ATP as a mediator of macula densa cell signalling

Within each nephro-vascular unit, the tubule returns to the vicinity of its own glomerulus. At this site, there are specialised tubular cells, the macula densa cells, which sense changes in tubular fluid composition and transmit information to the glomerular arterioles resulting in alterations in glomerular filtration rate and blood flow. Work over the last few years has characterised the mechanisms that lead to the detection of changes in luminal sodium chloride and osmolality by the macula densa cells. These cells are true “sensor cells” since intracellular ion concentrations and membrane potential reflect the level of luminal sodium chloride concentration. An unresolved question has been the nature of the signalling molecule(s) released by the macula densa cells. Currently, there is evidence that macula densa cells produce nitric oxide via neuronal nitric oxide synthase (nNOS) and prostaglandin E₂ (PGE₂) through cyclooxygenase 2 (COX 2)-microsomal prostaglandin E synthase (mPGES). However, both of these signalling molecules play a role in modulating or regulating the macula-tubuloglomerular feedback system. Direct macula densa signalling appears to involve the release of ATP across the basolateral membrane through a maxi-anion channel in response to an increase in luminal sodium chloride concentration. ATP that is released by macula densa cells may directly activate P2 receptors on adjacent mesangial cells and afferent arteriolar smooth muscle cells, or the ATP may be converted to adenosine. However, the critical step in signalling would appear to be the regulated release of ATP across the basolateral membrane of macula densa cells.     

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 normal juxtaglomerular apparatus in the human kidney. A morphological study.

Out of 49 serially studied juxtaglomerular apparatuses, 6 typical variants from two normal human kidneys were reconstructed graphically. The agranular Goormaghtigh cells filled the entire space between the macula densa, the afferent and the efferent arterioles and the glomerular mesangium. The Goormaghtigh cells were always in direct contact with all the other structures. They also invariably continued into the glomerular mesangium. The distal tubule regularly showed widening in the macula densa segment and, at this level, there was considerable variation in the shape of the distal tubule. Direct contact between the macula densa and the hilar arterioles was not always present, the area of contact was usually greater with the afferent than with the efferent arteriole.     

Histochemistry of the juxtaglomerular apparatus in the toad Bufo bufo

Summary An investigation regarding the question of whether there exists a macula densa as part of the juxtaglomerular apparatus in the kidney of amphibians has been carried out. With the aid of a histochemical reaction for glucose-6-phosphate dehydrogenase activity, the presence of a macula densa zone as a specialized part of the distal tubule in the toad Bufo bufo was demonstrated. The functional significance of the high glucose-6-phosphate dehydrogenase activity in the macula densa cells is discussed.     

Basic properties and potential regulators of the apical K+ channel in macula densa cells

These studies examine the properties of an apical potassium (K+) channel in macula densa cells, a specialized group of cells involved in tubuloglomerular feedback signal transmission. To this end, individual glomeruli with thick ascending limbs (TAL) and macula densa cells were dissected from rabbit kidney and the TAL covering macula densa cells was removed. Using patch clamp techniques, we found a high density (up to 54 channels per patch) of K+ channels in the apical membrane of macula densa cells. An inward conductance of 41.1 +/- 4.8 pS was obtained in cell-attached patches (patch pipette, 140 mM K+). In inside- out patches (patch pipette, 140 mM; bath, 5 mM K+), inward currents of 1.1 +/- 0.1 pA (n = 11) were observed at 0 mV and single channel current reversed at a pipette potential of -84 mV giving a permeability ratio (PK/PNa) of over 100. In cell-attached patches, mean channel open probability (N,Po, where N is number of channels in the patch and Po is single channel open probability) was unaffected by bumetanide, but was reduced from 11.3 +/- 2.7 to 1.6 +/- 1.3 (n = 5, p < 0.02) by removal of bath sodium (Na+). Simultaneous removal of bath Na+ and calcium (Ca2+) prevented the Na(+)-induced decrease in N.Po indicating that the effect of Na+ removal on N.Po was probably mediated by stimulation of Ca2+ entry. This interpretation was supported by studies where ionomycin, which directly increases intracellular Ca2+, produced a fall in N.Po from 17.8 +/- 4.0 to 5.9 +/- 4.1 (n = 7, p < 0.02). In inside- out patches, the apical K+ channel was not sensitive to ATP but was directly blocked by 2 mM Ca2+ and by lowering bath pH from 7.4 to 6.8. These studies constitute the first single channel observations on macula densa cells and establish some of the characteristics and regulators of this apical K+ channel. This channel is likely to be involved in macula densa transepithelial Cl- transport and perhaps in the tubuloglomerular feedback signaling process.     

