Prostaglandin synthesis in isolated rat kidney glomeruli.

Isolated rat kidney glomeruli converted octatritiated arachidonic acid to several prostaglandins whose production was inhibited by meclofenamate. These were, in order of decreasing abundance, prostaglandin F2 alpha, prostaglandin E2, 6-oxo-prostaglandin F1 alpha, thromboxane B2, and prostaglandin D2. These products were identified by thin-layer chromatography, before and after treatment with potassium hydroxide or sodium borohydride. Prostaglandins F2 alpha and E2 were also determined by radioimmunoassay. The major product made by glomeruli was an unidentified substance(s), whose appearance was partially inhibited by meclofenamate, and was likely to be a hydroxylated fatty acid(s). The specific activity of glomerular fatty acid cyclo-oxygenase (EC 1.14.99.1), based on radioimmunoassay for prostaglandins E2 and F2 alpha, was 10- to 40-fold higher than that of cortical tubular enzyme. These data demonstrate that glomeruli have the capability of synthesizing an array of end-products from arachidonic acid. These prostaglandins may exert important physiologic effects, because renin secretion and arteriolar resistance are regulated by the glomerulus and the afferent and efferent arterioles.     

Binding and degradation of 125I-insulin by renal glomeruli and tubules isolated from rats

Summary Isolated rat renal glomeruli and tubules were shown to exhibit specific binding of ¹²⁵I-insulin and enzymatic degradation of the hormone. Binding to both renal fractions reached a plateau by 1h at 22 °C and increased linearly with increasing protein concentrations. Binding was inhibited in both preparations by insulin and its analogues in the order of relative potency: insulin > despentapeptide insulin > proinsulin, but insulin was ten times more potent in inhibiting ¹²⁵I-insulin binding to glomeruli than that to tubules, indicating a different affinity of receptors for the hormone in the two renal fractions (about 17 versus 210 g unlabelled insulin/l inhibiting 50% of the ¹²⁵I-insulin binding to glomeruli and tubules, respectively). Bound ¹²⁵I-insulin dissociated at a faster rate from tubules than from glomeruli; this release was accelerated by unlabelled insulin in both renal fractions, but to a greater extent in glomeruli than in tubules. Two-thirds of the total bound material released from glomeruli was found to be intact insulin as measured by trichloroacetic acid precipitation, whereas only one-third of the material released from tubules was intact. No direct relationship between binding and degradation of ¹²⁵I-insulin in these renal fractions could be demonstrated, however, because of the release of proteolytic enzymes into the incubation medium resulting in almost all degradation being extracellular. Although differing in their affinity for ¹²⁵I-insulin the high affinity glomerular insulin receptor and the lower affinity tubular insulin receptor have characteristics similar to those of insulin receptors in insulin responsive tissues.     

Endogenous dopamine synthesis and DOPA-decarboxylase activity in rat renal cortex

The dopamine content of either cortical slices or isolated glomeruli prepared from rat kidneys and of their incubation medium was measured at different times both under basal conditions and in the presence of l-DOPA. Production of dopamine from l-DOPA by purified cytos olic proteins of the whole cortex was also measured. Dopamine synthesized by these 3 renal preparations accumulated linearly with time over 60 min. Dopamine produced by the glomeruli was more rapidly released into the incubation medium than that produced by the cortical slices. The dopamine synthetic rate was very low in the absence of l-DOPA but increased rapidly when l-DOPA was added to the incubation medium. No plateau was reached in the range of concentrations studied (0–100 μM) when cortical slices or cytosolic proteins were studied whereas dopamine production by isolated glomeruli reached an equilibrium above 10 μM l-DOPA. Dopamine synthesis in the presence of 100 μM l-DOPA was linearly related to the amount of renal protein. The synthetic rates were 43, 2.2 and 0.2 nmoles · h^?1 · mg^?1 for the cytosolic proteins, the cortical slices and the isolated glomeruli respectively. Dopamine synthesis by the cortical slices in the presence of 100 μM l-DOPA was progressively inhibited by increasing concentrations of α-methyl-DOPA. Cortical slices prepared from rats treated by benserazide, an inhibitor of l-DOPA decarboxylase, synthesized much less dopamine than those from control rats. These results show that rat renal cortex deprived of neuronal supply can synthesize dopamine in vitro from extracellular l-DOPA.     

