Distribution of the sodium/phosphate transporter during postnatal ontogeny of the rat kidney.

Renal phosphate reabsorption via the type II sodium/ phosphate cotransporter (NaPi-2) in the brush border membrane (BBM) of proximal tubules underlies alterations during aging. The ontogeny of NaPi-2 in kidneys from newborn to 6-wk-old rats was investigated. NaPi-2 protein distribution in the kidneys of neonatal, 13-d-old, 22-d-old, and 6-wk-old rats was immunohistochemically analyzed, and NaPi-2 mRNA distribution in neonatal and 6-wk-old rats was analyzed by in situ hybridization. In kidneys of newborn rats, the appearance of NaPi-2 protein and mRNA coincided with the development of the brush border (assessed by actin staining) on proximal tubular cells. NaPi-2 was not detectable in the nephrogenic zone or in the outgrowing straight sections of proximal tubules, which lack a brush border. In 13-d-old suckling rats, strong NaPi-2 staining was seen in the BBM of convoluted proximal tubules of all nephron generations. In contrast, in 22-d-old weaned rats, NaPi-2 staining in the BBM of superficial nephrons was weaker than that in the BBM of juxtamedullary nephrons. Western blotting demonstrated that the overall abundance of NaPi-2 protein in the BBM of 22-d-old rats was decreased to approximately 70% of that in 13-d-old rats. In kidneys of 6-wk-old rats, the internephron gradient for NaPi-2 abundance in the BBM corresponded to that in adult rats. The data suggest that the NaPi-2 system in the kidney is fully functional and possesses the capacity for regulation as soon as nephrogenesis is completed. The manifestation of NaPi-2 internephron heterogeneity immediately after weaning might be related to the change in dietary inorganic phosphate content.

Effects of phosphate intake on distribution of type II Na/Pi cotransporter mRNA in rat kidney.

BACKGROUND: Renal phosphate (Pi) reabsorption is regulated by dietary Pi intake, as well as in other ways. Changes in Pi reabsorption are associated with the modulation of sodium/Pi cotransporter type II (NaPi-2) protein abundance in the brush border membrane (BBM) of proximal tubules (PTs) and of renal NaPi-2 mRNA levels. In this study, we address whether the NaPi-2 protein and NaPi-2 mRNA distribution patterns in the renal cortex vary in parallel with changes of dietary Pi intake. METHODS: We investigated in cryosections of perfusion-fixed rat kidneys by in situ hybridization (ISH) and immunohistochemistry (IHC) the distribution patterns of NaPi-2 mRNA and of NaPi-2 protein one week, two hours, and four hours after changes in dietary Pi intake. RESULTS: NaPi-2 mRNA and NaPi-2 protein were present in PTs exclusively. In rats adapted to one week of high Pi intake, signals for NaPi-2 mRNA and NaPi-2 protein in cortical PTs were weak, except in the convoluted parts of PTs of juxtamedullary nephrons. After one week of low Pi intake, the ISH and IHC signals for NaPi-2 were high in PT segments in all cortical levels. The switch from a chronic high to a low Pi intake within two and four hours induced no increase and a slight increase, respectively, in the NaPi-2 mRNA signal in PTs of midcortical and of superficial nephrons, whereas in the BBM of these nephrons, NaPi-2 protein was markedly up-regulated. Two and four hours after switching from low to high Pi intake, the overall high ISH signal for NaPi-2 mRNA was unchanged, whereas NaPi-2 protein staining was drastically down-regulated in the BBM of PTs from superficial and midcortical nephrons. CONCLUSIONS: The marked changes in NaPi-2 protein abundance in the BBM, following altered dietary Pi intake, precede corresponding changes at the RNA level by several hours. Thus, the early adaptation to altered Pi intake involves mRNA-independent mechanisms. The up- or down-regulation of NaPi-2 protein abundance in the BBM and NaPi-2 mRNA in PT affects mainly midcortical and superficial nephrons.

Enhanced osteopontin expression and macrophage infiltration in MRL-Fas(lpr) mice with lupus nephritis.

