonstrated that MPA affected mesangial cell contractile response to angiotensin II or KCl. Aims of the present study were to evaluate if MPA can prevent CsA-induced contraction of human mesangial and aortic smooth muscle cells (ASMC). Using a morphoquantitative approach, we evidenced that pretreatment with MPA (1 microM) prevented the reduction of cell area induced by CsA within 30 min in both cell types. We then compared the expression of three main cytoskeleton proteins: tubulin, alpha-smooth actin (SMA) and basic calponin, in ASMC and in mesangial cells treated with MPA and/or CsA. CsA alone did not significantly change the expression level of these proteins neither in mesangial cells nor in ASMC. MPA decreased the expression level of tubulin in both mesangial cells and ASMC. Surprisingly, MPA, which stimulated SMA and calponin expression in mesangial cells, exerted an inhibitory effect on both contractile protein expression in ASMC. In conclusion, our results evidenced opposite effects of MPA on calponin and SMA protein expression in ASMC and in mesangial cells, despite similar antiproliferative properties, suggesting that sarcomeric protein expression is controlled by different intracellular mechanisms in mesangial and smooth muscle cells. However, MPA interferes in both cell types with the constrictive properties CsA, which may partially explain the protective effects of MPA against CsA nephrotoxicity.     

Inhibition of chymase protects against diabetes-induced oxidative stress and renal dysfunction in hamsters

Accumulating evidence suggests that the intrarenal renin-angiotensin system may be involved in the progression of diabetic nephropathy. Chymase is a potent local angiotensin II-forming enzyme in several species, including humans and hamsters. However, the pathophysiological role of chymase is not fully understood. Here, we report a causal role of chymase in diabetic nephropathy and the therapeutic effectiveness of chymase inhibition. In the present study, renal chymase expression was markedly upregulated in streptozotocin-induced diabetic hamsters. Oral administration of a specific chymase inhibitor, TEI-F00806, completely ameliorated proteinuria, the overexpression of transforming growth factor-β and fibronectin in glomeruli, and renal mesangial expansion, by normalizing the increase in intrarenal angiotensin II levels in diabetic hamsters independently of blood pressure levels. In contrast, ramipril did not show such sufficient effects. These effects occurred in parallel with improvements in superoxide production and expression of NAD(P)H oxidase components [NAD(P)H oxidase 4 and p22(phox)] in glomeruli. This study showed for the first time that chymase inhibition may protect against elevated intrarenal angiotensin II levels, oxidative stress, and renal dysfunction in diabetes. These findings suggest that chymase offers a new therapeutic target for diabetic nephropathy.     

Nitric oxide (NO) donor 3-morpholinosydnonimine antagonizes cyclosporin A-induced contraction in two in vitro glomerular models

Cyclosporin A induces in vivo a severe nephrotoxicity characterized by a large decrease in renal hemodynamics. The aim of this study is to establish the ability of the known NO donor 3-morpholinosydnomine (SIN-1) to prevent the cyclosporin A-induced contraction by using rat isolated glomeruli and cultured glomerular mesangial cells. Isolated rat glomeruli are obtained from the renal superficial cortex by a sieving method. Mesangial cells are cultured in RPMI 1640 with 15% fetal calf serum. The planar surface area (PSA) of either isolated glomeruli or mesangial cells is assessed using anage analyzer. Each glomerusus or mesangial cell serves as its own control through calculation of the area before any drug incubation and after incubation for 10, 20 and 30 min either in control solution or in control solution with cyclosporin A alone or cyclosporin A and SIN-1. Cyclosporin A (10^–6 mol/L) induces an important time-dependent contraction of either glomerulus or mesangial cell. When pretreated with different concentrations of SIN-1 (10^–4 to 10^–9 mol/L), only a slight size decrease is noted. In conclusion, a direct constrictive effect of cyclosporin A in isolated glomeruli and mesangial cells can be prevented bythe NO donor SIN-1, suggesting an important involvement of the nitric oxide pathway in the cyclosporin A-induced nephrotoxicity.Abbreviations CyA cyclosporin A - SIN-1 3-morpholinosydnonimine - FCS fetal calf serum - PSA planar surface area     

Signaling transduction pathway of angiotensin II in human mesangial cells: mediation of focal adhesion and GTPase activating proteins

Human mesangial cells (HMCs) respond to angiotensin II stimulation, which modulates their physiological activities, i.e., contraction and proliferation. It has been revealed that focal adhesion kinase (FAK) and paxillin participate in the angiotensin II-mediated signaling and cytoskeletal rearrangements at focal adhesion. We investigated the influences of cell adhesion upon angiotensin II effects in HMCs. In adherent cells, both FAK and paxillin were tyrosine phosphorylated by angiotensin II, while the cell detachment completely inhibited the tyrosine phosphorylation of paxillin. Activation of p44/42 mitogen-activated protein (MAP) kinase by angiotensin II was accentuated in suspended cells. Moreover, p190, a member of Rho GTPase activating protein (GAP), and RasGAP were coprecipitated with paxillin in adherent cells and angiotensin II stimulation reduced the formation of paxillin-p190 and paxillin-RasGAP complexes. These results suggest that the formation of focal adhesion complexes accelerated by accumulation of mesangial matrices may inhibit the proliferation of HMCs by modulating MAP kinase activity and be related to mesangial cell depletion.     

