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Although liposome-encapsulated clodronate has been used as a means to deplete macrophages from certain tissues, target leukocyte subtypes within the kidney are not clearly known under normal and pathologic conditions. The present study was therefore conducted to examine the effects of liposome clodronate on renal infiltrating cell type following unilateral ureteral obstruction (UUO) and tried to correlate these changes to the mechanisms of early development of renal fibrosis. Renal infiltrating leukocyte subtypes and counts were determined by using multicolor flow cytometric analysis of cell suspensions from obstructed kidneys. UUO for 5 days elicited renal tubular apoptosis and renal fibrosis and showed 4-fold increase in renal leukocytes including monocytes/macrophages, dendritic cells, and T-cells. Repeated administration of liposome clodronate selectively depleted F4/80+ monocytes/macrophages and F4/80+ dendritic cells but not F4/80(-) dendritic cells or other cell types in both obstructed and non-obstructed kidneys. Tubular apoptosis and renal fibrosis were also significantly attenuated by liposome clodronate. Increased gene expression of TNF-alpha and TGF-beta observed in obstructed kidneys were markedly attenuated by depletion of renal mononuclear phagocytes. These findings suggest that F4/80+ monocytes/macrophages and/or F4/80+ dendritic cells play a pivotal role in the development of obstruction-induced tubular apoptosis and renal fibrosis, possibly through TNF-alpha and TGF-beta dependent mechanisms.

Objectives: Renal interstitial inflammation is closely related to the progressive renal fibrosis. It has been reported that heme oxygenase-1 (HO-1) induction attenuated renal fibrosis in obstructive nephropathy. To elucidate the antifibrogenic mechanisms of HO-1, we examined the effect of HO-1 induction on renal interstitial inflammation., Methods: Adult male rats underwent unilateral ureteral obstruction (UUO). The rats were pretreated with cobalt protoporphyrin (CoPP, a potent HO-1 inducer; 15 or 50 mg/kg) subcutaneously on the day -6 and -1 before UUO. Sham-operated rats served as controls. Renal interstitial fibrosis, macrophage and T cell infiltration were immunohistochemically assessed on the day 5 after UUO. Gene expressions of HO-1 and profibrogenic molecules were determined by real-time reverse transcriptase-polymerase chain reaction., Results: CoPP dose-dependently induced HO-1 activity, protein, and messenger RNA (mRNA) expression in the renal cortices. CoPP significantly attenuated the renal fibrosis in a dose-dependent manner. Gene expressions of transforming growth factor-beta and extracellular matrix proteins were upregulated in UUO and were attenuated by CoPP. CoPP markedly inhibited T cell infiltration. Unexpectedly, it enhanced macrophage influx dose dependently. Double immunostaining of macrophage and HO-1 showed that CoPP elicited HO-1 overexpression in infiltrating macrophages, whereas UUO alone did not., Conclusions: HO-1 induction protected against the renal interstitial fibrosis in rat obstructive nephropathy. It is suggested that inhibition of T cell influx is, at least in part, involved in the protection. Increased macrophages that overexpress HO-1 may play an important role in attenuating renal fibrosis.

We previously reported that pyrrolidine dithiocarbamate blocked nuclear factor-kappaB (NF-kappaB) activation and attenuated interstitial inflammation and tubulointerstitial fibrosis in the rat obstructive nephropathy. Since pyrrolidine dithiocarbamate is an anti-oxidant and possesses additional biological properties, present experiment was conducted to clarify further the role of NF-kappaB in the development of tubulointerstitial fibrosis in obstructed kidney using a proteasome inhibitor that blocks NF-kappaB through stabilizing IkappaB, an endogenous inhibitor of NF-kappaB. At 5 days following unilateral ureteral obstruction (UUO) in rats, obstructed kidney exhibited tubulointerstitial fibrosis that was associated with macrophage infiltration. UUO decreased renal cortical IkappaB protein contents with concomitant increases in NF-kappaB DNA-binding activity and gene expression of monocyte chemoattractant protein-1. Administration of PSI, N-benzyloxy-carbonyl-Ile-Glu (O-t-Bu)-Ala-leucinal, a proteasome inhibitor, (3 mg/kg/day, s.c., b.i.d) to UUO rats inhibited proteasome activity and attenuated the changes in IkappaB content, NF-kappaB activity and MCP-1 mRNA expression observed in UUO rats. PSI also decreased macrophage influx and attenuated the development of fibrosis. Furthermore, up-regulated gene expression of pro-fibrogenic molecules observed in the obstructed kidney was attenuated by PSI. These results further support the notion that NF-kappaB plays an important role in the development of renal fibrosis in the obstructive nephropathy.