Quantitative measurement of glucose-6-phosphate dehydrogenase in cortical fractions of the rabbit nephron

A quantitative fluorimetric method is described for estimating the activity of glucose-6-phosphate dehydrogenase in isolated fractions of rabbit nephron from the superficial part of the renal cortex: macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. The mean activity in the macula densa region was 2.5 X 10(-18) mol/micrometers 3/min, which was about twice the mean activity of the proximal and distal tubular cells and four times that of the glomeruli. As glucose-6-phosphate dehydrogenase is located in the cytoplasm, the average cytoplasmic enzyme activity of the different tubular cells was calculated: macula densa activity was 4.0 X 10(-18) mol/micrometers 3/min whilst proximal tubular cells showed about a third, and distal tubular cells about a quarter of this activity.     

泰和鸡肾小球旁器的观察

本文用光镜和透射电镜对泰和鸡(乌骨鸡)的肾小球旁器进行了观察。结果表明,泰和鸡的肾小球旁器由球旁细胞、过渡型致密斑、球外间膜细胞和极周细胞所组成。极周细胞在鸟类还属首次报道,它位于肾小囊脏层与壁层上皮移行处,环绕着肾小体的血管极,其结构与哺乳动物的相似。本文还就泰和鸡肾小球旁器的结构与功能的关系作了讨论。     

Structural organization of the juxtaglomerular complex of the kidney and its changes in the modelling of renovascular hypertension]

While studying a normal juxtaglomerular complex (JGC) under light optic and electron microscope, histotopographic dependence in distribution of its components in the renal cortical substance was stated. In normal periglomerular cells, processes of synthesis and excretion of renine granules are taking place that is demonstrated by the presence of young maturing, mature forms and subsequent excretion of their contents. In 157 rats during the development of experimental renovascular hypertension, correlation of the JGC of various classes of granularity was increasing towards the latter. Hyperfunction of the periglomerular cells is dynamically performed transferring from the accumulative type into the secretory one; this is proved by both changing in granularity index and correlation in stages of granule formation and excretion of their contents. Juxtavascular cells (Goormaghtigh's cells) and mesangiocytes are the reserve for renine production. Cellular reaction of macula densa is manifested in metaplasia of epitheliocytes of the distal part of the nephron, in increasing index of macula densa and in ultrastructural changes.     

Studies on the histochemistry of phosphatase enzymes in the juxtaglomerular complex

Summary The localisation of alkaline-, adenosine tri-, glucose-6- and acid phosphatase was studied in the juxtaglomerular complexes of rat, mouse and human kidneys. An alkaline-and adenosine triphosphatase active region was observed between the macula densa, Goormaghtigh cell group and in the interstitium of the latter. The adenosine triphosphatase activity extended into the lateral cell membranes of the macular cells and in properly incubated sections it did not appear among other distal tubular cells. The granular juxtaglomerular cells were ATP-ase negative. The cells of the human macula densa and the granular juxtaglomerular cells of the rat and mouse showed acid phosphatase activity. The glucose-6-phosphatase reaction, accomplished at acid and alkaline pH, was negative in the JG complex of all three species. The possible role of these enzymes in the function of the JG complex also has been discussed.     

Histochemical studies on macula densa cells in the juxtaglomerular complex of rat ren in normal and experimental conditions.

The author pays particular attention to the JGA cells of a rat kidney macula densa in noraml and experimental conditions (a single injection of Unazid, Plive, in a dose of 15 mg/kg). The activity of alkaline phosphatase and adenosine-triphosphatase and acid and neutral mucopolysaccharides are negative in these cells, whereas, acid phosphatase and thiamine pyrophatase in the macula densa cells show an activity exclusively near the nucleus, usually in Golgi's zone. The functional meaning of results obtained from the authors own research and that of others was also discussed in the paper.     

Diffuse glomerulosclerosis--a dysfunction of the mesangium? A morphometric study of the rat's kidney.