MRL-Fas(lpr) mice spontaneously develop a chronic lupus-like renal disease, characterized by immune complex-mediated glomerulonephritis and abundant mononuclear cell infiltration in the interstitium. In the present study we have examined whether the macrophage chemoattractant osteopontin (Opn) could be important in the recruitment of macrophages in this murine model of autoimmune renal injury. We have examined the expression of Opn in the kidney of MRL-Fas(lpr) mice and have correlated Opn synthesis with the degree of macrophage infiltration. Immunofluorescence staining revealed prominent expression of Opn by proximal tubules in MRL-Fas(lpr) mice but not in MRL-++ control mice. Northern blot analysis demonstrated that steady-state transcript levels for Opn mRNA were also significantly increased in MRL-Fas(lpr) kidneys compared with control kidneys. Furthermore, in situ hybridization showed massive Opn mRNA transcripts in proximal tubules in MRL-Fas(lpr) mice but not in controls. The diffuse macrophage infiltration in the kidney of MRL-Fas(lpr) correlated with the enhanced Opn expression. Opn secretion in vitro by cultured renal tubular epithelial cells was upregulated by TNF-alpha and 1,25(OH)2-vitamin D3, whereas no regulation was observed in a control macrophage cell line. We conclude that the enhanced expression of the chemotactic molecule Opn by tubular cells is a prominent feature of murine lupus nephritis and might be promoted by the proinflammatory cytokine environment in MRL-Fas(lpr). The chronic upregulation of Opn could participate in the recruitment of monocytes in the kidney of MRL-Fas(lpr) mice, thereby contributing to the pathogenesis of autoimmune renal disease.

Characteristic matrix and tubular basement membrane abnormalities in the CBA/Ca-kdkd mouse model of hereditary tubulointerstitial disease.

CBA/CaH-kdkd mice develop hereditary tubulointerstitial disease with mononuclear cell infiltration and cyst formation, possibly representing a model of human nephronophthisis. The purpose of the present investigation was to examine the components of the fibrotic changes which typically develop in the kidneys of these mice. By conventional histology, kdkd mice displayed progressive interstitial fibroblast and matrix accumulation. Immunohistological analysis of kdkd kidneys showed marked deposition of fibronectin in the tubulointerstitial space and revealed prominent irregularities for laminin and collagen type IV in the tubular basement membrane (TBM), including thickening, widening and folding. Electron microscopy confirmed the TBM abnormalities and showed marked undulation and thickening in areas of proximal tubular (PT) degeneration. Immunofluorescence staining analysis for the fibronectin receptors VLA-4 and VLA-5 showed no expression on injured proximal tubules, whereas the expression of the laminin receptor VLA-6 was increased and irregular on altered PT. Analysis of RNA derived from kdkd kidneys revealed marked upregulation of steady-state mRNA levels for the fibrogenic growth factor TGF-beta. We conclude that TBM alterations, matrix accumulation and changes in integrin expression together with enhanced TGF-beta production are typical features of kdkd tubulointerstitial disease and suggest that characteristic TBM or matrix alterations could contribute to the pathogenesis of the disease in these mice.

Effects of hypoxia on renin secretion and renal renin gene expression.

Plasma renin activity (PRA) and renal renin mRNA levels were measured in male rats exposed to hypoxia (8% O2) or to carbon monoxide (CO; 0.1%) for six hours. PRA increased fourfold and 3.3-fold, and renin mRNA levels increased to 220% and 200% of control, respectively. In primary cultures of renal juxtaglomerular (JG) cells, hypoxia (lowering medium O2 from 20% to 3% or 1%) for 6 or 20 hours did not affect renin secretion or gene expression. Renal denervation did not prevent stimulation of the renin system by hypoxia. Because norepinephrine increased 1.7-fold and 3.2-fold and plasma epinephrine increased 3.9-fold and 7.8-fold during hypoxia and CO inhalation, respectively, circulating catecholamines might mediate the stimulatory effects of hypoxia on renin secretion and renin gene expression. Stimulation of beta-adrenergic receptors by continuous infusion of 160 microg/kg/hr isoproterenol increased PRA 17-fold and 20-fold after three and six hours, respectively, and renin mRNA by 130% after six hours. In rats with a stimulated renin system (low-sodium diet), isoproterenol did not stimulate PRA or renal renin mRNA further. In summary, both arterial and venous hypoxia can stimulate renin secretion and renin gene expression powerfully in vivo but not in vitro. These effects seem not to be mediated by renal nerves or by a direct effect on JG cells but might be mediated by circulating catecholamines.