Background: We have previously shown that correction of hypomagnesemia by magnesium (Mg) supplementation ameliorates chronic cyclosporine A (CsA) nephropathy via inhibiting gene expression of fibrogenic molecules. Experiments were conducted to further elucidate upstream mechanism of the beneficial effects upon CsA nephrotoxicity., Methods: CsA (15 mg/kg/day, subcutaneous [SC]) was administered daily to rats maintained on low sodium diet for 7, 14, and 28 days. Because blockade of renin-angiotensin system improves chronic CsA nephropathy, the effects of Mg supplementation and those of angiotensin-converting enzyme inhibitor (ACEI) were compared on renal function, renal histology, mononuclear cell infiltration, and gene expression profile., Results: CsA induced a decline in glomerular filtration and developed characteristic striped fibrosis that were mostly evident at day 28. Mg attenuated CsA-induced impaired renal function, whereas ACEI did not. Interstitial inflammation as evidenced by monocyte/macrophage infiltration preceded the renal fibrosis and increased progressively with the CsA treatment period. Concomitantly, CsA markedly up-regulated expression of chemoattractant proteins, osteopontin, and monocyte chemoattractant protein-1. These changes were abolished by Mg but were only partially affected with ACEI. CsA promoted renal mRNA expression of fibrogenic molecules and extracellular matrices that were almost completely abolished by Mg but partially suppressed by ACEI. Similarly, CsA-induced chronic fibrotic lesion was markedly attenuated by Mg supplementation but was partially attenuated by ACEI., Conclusion: Mg supplementation abolished CsA-induced precedent interstitial inflammation possibly via inhibition of chemoattractants expression and consequently attenuated tubulointerstitial fibrosis. In this protective mechanism, factors independent of the renin-angiotensin system appears to be mainly involved.

To examine the mechanism of nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a recently developed type II diabetic model, we compared the long-term effect of angiotensin-converting enzyme (ACE) inhibitor (imidapril, 1 mg/kg/day), calcium channel blocker (amlodipine, 10 mg/kg/day), and insulin (5-10 U/kg/day) on nephropathy of OLETF rats. Both imidapril and amlodipine, but not insulin, significantly reduced blood pressure of OLETF rats. Imidapril treatment significantly decreased urinary albumin excretions and improved glomerulosclerosis of OLETF rats, while amlodipine failed to improve nephropathy of OLETF rats despite lowering of blood pressure. Insulin treatment, which significantly decreased HbA1c throughout the treatment period, did not ameliorate nephropathy of OLETF rats. Serum ACE activity in OLETF rats was significantly lower than that in genetic control nondiabetic Long-Evans Tokushima Otsuka (LETO) rats. However, glomerular and aortic ACE activities in OLETF rats were significantly higher than those in LETO rats, and were significantly decreased by treatment with imidapril. Furthermore, immunohistochemical analysis of ACE in the kidney using specific antibodies indicated greater ACE immunostaining in the glomeruli and renal vessels of OLETF rats than in those of LETO rats. These observations demonstrate that ACE is involved in the development of nephropathy of OLETF rats and provide evidence that intrarenal ACE rather than circulating ACE may play an important role in nephropathy of this type II diabetic model.

Background: Hypomagnesemia is a common finding of cyclosporine (CsA)-treated patients and has been proposed as both a cause and a consequence of CsA-induced nephrotoxicity. This experiment was conducted to elucidate the role of hypomagnesemia in the pathogenesis of chronic CsA nephropathy., Methods: CsA (15 mg/kg/day subcutaneously) was administered to rats maintained on a low-sodium diet for 1, 2, and 4 weeks, and the effects of magnesium (Mg) supplementation on renal function, renal histology, and renal gene expression profile of fibrogenic molecules and vasoconstrictors was examined., Results: CsA elicited hypomagnesemia and induced a progressive decline in glomerular filtration. At 28 day, renal tubular atrophy and cortical striped interstitial fibrosis were evident with CsA treatment. Dietary supplementation of Mg ameliorated CsA-induced hypomagnesemia and almost completely abolished CsA-induced chronic fibrotic lesions. Neither CsA nor Mg supplementation affected blood pressure. Renal cortical mRNA of transforming growth factor beta, plasminogen activator inhibitor (PAI)-1, and extracellular matrix started to increase at 14 days and elevated further at 28 days. In contrast, the increase in mRNA of tissue inhibitor of matrix metalloproteinase-1 and renin was evident early at 7 days and reached peak at 14 days. These mRNA increases, except that of renin, were almost abolished when hypomagnesemia was corrected. Magnesium supplementation also improved glomerular dysfunction, at least in part, through inhibition of up-regulated mRNA of endothelin-1., Conclusion: CsA-induced hypomagnesemia contributes to chronic renal fibrotic lesions seen during CsA treatment through up-regulation of fibrogenic molecules, most notably early activation of tissue inhibitor of matrix metalloproteinase-1 expression.