Possible involvement of heat shock protein 25 in the angiotensin II-induced glomerular mesangial cell contraction via p38 MAP kinase.

In the rat kidney, mesangial cells (MCs), especially those in the extraglomerular mesangium (EGM) region of the juxtagomerular apparatus, express high amounts of heat shock protein 25 (HSP25). Because MCs are contractile in vivo and HSP25 is known to modulate polymerization/depolymerization of F-actin and to be involved in smooth muscle contraction, it is possible that HSP25 participates in the contraction process of MCs. We analyzed a permanent mouse MC line using Northern and Western blot analyses, and observed that similar to the MCs in the glomerulus, these cells also express high amounts of HSP25 constitutively. Exposure of these cells to angiotensin II (ANG II: 2 x 10(-7) M) evoked contraction and a concomitant increase in HSP25 phosphorylation, while the cytoplasmic fraction of HSP25 was transiently reduced. Because phosphorylation of HSP25 is essential for its actin-modulating function, we suppressed the activity of p38 MAP kinase, the major upstream activator of HSP25 phosphorylation, with the specific inhibitor SB 203580. This maneuver reduced HSP25 phosphorylation dramatically, abolished cell contraction, and prevented the decrease of the cytoplasmic HSP25 content. This suggests that HSP25 might be a component of the contraction machinery in MCs and that this process depends on p38 MAP kinase-mediated HSP25 phosphorylation. The decrease of cytoplasmic HSP25 content observed after ANG II exposure is probably the result of a transient redistribution of HSP25 into a buffer-insoluble fraction, because the whole cell content of HSP25 did not change, a phenomenon known to be related to the actin-modulating activity of HSP25. The fact that this function requires phosphorylation of HSP25 would explain the observation that HSP25 does not redistribute in SB 203580-pretreated cells.     

Interleukin-1 production by mononuclear leukocytes and mesangial cells in experimental nephrotoxic nephritis

We investigated the intensiveness of the cell proliferation, number of mononuclear leucocytes (MNL) and production of interleukin-1 (IL-1) in glomerular cell cultures of rats with nephrotoxic serum (NTS) nephritis and control (C) rats. Five days after intravenous injection of nephrotoxic serum from rabbits the glomeruli were isolated, treated with collagenase and cultured over a period of 20 days. In cultures the number and intensity of mesangial cell proliferation in the NTS group were significantly higher than in the C group. Thus, the intensive mesangial cell proliferation in experimental nephrotoxic serum nephritis is related to infiltration of glomerulus by MNL of bone marrow origin with increased production of IL-1.     

The juxtaglomerular apparatus in the avian kidney

Summary The domestic fowl has two types of glomerulus (mammalian and reptilian type). 30 of each were studied morphometrically in semi thin and PAS-stained sections. The juxtaglomerular apparatus was larger in the mammalian type, but complete in both, containing macula densa, Goormaghtigh (lacis) cells and hilar arterioles. Granular epithelioid cells were occasionally found in the afferent arterioles and within the glomerulus in the mammalian type only.All glomeruli studied had a prominent mesangial cell mass (MCM), which was larger in mammalian type glomeruli. Hilar arterioles often penetrated the mesangial cell mass and regularly ramified within it. There was always extensive direct contact between the Goormaghtigh cell mass and the macula densa on the one side and the MCM on the other. In mammalian type glomeruli, the afferent arterioles were invariably found centrally within the MCM, but in the reptilian type no distinct pattern was found. The close relationship between the MCM and the hilar arterioles, especially in mammalian type glomeruli, suggests that the MCM regulates the glomerular filtration rate.     

The role of the mesangial cell and its matrix in the pathogenesis of diabetic nephropathy.