The phagocytic capacity of the glomerular mesangial cells in 42 rats was inhibited by the prolonged injection of PVA. Morphometric evaluation of electron microscopic sections showed that this treatment leads to an accelerated thickening of the lamina densa of the glomerular basement membrane and of the mesangial matrix (early glomerulosclerosis). This suggests that a disturbance of lysosomal degradation can lead to glomerulosclerosis, and that the possibility of mesangial dysfunction should always be considered as a possible factor in the pathogenesis of all types of diffuse glomerulosclerosis.     

The glomerular mesangium: a comparative study of mesangial handling of iron dextran complex and endogenous IgG in mice and rats

The function and channel system of the glomerular mesangium in mice and rats was investigated by studying the uptake and transport of intravenously injected iron-dextran particles, and the localization of endogenous IgG. Animals were killed at 30 min, 8 h, 1 and 3 days and 1 and 2 weeks after intravenous injection of iron dextran complex. It was found that the tracer was present maximally in the mesangium of the mouse at one day after injection whereas a maximum was not reached until the third day in the rat. Maximal levels of tracer particles in the extra-glomerular lacis area were found at three days in the mouse and at 2 weeks in the rat. Disappearance of the tracer from the blood as indicated by the measured serum iron levels did not seem to differ significantly in the two species. Using an ultrastructural immunoperoxidase technique, considerable amount of endogenous IgG were localized in the mesangial channel system in the stalk region and in the extraglomerular lacis area of mice, whereas in rats only very scanty endogenous IgG was present in these locations. It is suggested that the difference in mesangial handling of macromolecular material in mice and rats is more likely to be due to a different rate of transport through the mesangial channel system than to primary differences in mesangial phagocytotic activity.     

Immunolocalization of cyclooxygenase-2 in the macula densa of human elderly.

To gain insight into the role of prostanoids in human kidney function, we examined the distribution of cyclooxygenase (COX) 1 and COX-2 by immunofluorescence and immunohistochemistry in human kidneys from adults of various age groups. COX-1 was detected in the collecting ducts, thin loops of Henle and portions of the renal vasculature. COX-2 was detected in the renal vasculature, medullary interstitial cells, and the macula densa. In addition, COX-2 immunoreactivity was noted in afferent arteries and the macula densa of the renal cortex and was more evident in the kidneys of older adults.     

Quantitation of glucose-6-phosphate dehydrogenase activity in cortical fractions of the nephron in sodium-depleted and sodium-loaded rabbits

Summary Glucose-6-phosphate dehydrogenase activity was measured quantitatively in isolated cortical fractions of the nephron in sodium-depeleted and sodium-loaded rabbits. The samples consisted of isolated fractions of macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. In sodium-depleted rabbits enzyme activity was identical to that of normal rabbits. In sodium-loaded rabbits a significant decrease in enzyme activity was found in the macula densa and proximal convoluted tubule. However, using conventional histochemical incubation methods semiquantitative estimation of glucose-6-phosphate dehydrogenase showed a slight decrease in enzyme activity in the macula densa and distal convoluted tubule, and a slight increase in the proximal convoluted tubule during sodiumdepletion. During sodium-load a pronounced decrease in enzyme activity was seen in the macula densa and distal convoluted tubule. These results show that semiquantitative histochemical evaluation of changes in enzyme activity is less reliable than the more precise quantitative method especially when there are only slight changes in enzyme activity. Only where there were marked changes in histochemical enzyme activity might the results of quantitative and semiquantitative methods be in accord.     

An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.     

A major role for the EP4 receptor in regulation of renin

Prostaglandins have been implicated as paracrine regulators of renin secretion, but the specific pathways and receptor(s) carrying out these functions have not been fully elucidated. To examine the contributions of prostanoid synthetic pathways and receptors to regulation of renin in the intact animal, we used a panel of mice with targeted disruption of several key genes: cyclooxygenase-2 (COX-2), microsomal PGE synthases 1 and 2 (mPGES1, mPGES2), EP2 and EP4 receptors for PGE(2), and the IP receptor for PGI(2). To activate the macula densa signal for renin stimulation, mice were treated with furosemide over 5 days and renin mRNA levels were determined by real-time RT-PCR. At baseline, there were no differences in renin mRNA levels between wild-type and the various strains of mutant mice. Furosemide caused marked stimulation of renin mRNA expression across all groups of wild-type control mice. This response was completely abrogated in the absence of COX-2, but was unaffected in mice lacking mPGES1 or mPGES2. The absence of G(s)/cAMP-linked EP2 receptors had no effect on stimulation of renin by furosemide and there was only a modest, insignificant reduction in renin responses in mice lacking the IP receptor. By contrast, renin stimulation in EP4(-/-) mice was significantly reduced by ～70% compared with wild-type controls. These data suggest that stimulation of renin by the macula densa mechanism is mediated by PGE(2) through a pathway requiring COX-2 and the EP4 receptor, but not EP2 or IP receptors. Surprisingly, mPGES1 or mPGES2 are not required, suggesting other alternative mechanisms for generating PGE(2) in response to macula densa stimulation.     