In an investigation of the involvement of prostanoids in the pathogenesis of nephropathy in type 2 diabetes, we repeatedly measured the urinary excretion of prostanoids in both diabetic and healthy rats as the rats aged. Seven rats of the Otsuka Long-Evans Tokushima Fatty strain were used as rats with a model of type 2 diabetes and seven rats of the Long-Evans Tokushima Otsuka strain were used as rats without diabetes. Thromboxane (TX) B2 and 6-keto-prostaglandin (PG) F1alpha, the amounts of which reflect renal production of TXA2 and PGI2, respectively, and PGE2 in urine collected in metabolic cages were assayed when rats were 14, 30, 46, and 54 weeks old. Plasma glucose and urinary protein excretion also were measured periodically. The mean plasma glucose concentration of the diabetic rats was higher than that of the healthy rats throughout the study. At 30 weeks and later, urinary protein excretion by the diabetic rats was greater than that of the healthy rats, and it increased with age. Urinary excretion of TXB2 by the diabetic rats was higher than that of the healthy rats at 14 weeks (52.4+/-23.5 vs. 27.0+/-2.6 ng/day; mean +/- SD, P = .015) and the difference continued to the end of the experiment. Urinary excretion of 6-keto-PGF1alpha by the diabetic rats was high at 14 weeks (52.3+/-12.8 vs. 26.9+/-4.6 ng/day; mean +/- SD, P<.001) but decreased with age and was the same as that of the healthy rats at 54 weeks. The urinary excretion of PGE2 by the two groups of rats was not significantly different. These results suggest that altered renal production of TXA2 and PGI2 is involved in the pathogenesis of diabetic nephropathy in rats with type 2 diabetes.

Direct effects of adenosine A2 receptor activation on renal function were examined in dogs. When renal perfusion pressure was maintained constant at 100 mmHg, renal administration of a selective A2 receptor agonist, CGS 21680C (sodium salt of CGS 21680, 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamido adenosine) (1 microg x kg(-) x min (-1)), although it decreased blood pressure by 20 mmHg, increased renal blood flow and renin release, whereas glomerular filtration rate and urine flow were unaffected. These results suggest that stimulation of renal A2 receptor led to both afferent and efferent arteriolar dilatation, whereas renal A2 receptor plays a minor role in urine formation. CGS 21680C induced renin release and tachycardia that were blocked by propranolol, indicating these effects of A2 receptor stimulation appeared to be indirect.

FK506, a major immunosuppressive agent, often causes nephrotoxicity accompanied by renal vasoconstriction. It is recognized that endothelin (ET) plays a role in the cyclosporin A-induced nephrotoxicity, but the involvement of ET in the FK506-induced renal dysfunction is still poorly understood. We elicited nephrotoxicity by daily administration of FK506 in spontaneously hypertensive rats, and we examined the renal gene expression of ET and its receptors and the effects of an ET receptor antagonist on FK506-induced renal dysfunction. FK506 administration (4 mg/kg/day, i.m.) for 14 days induced nephrotoxicity, including a renal vasoconstriction and a decrease in glomerular filtration rate. The renal dysfunction was accompanied by an increase in ET-1 mRNA levels, while ET(B)-receptor mRNA was unaffected. Continuous administration of an ET(A)/ET(B) antagonist, TAK-044 (3 mg/day, s.c.), which effectively blocked systemic and renal vascular responses to exogenously administered ET-1, partially attenuated the FK506-induced renal vasoconstriction. However the reduced glomerular filtration rate were not affected by TAK-044. Thus, although enhanced gene expression of ET-1 in the kidney is involved in the renal vasoconstriction, ET does not play a major role in the FK506-induced renal dysfunction.