Effects of the angiotensin II type-1 receptor antagonist ZD7155 on angiotensin II-mediated regulation of renin secretion and renal renin gene expression, renal vasoconstriction, and blood pressure in rats.

Angiotensin II receptors have recently been subclassified as type-1 or type-2 receptors. The in vitro and in vivo effects of blocking the angiotensin II type-1 receptor with ZD7155, an angiotensin II type-1 selective receptor antagonist, have been studied in angiotensin II-mediated increases in cytosolic calcium in rat mesangial cells, in angiotensin II-induced renal and systemic vasoconstriction, and in angiotensin II-mediated regulation of renin secretion and renal renin gene expression. ZD7155 completely blocked the ability of angiotensin II to elicit an increase in free intracellular calcium concentrations in rat mesangial cells. In isolated perfused rat kidneys, ZD7155 completely abolished the angiotensin II-induced vasoconstriction and increased renin secretion to 700% of baseline levels. Furthermore, ZD7155 decreased systolic blood pressure by 16 mm Hg, increased plasma renin activity 3.7-fold, and stimulated renal renin gene expression 4.2-fold in Sprague-Dawley rats in vivo. Our results suggest that ZD7155 is a potent antagonist of the angiotensin II type-1 receptor, which mediates angiotensin II-induced increases of free intracellular calcium concentrations in (e.g., renal mesangial cells), constriction of the renal and systemic vasculature, and inhibition of renin secretion and synthesis.

Acute hypoxia stimulates renin secretion and renin gene expression in vivo but not in vitro.

This study aimed at examining the influence of acute hypoxia on renin secretion and renin gene expression in the kidney. To this end, male Sprague-Dawley rats were exposed to severe hypoxic stress (8% O2) or to carbon monoxide (0.1% CO) for 6 h, and plasma renin activity (PRA) and renal renin mRNA levels were determined. PRA values increased from 3 to 13 and 10 ng angiotensin I x h(-1) x ml(-1), and renin mRNA levels increased by 120 and 100% during hypoxia and CO, respectively. Lowering the PO2 from 150 to 20 or 7 mmHg in the gas atmosphere of primary cultures of renal juxtaglomerular cells had no influence on renin secretion and renin gene expression after 6 and 20 h. Our findings thus suggest that both arterial and venous hypoxia can be powerful stimulators of renin secretion and renin gene expression in vivo. Because renal denervation did not prevent stimulation of the renin system by hypoxia, the effect could be indirectly mediated via the baroreceptor-macula densa mechanism. Another potential mediator of the effect could be circulating catecholamines, since we found that plasma norepinephrine increased from 0.7 to 1.5 and 2.4 ng/ml and plasma epinephrine increased from 0.3 to 1.4 and 2.7 ng/ml during hypoxia and CO inhalation, respectively.

Effects of hypoxia on growth factor expression in the rat kidney in vivo.

There is accumulating evidence from in vitro studies suggesting that the genes of endothelin-1, PDGF, and VEGF are, like the erythropoietin gene, regulated by oxygen tension and by divalent cations. Hypoxia-induced stimulation of, such as endothelin-1, PDGF or VEGF might be involved in the pathogenesis of acute or chronic renal failure, and in renal "inflammatory" diseases (glomerulonephritis, vasculitis, allograft rejection). Hypoxia (8% O2) for six hours caused a 55-fold/1.6-fold increase of renal erythropoietin/endothelin-1 gene expression, whereas endothelin-3, PDGF-A, PDGF-B, and VEGF gene expression was unchanged. Carbon monoxide (0.1%) treatment for six hours stimulated renal erythropoietin gene expression 140-fold; however, endothelin-1, endothelin-3, PDGF-A, PDGF-B, and VEGF gene expression was not affected. Finally, cobalt treatment (60 mg/kg CoCl2) increased only renal erythropoietin/PDGF-B gene expression 5-fold/1.65-fold. These findings suggest that hypoxia is a rather weak stimulus for renal endothelin-1 gene expression, and that renal PDGF and VEGF gene expression in vivo is not sensitive to tissue hypoxia, in contrast to cell culture experiments. The in vivo regulation of endothelin-1, PDGF, and VEGF differs substantially from that of erythropoietin, suggesting that the basic gene regulatory mechanisms may not be the same.