Annexin A1 modulates macula densa function by inhibiting cyclooxygenase 2

Annexin A1 (ANXA1) exerts anti-inflammatory effects through multiple mechanisms including inhibition of prostaglandin synthesis. Once secreted, ANXA1 can bind to G protein-coupled formyl peptide receptors (Fpr) and activate diverse cellular signaling pathways. ANXA1 is known to be expressed in cells of the juxtaglomerular apparatus, but its relation to the expression of cyclooxygenase 2 (COX-2) in thick ascending limb and macula densa cells has not been elucidated. We hypothesized that ANXA1 regulates the biosynthesis of COX-2. ANXA1 abundance in rat kidney macula densa was extensively colocalized with COX-2 (95%). Furosemide, an established stimulus for COX-2 induction, caused enhanced expression of both ANXA1 and COX-2 with maintained colocalization (99%). In ANXA1-deficient mice, COX-2-positive cells were more numerous than in control mice (+107%; normalized to glomerular number; P < 0.05) and renin expression was increased (+566%; normalized to glomerular number; P < 0.05). Cultured macula densa cells transfected with full-length rat ANXA1 revealed downregulation of COX-2 mRNA (-59%; P < 0.05). Similarly, treatment with dexamethasone suppressed COX-2 mRNA in the cells (-49%; P < 0.05), while inducing ANXA1 mRNA (+56%; P < 0.05) and ANXA1 protein secretion. Inhibition of the ANXA-1 receptor Fpr1 with cyclosporin H blunted the effect of dexamethasone on COX-2 expression. These data show that ANXA1 exerts an inhibitory effect on COX-2 expression in the macula densa. ANXA1 may be a novel intrinsic modulator of renal juxtaglomerular regulation by inhibition of PGE(2) synthesis.     

An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system.

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.     

Quantitative measurement of glucose-6-phosphate dehydrogenase in cortical fractions of the rabbit nephron

Summary A quantitative fluorimetric method is described for estimating the activity of glucose-6-phosphate dehydrogenase in isolated fractions of rabbit nephron from the superficial part of the renal cortex: macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. The mean activity in the macula densa region was 2.5×10^–18 mol/m³/min, which was about twice the mean activity of the proximal and distal tubular cells and four times that of the glomeruli. As glucose-6-phosphate dehydrogenase is located in the cytoplasm, the average cytoplasmic enzyme activity of the different tubular cells was calculated: macula densa activity was 4.0×10^–18 mol/m³/min whilst proximal tubular cells showed about a third, and distal tubular cells about a quarter of this activity.     

Morphogenesis of the renal juxtaglomerular apparatus and peripolar cells in the sheep

Summary The morphogenesis of the juxtaglomerular apparatus and peripolar cells was studied in the metanephros of fetal sheep (from 24 to 147 days of gestation) using light and electron microscopy. The first juxtaglomerular apparatus was detected at 45 days of gestation, following constriction of the edges of Bowman's capsule and formation of the vascular pole of the renal corpuscle. Mesenchymal cells gave rise to lacis cells and to smooth muscle and epithelioid cells of the juxtaglomerular arterioles. Epithelioid cells developed only sparse cytoplasmic granulation, first detectable at 92 days. The macula densa developed from tubular cells at the junction of the middle and upper limbs of the S-shaped body of the developing nephron. Peripolar cells arose from epithelial cells in the lower limb of the S-shaped body, at the constricting edges of Bowman's capsule, and formed a cuff around the origin of the glomerular tuft. Cytoplasmic granules were first detected in peripolar cells at 53 days, and remained more prominent than epithelioid cell granulation throughout gestation.