1. Endothelin (ET) is a potent vasoconstrictor peptide which has been shown to have an important role in the regulation of systemic and renal haemodynamics. In order to elucidate the role of endogenous ET in the kidney, we examined the effects of ET receptor antagonists on systemic and renal vasculature in normotensive anaesthetized rats. 2. Intravenous injection of a selective ETA receptor antagonist, FR139317 (0.5 mumol kg-1, for 20 min) induced a very small fall in blood pressure. Similarly, a non-selective ETA/ETB receptor antagonist, TAK-044 (12.5 mumol kg-1, for 20 min) slightly decreased blood pressure. A selective ETB receptor antagonist, BQ-788 (0.5 mumol kg-1, for 20 min) had no effect of blood pressure. 3. FR139317 and TAK-044 did not affect renal blood flow or calculated renal vascular resistance. In contrast, BQ-788 significantly reduced renal blood flow by 18.2 +/- 2.4% and increased renal vascular resistance. Furthermore, the renal vascular action of BQ-788 was not observed when combined with FR139317. 4. Pretreatment with a nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 37 mumol kg-1, i.v.) and a cyclo-oxygenase inhibitor ibuprofen (44 mumol kg-1, i.v.) completely abolished the BQ-788-mediated renal vasoconstriction. 5. These results indicate that activation of ETB receptors by endogenous ET acts as a physiological brake for the ETA-mediated renal vasoconstriction; this effect appears to be mediated by stimulation of NO and/or vasodilator prostaglandin(s) release.

1. The present study was conducted in order to elucidate the in vivo contribution of nitric oxide (NO) and the glibenclamide-sensitive potassium channel in the renal action of adrenomedullin in anaesthetized dogs. 2. Intrarenal arterial infusion of adrenomedullin (20 ng kg-1 min-1) elicited a pronounced increase in renal blood flow with no changes in systemic blood pressure. The renal vasodilator action of adrenomedullin was markedly attenuated by pretreatment with NG-nitro L-arginine (L-NOARG), but this was reversed by continuous infusion of L-arginine. 3. Pretreatment with glibenclamide almost completely blocked the renal vasodilatation induced by lemakalim, but had no effect on the renal vasodilator and diuretic action of adrenomedullin. 4. Intrarenal arterial infusion of adrenomedullin induced diuresis and natriuresis. Diuretic and natriuretic action of adrenomedullin was also attenuated by L-NOARG. L-Arginine partly reversed the effect of L-NOARG and adrenomedullin-induced diuresis and natriuresis. 5. These data indicate that the in vivo renal vasodilator action of adrenomedullin is mediated by the release of NO. The glibenclamide-sensitive potassium channel is not involved in the renal action of adrenomedullin, at least, not in anaesthetized dogs. Since the inhibition of L-NOARG of adrenomedullin-induced diuresis occurred concomitantly with the attenuation of the renal vasodilator action of adrenomedullin, direct involvement of NO in adrenomedullin-induced diuresis remains to be established.

We have previously reported that renal mRNA levels for transforming growth factor-beta 1, fibronectin, and collagens were increased in 32-week-old stroke-prone spontaneously hypertensive rats (SHRSP) with severe nephrosclerosis. To elucidate the mechanism of hypertension-induced nephrosclerosis, we examined gene expression and localization of transforming growth factor-beta 1 and cellular phenotype in the kidney of 25-week-old SHRSP with moderate renal damage. Renal mRNA was measured by Northern blot analysis. The localization of transforming growth factor-beta 1 and cellular phenotype was determined by immunohistochemistry. In the kidney of 25-week-old SHRSP, renal transforming growth factor-beta 1 mRNA was elevated compared with Wistar-Kyoto rats (WKY), whereas renal collagen mRNAs of SHRSP were not increased. Immunoreactive transforming growth factor-beta 1 in SHRSP was mainly localized in glomerular cells. Furthermore, alpha-smooth muscle actin and desmin were significantly expressed in SHRSP glomerular cells, in contrast to negligible expression of these proteins in WKY. alpha-Smooth muscle actin staining was also observed in interstitial cells, and vimentin, another phenotypic marker, was expressed in atrophic tubular cells of SHRSP, despite no staining of these proteins in WKY. Furthermore, all these phenotypic changes in SHRSP were associated with increased cell proliferation, as shown by the increased number of proliferating cell nuclear antigen-positive cells. Treatment of SHRSP with cilazapril and nifedipine (from the age of 13 to 25 weeks) prevented the increase in transforming growth factor-beta 1 expression and the cellular phenotypic modulation and was accompanied by a reduction of urinary albumin excretion and inhibition of cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of a selective agonist for endothelin ETB receptors, Suc-[Glu9,Ala11,15]endothelin-1-(8-21), IRL1620, on renal hemodynamics and urine formation were studied in anesthetized dogs. Intrarenal arterial infusion of IRL1620 at a dose of 50 ng/kg/min increased renal blood flow from 3.37 +/- 0.30 (mean +/- S.E.) to a maximal value of 4.43 +/- 0.45 ml/g kidney weight per min (ml/g/min) at 9.1 +/- 1.0 min after the start of infusion, with no change in systemic blood pressure and heart rate. Urine flow rate increased and urine osmolality, osmolar clearance and free water reabsorption decreased significantly whereas glomerular filtration rate remained unchanged. In dogs given ibuprofen (12.5 mg/kg, i.v.) after the start of infusion of the peptide, renal blood flow increased slightly but significantly from 3.78 +/- 0.82 to 4.17 +/- 0.96 ml/g/min (1.0 +/- 0.1 min), followed by a gradual reduction in renal blood flow. In dogs given L-NG-nitroarginine (75 micrograms/kg/min), the renal blood flow decreased following intrarenal administration of IRL1620 (50 ng/kg/min). It is suggested that IRL1620 enhances the release of nitric oxide and prostaglandins in the kidney and promotes renal vasodilation. The IRL1620-induced reduction of urine osmolality and free water reabsorption was affected by neither ibuprofen nor L-NG-nitroarginine, thereby suggesting that the suppression of urine concentration did not seem to be linked to the enhanced production of nitric oxide or prostaglandins in the kidney.