Mesangial cells are pericyte-like cells which are found the glomeruli of the kidney. It is well known that they have important contractile and synthetic properties regulating the function of the glomerulus. During diabetes the synthesis of various extracellular matrix (ECM) components by mesangial cells are increased. In recent years it has been recognized that degradation of ECM may also be decreased in diabetes, contributing to the process of mesangium accumulation. The major enzymes responsible for ECM degradation are a large group of enzymes collectively known as matrix metalloproteinases (MMPs). The physiology of MMPs is complex and their activity is tightly regulated at many levels. The MMPs are synthesized as proenzymes and require activation via catalytic cleavage to become fully active. In this regard it is of importance that the mesangial cell and its pericellular matrix have a very active plasminogen cascade that can liberate plasmin locally to mediate matrix degradation both directly and indirectly, by activating the MMPs. In addition, the MMPs are regulated by transforming growth factor beta (TGF-beta). There is evidence that each of these pathways regulating the matrix degradation is affected by the diabetic environment and this will be the subject of this contribution.     

Role of 12-lipoxygenase in decreasing P-cadherin and increasing angiotensin II type 1 receptor expression according to glomerular size in type 2 diabetic rats

12-lipoxygenase (12-LO) was implicated in the development of diabetic nephropathy (DN), in which the proteinuria was thought to be associated with a decreased expression of glomerular P-cadherin. Therefore, we investigated the role of 12-LO in the glomerular P-cadherin expression in type 2 diabetic rats according to the glomerular sizes. Rats fed with high-fat diet for 6 wk were treated with low-dose streptozotocin. Once diabetes onset, diabetic rats were treated with 12-LO inhibitor cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) for 8 wk. Then glomeruli were isolated from diabetic and control rats with a sieving method. RT-PCR, Western blotting, and immunofluorescent staining were used for mRNA and protein expressions of P-cadherin and angiotensin II (Ang II) type 1 receptor (AT1). We found that CDC did not affect the glucose levels but completely attenuated diabetic increases in glomerular volume and proteinuria. Diabetes significantly decreased the P-cadherin mRNA and protein expressions and increased the AT1 mRNA and protein expressions in the glomeruli. These changes were significantly prevented by CDC and recaptured by direct infusion of 12-LO product [12(S)-HETE] to normal rats for 7 days. The decreased P-cadherin expression was similar between large and small glomeruli, but the increased AT1 expression was significantly higher in the large than in the small glomeruli from diabetic and 12(S)-HETE-treated rats. Direct infusion of normal rats with Ang II for 14 days also significantly decreased the glomerular P-cadherin expression. These results suggest that diabetic proteinuria is mediated by the activation of 12-LO pathway that is partially attributed to the decreased glomerular P-cadherin expression.     

A re-evaluation of the mesangial cells of the renal glomerulus

Summary The topographical localization of the mesangial cells in renal glomeruli of rats, and their relationships with the structures at the hilus of the glomerulus were studied in this investigation. It was observed that the mesangial cells occupy a parietal position in the wall of the glomerular capillaries, and that they are anatomically continuous with the smooth muscle cells of the tunica media of the glomerular arterioles.This study was supported by a United States Public Health Service Grant AM 08628 (Institute of Arthritis and Metabolic Diseases).     

Synthesis of the glomerular basement membrane in the rat kidney

To study the origin and the formation of the glomerular basement membrane, autoradiographic investigations with³H-proline and³H-leucine have been performed in ultrathin and semithin sections of the glomeruli of 42 male rats. The results of this study indicate that, of the three cell types of the glomerulus, the epithelial cells (=podocytes) synthesize the proline-rich scleroproteins of the glomerular basement membrane. Our autoradiographic studies have yielded no evidence for participation of the endothelial or mesangial cells in the formation of the basement membrane. The mesangial cells appear to be responsible for the synthesis of the mesangial matrix only.     

Circadian Changes in the Surface Area of Renal Glomeruli from Normal Rats

In an attempt to further investigate circadian changes in kidney function, the planar surface area (PSA) (µm 2) of renal isolated glomeruli from normal rats was monitored using a computerized image analyzer method. Eight male Sprague-Dawley (SPRD) rats, aged 12û14 weeks, were entrained to a 12-h light/12-h dark cycle in controlled environmental conditions of temperature (22±2°C) two weeks before the experiments started. Isolated glomerular preparations were obtained, using a mechanical sieving technique, at four different circadian times: 07:00, 13:00, 19:00 and 01:00. It was the finding of the present study that the PSA of the glomeruli varied significantly over the 24-h period, but showed a weak amplitude. The size of the glomeruli reached the highest values at night (21358.59±456.72 µm 2 at 21:17) with an acrophase at 21:39, as do all the other renal parameters, but also blood pressure and many vasoactive compounds involved in the regulation of the mesangial cell physiology, contractile element of the glomerulus. Such chronobiological data not only provided a clear example of complementarity between in vivo and ex vivo experiments, but evidenced that temporal changes do exist in kidney structure and could be correlated with rhythms in renal physiology.