Increased phosphorylation of focal adhesion kinase in diabetic rat kidney glomeruli

Summary Altered extracellular matrix production by the glomerular mesangium is a feature of diabetes mellitus. Matrix proteins, including fibronectin, via interaction with cell-surface receptors (the integrins) may activate intracellular pathways such as prostaglandin production, shown previously to be stimulated by addition of fibronectin to glomerular cores. However, the signalling pathways involved are unclear. An intracellular tyrosine kinase (focal adhesion kinase), associated with focal adhesions, is known to be phosphorylated after interaction with matrix proteins. We now show for the first time, in glomeruli from diabetic rats, that focal adhesion kinase has increased phosphorylation on tyrosine, when compared with non-diabetic control rats. This phosphorylation was labile and disappeared with extended time of sample preparation or digestion of glomeruli to glomerular cores. Cultured mesangial cells, from non-diabetic rats, plated onto fibronectin also showed increased tyrosine phosphorylation of focal adhesion kinase accompanied by a twofold increase in prostaglandin production. However, it may not be possible to replicate fully the diabetic state in vitro merely by use of raised glucose concentrations, as these conditions (for 3 weeks) resulted in decreased focal adhesion kinase phosphorylation, despite increased fibronectin and prostaglandin levels. A role for increased focal adhesion kinase phosphorylation in kidney glomeruli isolated from diabetic rats, and any linkage to intracellular signalling pathways remains to be determined.Abbreviations FAK Focal adhesion kinase - STZ streptozotocin - DMEM Dulbecco's modified Eagle's medium - HRP horseradish peroxidase - ABTS 2,2-azinodi-3-ethylbenzthiazoline sulfonic acid - PGE₂ prostaglandin E₂ - PMSF phenylmethanesulphonyl fluoride - FCS fetal calf serum - ECL enhanced chemiluminescence - PVDF polyvinylidene difluoride     

Evidence for parathyroid hormone sensitive adenylate cyclase in rat glomeruli

Isolated rat glomeruli contain an adenylate cyclase system. The amount of cyclic AMP formed increased progressively with incubation time. The rate of cyclic AMP formation increased linearly with glomerular protein concentration. This adenylate cyclase system was temperature and pH dependent. There was no evidence for saturation of the enzyme with substrate up to 10^?2 M ATP. Adenylate cyclase was strikingly activated by fluoride (10^?2 M). Purified bovine parathyroid hormone (PTH) and synthetic 1–34 bovine PTH fragment both stimulated adenylate cyclase activity: maximum activity was 3.9 to 5.4 basal activity and k_m close to 10^?7 M. Epinephrine and isoproterenol produced a slight stimulation whereas salmon calcitonin, glucagon, norepinephrine and antidiuretic hormone were inactive. The demonstration of PTH activated adenylate cyclase in glomeruli raises the possibility of a role for this hormone in regulation of glomerular activity.     

Hormone-sensitive adenylate-cyclase in isolated rabbit glomeruli

Adenylate cyclase activity and its hormonal control was measured in glomeruli isolated from collagenase treated rabbit kidneys. Experiments were performed on glycerol treated frozen glomeruli since the freezing procedure was shown to greatly enhance adenyl-cyclase activity. It was found that cAMP generation was linear 1) as a function of time up to 60 min and 2) as a function of the number of glomeruli up to 50 per sample. All further determinations were made using samples of ten glomeruli each, incubated for 30 min at 30 °C. Under such conditions, the mean control adenylate cyclase activity was found to be equal to 0.65 ± 0.04 SEM pmoles/10 gl/30 min. Glomerular adenylate cyclase was stimulated by PTH. From the mean dose response curve, an apparent k_m value (2.3 × 10^?7 M) and a maximal stimulation ratio (V_max equal to 272% of the control) were calculated. PTH stimulation up to 5-fold could be observed in the most sensitive preparations. Low concentrations of arginine vasopressin produced a small but significant stimulatory effect with a mean value equal to 139 per cent of that measured under the control conditions. No effect on adenylate cyclase activity was obtained either with angiotensin II or isoproterenol.     