In order to elucidate the role of adrenomedullin in the kidney, we investigated the effects of adrenomedullin on renal hemodynamics and urine formation in anesthetized dogs. Intrarenal arterial infusion of adrenomedullin (0.8, 4 and 20 ng.kg-1.min-1) elicited dose-dependent increases in renal blood flow (by 10, 26 and 37%, respectively) with no change in blood pressure or heart rate, indicating a renal vasodilatory action of adrenomedullin. The glomerular filtration rate did not increase with the lower two doses, but increased marginally by 9% at the highest dose. Infusion of adrenomedullin at the rates of 4 and 20 ng.kg-1.min-1 increased urine flow and the urinary excretion of sodium and potassium dose dependently. Arterial and renal venous plasma renin activity was unaffected by adrenomedullin. These findings indicate that adrenomedullin is a potent renal vasodilatory peptide with a diuretic action. Since the threshold for the renal vasodilatory action of adrenomedullin is close to its physiological concentration in human plasma, adrenomedullin may play an important role in the regulation of renal function.

We examined the effects of intrarenal infusion of EXP3174, a non-peptide angiotensin II receptor antagonist, in order to evaluate the physiological role of endogenous angiotensin II in regulating renal hemodynamics and urine formation and to assess the possibility of a tubular site(s) of action of endogenous angiotensin II in anesthetized dogs. Intrarenal infusion of EXP3174 at 15 micrograms/kg per min caused increases in renal blood flow (RBF), glomerular filtration rate (GFR), urine, flow and urinary electrolyte excretion. The lower dose of EXP3174 (0.5 micrograms/kg per min) did not change mean arterial pressure, RBF and GFR, but did increase urine flow. The fractional excretion of sodium, the fractional proximal excretion of sodium and the fractional distal excretion of sodium increased with lower doses of EXP3174 infusion. EXP3174 did not affect the linear relationship between the free water reabsorption rate and osmolar clearance. These data suggest that endogenous angiotensin II plays a significant role in regulating renal hemodynamics and urine formation and endogenous angiotensin II stimulates sodium reabsorption in the proximal and the distal portions of the tubules, with the exception of the medullary portion of the ascending limb of Henle.

Studies were carried out to define the effect of semotiadil on renal hemodynamics, renal function and renin release in pentobarbital-anesthetized dogs. Intrarenal arterial infusion of semotiadil resulted in a significant increase in renal blood flow (RBF), glomerular filtration rate (GFR), urine flow, urinary excretion of electrolytes and renin release. Semotiadil did not affect the linear relationship between osmolar clearance and the free water reabsorption rate, and increased the urinary excretion of sodium and calcium to the same extent. These results suggest that the main tubular site of action of semotiadil is the proximal tubule. Intrarenal infusion of a potent non-peptide angiotensin II antagonist, DuP753 (15 micrograms/kg per min), resulted in an increase in RBF, GFR, urine flow and UNaV. In spite of the blockade of the intrarenal renin angiotensin system(RAS) with DuP753, semotiadil caused almost the same effects as it did in the absence of DuP753. These results suggest that the renal effects of semotiadil are independent of the intrarenal RAS.

The renal effects of manidipine, a new dihydropyridine calcium antagonist, were compared with those of nifedipine in anesthetized dogs. Intrarenal administration of manidipine 1 and 5 micrograms/kg resulted in prolonged (120 min) increases in renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (UF), and urinary excretion of electrolytes. In contrast, the renal effects of nifedipine disappeared within 5 to 10 min after administration. These results demonstrate that manidipine is a long-acting calcium antagonist. Manidipine proportionally increased RBF and GFR. As a result, filtration fraction was kept constant. The changes in afferent and efferent arteriolar resistance suggested that manidipine increases GFR via dilation of afferent arterioles with subsequent elevation of filtration pressure in the glomeruli. Manidipine also proportionally increased Tc H2O and Cosm; Tc H2O/Cosm did not change throughout the experiments. In addition, manidipine increased the fractional excretion of sodium from the distal tubule which was calculated from the lithium and sodium clearance. The mode of diuretic action of manidipine most likely involves inhibition of sodium reabsorption in the proximal and distal tubules, except the medullary portion of the ascending limb of Henle. The renal vascular site of action of manidipine might be the afferent arteriole.