Endothelins inhibit cyclic-AMP induced renin gene expression in cultured mouse juxtaglomerular cells.

We have recently described that endothelins-1 to -3 equipotently inhibit cAMP stimulated renin secretion from cultured mouse juxtaglomerular cells by a process involving phospholipase C activation. This study examined the influence of endothelin-2 on renin gene expression in renal juxtaglomerular cells. To this end we semiquantitated renin mRNA levels by competitive RT-PCR in primary cultures of mouse renal juxtaglomerular cells after 20 hours of incubation. We found that endothelin-2 (0.1 to 100 nmol/liter) did not change basal renin gene expression. The adenylate cyclase activator forskolin (3 mumol/ liter) increased renin mRNA levels to 400% of the controls and this stimulation was dose-dependently attenuated by ET-2 to 250% of the control value. The effect of ET-2 was mimicked by the ETB-receptor agonist sarafotoxin S6c. The kinase inhibitor staurosporine (100 nmol/ liter) increased renin secretion and renin mRNA levels. Combination of staurosporine with forskolin produced the same effects on renin secretion and renin mRNA levels as did staurosporine alone. In the presence of both forskolin and staurosporine ET-2 had no significant effect on renin secretion and renin gene expression. The phorbol ester PMA (30 nmol/ liter), which was used to stimulate protein kinase C activity, attenuated cAMP stimulated renin secretion and renin mRNA levels. Lowering the extracellular concentration of calcium by the addition of 1 mmol/liter EGTA did not inhibit the effect of ET-2 on cAMP induced renin secretion and renin gene expression. These findings suggest that endothelins inhibit cAMP stimulated renin gene expression by an event that is mediated via ETB receptors. This inhibitory effect may in part involve protein kinase C activation.

Influence of hypoxia on hepatic and renal endothelin gene expression.

This study aimed to investigate the influence of different forms of tissue hypoxia on the expression of the endothelin genes in kidneys and livers. Tissue hypoxia in rats was induced by five different manoeuvres, namely hypoxia (8% O2), functional anaemia (0.1% CO), haemorrhage (haematocrit, hct = 0.12), cobalt treatment (60 mg/kg) for 6 h each and renal artery stenosis (0.2-mm clips) for 2 days. Endothelin-1 (ET-1) mRNA levels in the kidneys were increased by 200% with renal artery stenosis, 70% by hypoxia, 50% by anaemia, 30% by CO, but were not changed by cobalt. ET-3 mRNA in the kidneys decreased during renal artery clipping and cobalt treatment and were not significantly changed under the other conditions. ET-2 mRNA was not detected in the kidneys and livers. The abundance of ET-1 in the livers of normoxic animals was about 15% of that found in the kidney. Hypoxia increased ET-1 mRNA by 200%, haemorrhage by 400%, whilst CO and cobalt did not change hepatic ET-1 gene expression. The abundance of ET-3 mRNA in the livers of normoxic animals was about 6% of that found in the kidneys. The expression of the ET-3 gene in the livers was decreased by CO, but was not changed by any of the other experimental conditions used. These findings suggest that hyoxaemia and tissue hypoxia are moderate stimuli for the expression of the ET-1 gene but not for the ET-3 gene in the kidney and more potent stimuli in the liver, whilst cobalt does not activate ET-1 gene expression in the kidneys nor the livers.

Effects of endothelins on renin secretion from isolated mouse renal juxtaglomerular cells.

This study was performed to examine the effects of endothelin (ET)-1, ET-2, and ET-3 on renin secretion from cultured mouse renal juxtaglomerular (JG) cells. Although different ETs had no consistent effect on basal renin secretion, they equipotently inhibited adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated renin release with a concentration of approximately 3 nM inhibiting 50% of maximal response. ETs did not significantly affect renin release stimulated by the nitric oxide donor sodium nitroprusside (100 microM) or that stimulated by low [2 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] or high (3 mM CaCl2) extracellular calcium. The inhibitory effect of ETs on cAMP-dependent renin secretion was abolished by lowering extracellular calcium concentration to the nanomolar range. However, the action of ETs was not changed by the ETA receptor antagonist BQ-123 (100 nM) and was mimicked by ETB receptor agonists IRL-1620 (1 microM), sarafotoxin S6b (1 microM), and [Ala1,3,11,15]ET-1 (1 microM). All ETs induced calcium oscillations in JG cells that were dependent on extracellular calcium and were associated with prominent calcium-activated chloride currents. These findings suggest that ETs inhibit rather selectively the cAMP-activated pathway of renin secretion through a calcium-sensitive process. The action of ETs on renal JG cells appears to be mediated via ETB receptors and is presumably related to activation of phospholipase C and subsequent events.