PGE2 binding sites and PG-stimulated cyclic AMP accumulation in rat isolated glomeruli and glomerular cultured cells

[3H]PGE2 specifically bound to isolated glomeruli. The KD value and the number of sites were 80 nM and 528 fmoles/mg respectively. PGE1 and PGE2 resulted in equipotent inhibition of binding whereas PGI2 was markedly less active. It was not possible to demonstrate specific receptors for PGE2 in glomerular mesangial and epithelial cultured cells. PGE1, PGE2 and PGI2 (0.1-100 microM) stimulated cyclic AMP concentration both in isolated glomeruli and glomerular cultured cells. Basal cyclic AMP in epithelial cells was greater than in mesangial cells or glomeruli. The cyclic AMP accumulation in the presence of PGs was greatest in mesangial cells. Maximum stimulation was in the range 300-1400%. For the three preparations, PGE2 and PGE1 produced a greater effect than PGI2. ED50 values were identical for PGE1 and PGE2 (5 microM for epithelial cells and glomeruli, 20 microM for mesangial cells). ED50 value for PGI2 were lower than those for PGE1 or PGE2 (0.2, 2 and 5 microM for glomeruli, epithelial cells and mesangial cells, respectively). The effects of the three PGs were not additive when tested at maximally effective concentrations. These results demonstrate that PGE1, PGE2 and PGI2 stimulate glomerular and cellular cyclic AMP. A relationship between [3H]PGE2 binding sites and this biological effect has not been established. The physiological events secondary to the increase in glomerular cyclic AMP are also yet to be determined.     

Identification and characterization of leukotriene C4 receptors in isolated rat renal glomeruli

The immediate reduction of renal blood flow and glomerular filtration rate in response to intravenous infusion of leukotriene C4 in the rat prompted an analysis of isolated rat renal glomeruli for the presence of specific receptors for leukotriene C4. Specific binding of [3H]leukotriene C4 to glomeruli increased in a time-dependent manner, reached equilibrium after 60 minutes of incubation at 4 degrees C, and was 80% reversible upon addition of excess unlabeled leukotriene C4 at equilibrium. Specific binding of [3H]leukotriene C4 to glomeruli increased in a dose-dependent manner, approaching saturation at concentrations of 40-60 nM. Inhibition of binding of [3H]leukotriene C4 with increasing concentrations of unlabeled leukotriene C4 was dose dependent. The equilibrium dissociation constant for [3H]leukotriene C4 binding to glomeruli, calculated from saturation and competitive binding-inhibition studies, was 25 +/- 7 nM and 35 +/- 16 nM (mean +/- SEM), respectively, and glomerular leukotriene C4 receptor density was 8.5 +/- 1.5 and 9.0 +/- 3.0 pmol/mg protein, respectively. The other natural vasoactive sulfidopeptide leukotrienes, leukotriene D4 and leukotriene E4, the chemotactic agent, leukotriene B4, and the sulfidopeptide leukotriene antagonist, FPL 55712, competed for the receptor at concentrations 2-3 orders of magnitude higher than the homoligand, leukotriene C4. The binding and specificity characteristics of the glomerular leukotriene C4 receptor are similar to those previously reported for the DDT1 nonvascular smooth muscle cell line derived from hamster vas deferens, for guinea pig ileum smooth muscle, and for a subcellular fraction of rat lung homogenate, and represent the first characterization of such a receptor in a vascular tissue.     