The effects of cromakalin, a potassium channel opener, on renal hemodynamics and renal function were investigated in pentobarbital-anesthetized dogs. Intrarenal infusion of cromakalim at 0.5 micrograms/kg/min resulted in significant increases in renal blood flow (RBF), glomerular filtration rate (GFR), urine flow, and renin release. The urinary excretion rate of sodium increased by the same proportion as that of calcium. Free water reabsorption rate/osmolar clearance did not significantly change throughout the experiments. These data suggest that cromakalim did not inhibit sodium transport at the medullary portion of the ascending limb of Henle's loop and may have increased the delivery of sodium to Henle's loop. Intrarenal infusion of nicardipine increased RBF, GFR, urine flow, and the urinary excretion of electrolytes (Na, K, and Ca). The renal effects of cromakalim were very similar to those of nicardipine. Cromakalim was superimposed on a nicardipine infusion of a maximal effective dose. Superimposition of cromakalim to the nicardipine infusion did not cause any additional changes in renal hemodynamics and renal function. These data suggest that cromakalim and nicardipine exert their effects on renal hemodynamics and function via the same pathway.

Intrarenal infusion of forskolin (0.2 microgram/kg/min) in anesthetized dogs increased renal blood flow (RBF) and glomerular filtration rate (GFR) to the same degree but did not change the mean arterial pressure or heart rate. Forskolin also increased urine flow and urinary sodium excretion. The proportional increases in RBF and GFR may result from a preferential action of forskolin to dilate the afferent arteriole.

The current study was designed to evaluate the role of angiotesin II (ANG II) in the regulation of renin mRNA and angiotensinogen mRNA levels. We investigated the changes in renin mRNA levels in the kidney and angiotensinogen mRNA levels in the liver induced by ANG II infusion and by inhibition of the endogenous renin-angiotensin system with enalapril or saralasin in rats. mRNAs for angiotensinogen and renin were measured by densitometric analysis of Northern blot hybridization. After a 4-h intravenous infusion of ANG II (0.2 micrograms.kg-1.min-1), angiotensinogen mRNA level in the liver increased 2.5-fold to the control level, and renin mRNA level in the kidney decreased by 45%. Four hours after treatment with a single dose of enalapril (3 mg/kg po), angiotensinogen mRNA level in the liver decreased by 50%, and renin mRNA level in the kidney increased 1.5-fold to the control level. These mRNA levels returned to control levels 8 h after treatment. After a 4-h infusion of saralasin (2.5 micrograms.kg-1.min-1) in the low-sodium state, angiotensinogen mRNA level in the liver decreased by 45%, and renin mRNA level in the kidney increased twofold to the control level. These findings suggest that ANG II is one of the factors that regulate angiotensinogen mRNA level in the liver and renin mRNA level in the kidney.

We studied, by whole-body autoradiography, the distribution of exogenously administered renal renin in rat. Rat renal renin was completely purified and labeled with 125I ([125I]-renin) and was then injected into the tail veins of conscious rats at a dose of 30 microCi, 430 ng. After various intervals, rats were killed by an overdose of ether, the whole body rapidly frozen in acetone-dry ice, and autoradiography performed on sagittal whole-body sections. To remove breakdown products ([125I]-tyrosine and free 125I) from [125I]-renin, sections were treated with perchloric acid solution. The main accumulation of [125I]-renin acid-insoluble radioactivity was observed in liver and renal cortex. The accumulation in these organs was already evident 2 min after the injection, reached a maximum level by 15 min, then gradually decreased. A small amount of [125I]-renin was also evident in spleen, bone marrow, and adrenal gland. Thirty min after the injection, radioactivity began to appear in the thyroid gland, stomach, and small intestine, but disappeared with acid treatment, except in the thyroid. Radioactivity was negligible in other organs including brain, submaxillary gland, lung, heart, and testis. These autoradiographs clearly demonstrate that exogenously administered renal renin is distributed mainly in the liver and renal cortex.