Endothelin inhibits cAMP-induced renin release from isolated renal juxtaglomerular cells.

We examined the effects of endothelin (ET)-1, -2, and -3 on renin secretion from cultured mouse renal juxtaglomerular cells. Although different ETs had no consistent effect on basal renin secretion, they equipotently inhibited cAMP-stimulated renin release with an IC50 value of approximately 3 nM. ETs did not significantly affect renin release stimulated by the nitric oxide (NO)-donor nitroprusside (100 microM) or that stimulated by low (2 mM EGTA) or high (3 mM CaCl2) extracellular calcium. The inhibitory effect of ETs on cAMP-dependent renin secretion was abolished by lowering extracellular calcium concentration to the nanomolar range. However, the action of ETs was not changed by the ETA receptor antagonist BQ 123 (100 nM) and was mimicked by the ETB receptor agonists IRL 1620 (1 microM), sarafotoxin S6c (1 microM), and [Ala1,3,11,15]endothelin-1 (4 Ala-ET-1, 1 microM). These findings suggest that ETs selectively inhibit the cAMP-activated pathway of renin secretion through a calcium-sensitive process. The action of ETs on renal juxtaglomerular cells appears to be mediated via ETB receptors.

Endothelium-mediated regulation of renin secretion.

The aim of the present study was to investigate the endothelial influence on renin secretion of isolated juxtaglomerular cells. Specifically the role of nitric oxide (NO) and of endothelin was studied. Coculture of primary cultures of juxtaglomerular cells with aortic and microvascular endothelial cells decreased renin secretion. Inhibition of NO formation by absence of l-arginine or presence of N omega-nitro-l-arginine caused a marked decrease in cGMP accumulation and a reduction in renin secretion in cocultures. Exogenous NO (NO liberators sodium nitroprusside/SIN 1) stimulated the 20-hour renin secretion from juxtaglomerular cells markedly, too. The effect of NO on renin secretion was biphasic: short-time inhibition and long-time stimulation of renin release. NO's stimulatory effect on renin secretion is dependent on extracellular calcium, but independent on cAMP or cGMP accumulation. Endothelin 1, 2, and 3 did not affect basal renin secretion, but inhibited cAMP stimulated renin release to a similar extent. Endothelin's action is not mediated via the subtype A endothelin receptor, but seems to involve calcium mobilization in juxtaglomerular cells that is dependent on extracellular calcium and associated with prominent calcium activated chloride channels. Taken together, coculture of juxtaglomerular cells with endothelial cells inhibits renin secretion despite the stimulatory effect of native NO released from endothelial cells. cAMP stimulated renin secretion is inhibited by all three endothelin isoforms thus contributing to the inhibition of renin secretion in coculture.

Effect of endothelium-derived relaxing factor on renin secretion from isolated mouse renal juxtaglomerular cells.

This study aimed to examine the direct influence of native endothelium derived relaxing factor (EDRF) on renin secretion. To this end isolated mouse renal juxtaglomerular cells were cocultured with bovine aortic endothelial cells which produced and released significant amounts of EDRF as assayed by guanylate cyclase activities which were measured separately in endothelial and juxtaglomerular cells as well as in the cocultures of juxtaglomerular with endothelial cells. EDRF production was blunted in the absence of extracellular L-arginine and in the presence of N omega-nitro-L-arginine (L-NAG; 200 microM). Inhibition of endothelial EDRF production by removal of arginine or addition of L-NAG was associated with a significant decrease of renin secretion from the cocultures while the same regimen had no effect on renin secretion from JG cells alone. Exogenous generation of nitric oxide by the addition of sodium nitroprusside (100 microM) stimulated renin secretion in the cocultures both at normal and inhibited EDRF formation as well as from juxtaglomerular cells alone. These findings suggest that native EDRF released from vascular endothelial cells is a stimulatory signal for renin secretion from renal juxtaglomerular cells.