Angiotensin II receptors in human isolated renal glomeruli

125I-Labeled angiotensin II ([125I]A II) binds specifically to glomeruli isolated from human kidneys that were obtained at nephrectomy or early autopsy. Equilibrium was reached after 30 min, and specific binding represented more than 90% of the total binding. Dissociation after dilution with the addition of an excess of unlabeled hormone was more rapid than after dilution alone. The effect increased as a function of the A II concentration. The Scatchard plot derived from saturation experiments was curvilinear, with an upward concavity. Two groups of receptor sites could be defined by the Kd values (0.1 and 2 nM, respectively) and the number of receptor sites (40 and 300 fmol mg glomerular protein-1, respectively). Alternatively, binding could be considered to follow a negative cooperative type of hormone-receptor interaction. [Asn1, Val5]A II, [Asp1,Ile5]A II, [des, Asp1,Ile5]A II, [Sar1, Ala8]A II, and [Sar1, Ile8]A II were all equally effective as competitive inhibitors of [125I]A II binding. Both calcium and magnesium (0.5-5 mM) produced an increase in [125I]A II specific binding, whereas guanylylimidodiphosphate, an analog of GTP, inhibited it. Degradation of the [125I]A II present in the incubation medium was estimated by three different techniques. It increased linearly with time and reached 20% at 30 min. Specific binding of A II to human glomeruli at plasma concentrations observed in man under physiological conditions and during the iv administration of A II demonstrates that human renal glomeruli include target cells for A II and thus suggests a role for A II in regulation of the glomerular filtration rate in man.     

Prostaglandin synthesis and release by subpopulations of rat interstitial macrophages

Recent data indicate that interstitial macrophages are not functionally homogeneous, but are heterogeneous with several subpopulations that differ both morphologically and functionally. Furthermore, interstitial macrophages are believed to be precursor to alveolar macrophages, which have recently been shown to be heterogeneous in their ability to synthesize and release prostaglandins. Considering the apparent importance of prostaglandin synthesis and release in inflammatory and immune responses, the current study was undertaken to determine if interstitial macrophage subpopulations differ in their ability to synthesize and release prostaglandin (PG) E, PGI2, and thromboxane (Tx) A2 after stimulation by calcium ionophore A23187, zymosan, or aggregated IgG. Interstitial macrophages were harvested and separated into 18 density-defined fractions. Density-defined interstitial macrophages (DD-IM) showed marked heterogeneity in prostaglandin synthesis and release. Maximal PGE synthesis and release was seen as a single broad peak after calcium ionophore A23187 and zymosan stimulation. In contrast, no peak in PGE synthesis was seen after aggregated IgG stimulation. PGI2 synthesis also was seen as a single broad peak generated by the lower density interstitial macrophage subpopulations after all stimuli. Similarly, TxA2 synthesis and release was maximal from a broad range of various DD-IM after calcium ionophore A23187, zymosan, and aggregated IgG stimulation. Furthermore, the synthesis and release of TxA2 correlated with the presence of zymosan and IgG receptors on DD-IM. The results demonstrate that DD-IM are heterogeneous in ability to synthesize and release prostaglandins, which are dependent on the stimuli.     

Extraction of erythropoietin from isolated renal glomeruli of hypoxic rats

We investigated whether erythropoietin (Ep) can be extracted from renal glomeruli of hypoxic rats. Hypoxia was induced in a hypobaric chamber at 0.42 atmospheres for usually 6 h. Glomeruli were isolated with a sieving technique from kidneys that were flushed free of blood. Ep activities in glomeruli homogenates were determined in the fetal mouse liver cell assay and in the hypoxia exposed mouse assay. We found in vitro erythropoietic activity of glomerular extracts, which increased about 25-fold during hypoxia. Incubation of the extracts with anti-Ep serum abolished the activity. Its binding characteristics on Wheat germ lectin. Ricinus communis lectin and DEAE-cellulose suggested that glomeruli contained Ep precursor(s) that lacked terminal sugar residues. Such an incomplete carbohydrate portion seems also compatible with the finding that glomerular extracts did not stimulate erythropoiesis in hypoxia exposed mice.     