Rat renal renin, highly purified and labeled with 125I, was intravenously given to conscious rats, to study the fate of the circulating renin. Rat antirenin anti-serum was used to identify the labeled renin. The disappearance of [125I]-renin from the plasma showed two exponential components and the metabolic clearance rate was 11.4 +/- 1.0 ml/min/kg. Both 70% hepatectomy and bilateral nephrectomy decreased the clearance rate by about 50%. [125I]-renin accumulated mainly in the liver and kidney, and high performance liquid chromatography (HPLC) analysis indicated the degradation of [125I]-renin by these organs. Biliary excretion of [125I]-renin was negligible and urinary excretion accounted for 2% of the injected dose. Light- and electron-microscopic autoradiography indicated that [125I]-renin is taken up mainly by Kupffer cells and proximal convoluted tubular cells in the liver and kidney, respectively, and thereafter distributes to the lysosomes. In conclusion, both the liver and kidney are responsible for the clearance of circulating renin.

125I labelled mouse submaxillary renin was administered intravenously to anaesthetized male Institute of Cancer Research, USA (ICR) mice in an attempt to determine the organ-related degradation of circulating renin. A specific antirenin antiserum was used for identification. The disappearance rate of labelled renin from plasma was estimated on the basis of a two-compartmental model. The mean half-time of clearance of labelled renin from plasma in intact control and 70% hepatectomized mice was 13.3 +/- 0.8 and 12.0 +/- 0.9 min respectively; these values were not significantly different. The mean half-time of clearance of labelled renin from plasma in nephrectomized mice was 40.0 +/- 2.4 min; this was significantly longer than intact controls and 70% hepatectomized mice. Fifty per cent of the administered renin accumulated in the kidney and 9% in the liver. A small amount of 125I labelled renin was identified in urine, and a large amount of 125I liberated from labelled renin was detected. Since the high performance liquid chromatography profiles showed that plasma renin rapidly moves into the kidney, clearance of circulating renin may take place mainly in this organ.

Using highly purified 125I-labeled rat renal renin, we have demonstrated that both the liver and kidney are responsible for clearance of renal renin. In the present study, light and electron microscope autoradiography of intravenously administered 125I-labeled rat renal renin was performed in rat liver and kidney to observe the cellular and subcellular distribution of the renin. Fifteen and 60 min after the injection, the liver and kidneys were removed and fixed with 0.1 M sodium phosphate buffer, pH 6.8, containing 2.5% glutaraldehyde and 0.5% tannic acid, which allowed for the removal of breakdown products of the labeled renin, and then autoradiography was performed. In the liver, silver grains were mainly localized in Kupffer cells and not in hepatocytes. In the kidney, silver grains were evident in the proximal tubule cells. In both liver and kidney cells, silver grains were mainly located over the lysosomes. Gel permeation high-performance liquid chromatography analysis of the liver and kidney extracts indicated two main fractions, including immunoreactive 125I-renin and the breakdown products (free 125I and 125I-tyrosine). In conclusion, circulating renal renin is mainly taken up by Kupffer cells and proximal tubule cells and is subsequently transported to the lysosomes to be degraded. These results, taken together with our previous finding that nonglycosylated submaxillary renin does not distribute in the liver, suggest that the carbohydrate moieties of renal renin are necessary for the recognition by Kupffer cells.

Effects of 4-phenoxy-3-(1-pyrrolidinyl)-5-sulfamoyl benzoic acid (piretanide, Arelix) on renal function and hemodynamics were investigated in anesthetized dogs by clearance, stop-flow and radioactive microsphere methods. Marked diuresis occurred within 10 min after intravenous injection of piretanide (0.01, 0.05 and 0.25 mg/kg) and it was accompanied b natriuresis. Piretanide (0.25 mg/kg) clearly inhibited the reabsorption of solute-free water during saline diuresis, suggesting that the drug acts in the ascending limb of Henle's loop. Stop-flow experiments revealed that sodium reabsorption was reduced by piretanide (0.25 mg/kg) at the distal tubule including the ascending limb of Henle's loop. Piretanide (0.25 mg/kg) caused a maximum increase of about 30% in renal blood flow at 10-15 min after the injection; within the renal cortex the increase in blood flow was greater in the juxtamedullary area than in the superficial area. The increased blood flow in the inner cortex conceivably resulted in a washout of the osmotic substances in the medulla and inhibited the concentrating mechanisms of the kidney. The effect of piretanide on intrarenal hemodynamics may also contribute to its diuretic action.