Amino acids release somatostatin-like immunoreactivity from isolated rat glomeruli

We reported recently the presence of somatostatin-like immunoreactivity (SLI) in the glomerulus of rat kidney. In the present study, we examined factors affecting SLI release from isolated rat glomeruli using a perifusion system. Perifusate containing a mixture of essential amino acids stimulated SLI release, while other hormonal agents such as parathyroid hormone, vasopressin, angiotensin II, bradykinin, epinephrine, PGE2, known to have direct actions on the glomerulus, had no discernible effect on SLI release. Addition of somatostatin to the perifusate did not affect either basal or angiotensin II-stimulated PGE2 release from isolated glomeruli. Our preliminary results demonstrate the stimulatory effect of mixed amino acids on somatostatin release from isolated glomeruli. Further studies are needed to elucidate the possible physiological significance of the present findings.     

Lesions of renal glomeruli in mice with virus-induced diabetes mellitus-like disease

Summary Encephalomyocarditis (EMC) virus infected DBA/2 mice develop a diabetes mellitus-like disease. Many animals survive the acute viral infection and exhibit hyperglycemia and glycosuria for varying periods thereafter. Accumulations of homogeneous, electron dense, basement membrane-like material are observed in the mesangium of the glomerulus of these animals as early as three months after inoculation. The peripheral capillary basement membranes are not affected. Since the alterations are not found in uninfected animals, it is assumed that the abnormal metabolic state or the virus infection, or both processes, are responsible for the glomerular changes. Further investigation will be required to elucidate the pathogenesis of this obscure lesion.     

大黄对糖尿病大鼠肾小球形态改变的影响及定量分析

用先进的图像分析系统，观察了中药大黄提取物Ⅰ号对链脲菌素（ＳＴＺ）所致糖尿病肾病大鼠模型肾小球形态学的影响，结果提示，在病程第三天糖尿病组大鼠肾小球干均体积较大黄提取物Ⅰ号治疗组大鼠明显增大（Ｐ＜０．０１）；病程的第三、七和第１４天糖尿病组大鼠肾小球系膜区平均面积较治疗组扩大，有统计学差异（分别为Ｐ＜０．０１．Ｐ＜０．０１和Ｐ＜０．００１）．本研究在实验性大鼠的体内进一步证实大黄提取物Ⅰ号抑制肾脏肥大的作用。     

Bothrops moojeni snake venom-induced renal glomeruli changes in rat

The venom of Bothrops moojeni has potent proteolytic and phospholipase A2 activities. In previous work, we showed that intravenous injection of this venom in rats decreased creatinine clearance and caused tubular dysfunction and histopathological changes with no alterations in blood pressure. The current study used scanning and transmission electron microscopy to assess the ultrastructural changes caused by B. moojeni venom (0.4 mg/kg i.v.) in rat renal glomeruli and correlated these alterations with the severity of proteinuria 5 hours, 16 hours, and 48 hours after venom injection. The changes included mesangiolysis, glomerular microaneurysms, and glomerular basement membrane (GBM) abnormalities. In addition, there was a reduction in the number and width of podocyte pedicels, which caused a reduction in the number of filtration slits. Electron-dense amorphous material, which may be proteinaceous in origin, was found in the pedicels. The severity of the ultrastructural abnormalities correlated with the level of proteinuria. These morphophysiological changes were attributed to biochemical and physiological disturbances in the components of the GBM and mesangial matrix as well as in cytoskeleton-associated proteins of podocytic processes, and could account for the breakdown of optimal glomerular filtration barrier functioning. These results, together with the absence of appreciable glomerular fibrin deposits, support the hypothesis of a direct activity of B. moojeni venom on rat kidneys. Proteolytic activity of the venom on renal glomeruli could then contribute to the onset of acute renal failure, and would explain the clinical manifestations of renal injury after bites by this and other Bothrops species.     

尿中前列腺素变化在肾移植排异反应中的诊断价值

为了探讨肾移植术后尿中前列腺素的变化及其临床意义，我们测定了３４例肾移植术后患者、１６例尿毒症患者尿中前列腺素的变化。结果表明，尿中前列腺素的变化，尤其是ＴＸＢ２的变化是监测肾移植早期排异反应的较好指标。