The present studies were undertaken to explore further the role of prostaglandins in the release of renin from the renal cortex. To provide the best assessment of renin release, renin was determined by a radioimmunoassay for the direct measurement of renin. Slices of mouse renal cortex were incubated at 37 degrees C with arachidonic acid (AA), 5,8,11,14-eicosatetraenoic acid (ETA), indomethacin, prostaglandins, and synthetic prostaglandin endoperoxide analogue (EPA). Our results showed that AA at 1.5 X 10(-8) M significantly increased renin release at 10 and 30 min of incubation. This renin increase ws abolished by either ETA or indomethacin. Prostaglandin F2 alpha (PGF2 alpha) also significantly stimulated renin release at 10 and 60 min. PGE2 and 16,16-dimethyl PGE2 (DMPGE2) showed much less renin release-stimulating activity. EPA and PGI2 on the other hand very strongly stimulated renin release. However, at higher concentrations the stimulating effect of PGI2 and EPA disappeared and even became inhibitory in the case of EPA. Other prostaglandins were found to have no effect on renin release. The results suggest that the prostaglandin system directly affects renin release from the juxtaglomerular cells independent of systemic neurogenic and hemodynamic influences.

In an attempt to define the effects of a calcium antagonist, nitrendipine, on renal function, intrarenal hemodynamics, and renin release, we infused the drug into the renal artery of anesthetized dogs. Infusion of nitrendipine at a rate of 5 micrograms/min in both hydropenic and hydrated dogs resulted in a significant increase in renal blood flow (RBF), glomerular filtration rate (GFR), urine flow and renin release, with no change in systemic blood pressure, indicating a significant renal vasodilation. The urinary excretion rate of sodium increased by the same proportion as that of calcium. The intrarenal blood flow as measured by hydrogen washout rate showed that the outer cortical flow increased by the same proportion as the inner cortical flow. During nitrendipine infusion, free water reabsorption rate (TcH2O) in hydropenic dogs or free water clearance (CH2O) in hydrated dogs increased in proportion to the urine flow. Neither the ratio of TcH2O nor that of CH2O to osmolar clearance changed throughout the experiment. These data suggest that nitrendipine may not inhibit sodium transport in the medullary portion of the ascending limb of Henle and may induce an enhancement of the delivery of sodium to the Henle loop. Thus, nitrendipine exerts its diuretic and natriuretic actions via the alteration of renal hemodynamic and the inhibitory effect on proximal sodium reabsorption.

We investigated the effects of leukotrienes (LTs) C4 and D4 on renal function and hemodynamics of dogs. LTC4 (0.5 microgram/min, infused into the renal artery), but not LTD4 (1 microgram/min), caused an increase in renal blood flow, urine flow, urinary excretion of sodium and potassium, and rate of free water reabsorption, with a concomitant rise in systemic blood pressure. Intrarenal infusion of LTC4 under conditions of constant renal perfusion pressure induced no significant effect on renal blood flow or urine formation. Infusions or LTC4 and LTD4 into the renal artery induced no change in the release of prostaglandins, PGE2, PGF2 alpha, 6-keto-PGF1 alpha or thromboxane B2 into renal venous blood. From these findings, we concluded that the renal effects of LTC4 were secondary responses to a rise in systemic blood pressure.

Purified mouse submaxillary gland renin was labelled with iodine(125I) by the chloramine-T method and this 125I labelled renin was given to male ICR mice (6-7 wks. old), intravenously in a dose of 20 ng(2 microCi)/40 g body weight. At a specified time, the kidneys of these treated mice were excised, the radioactivity determined and the tissues immediately prepared for microscope and electron microscope autoradiography(micro-ARG and EM-ARG). Silver grains accumulated mainly at the apical site of the proximal convoluted tubules but were not identified in other areas of the nephron, including macula densa or in the arterial wall, glomeruli, and juxtaglomerular cells (micro-ARG). Silver grains were seen in the pinocytotic vesicles, vacuoles and lysosomal granules(EM-ARG). This is the first demonstration that exogenous renin is ultrafiltered through glomerular capillaries and is reabsorbed by pinocytosis in the one-third upper area of the proximal convoluted tubule.

The study was designed to define the serotonin (5-HT) receptor subtypes in the canine kidney. An intrarenal infusion of 5-HT at a dose of 5 micrograms/min in anesthetized dogs resulted in a biphasic response of renal blood flow which decreased transiently then increased above the control level during prolonged infusion. The decrease of renal blood flow was abolished by infusion of methysergide but not by ketanserin, and the subsequent increase was abolished by infusion of either ketanserin or methysergide. Terazosin, an alpha1-adrenoceptor antagonist, did not modify the renal action of 5-HT. These findings suggest that the renal blood flow response induced by 5-HT did not depend on an indirect effect via the sympathetic nervous system, the initial vasoconstriction was mediated via a 5-HT1-like receptor, and that the latter vasodilatation was mediated via a 5-HT2 receptor. The infusion of 5-HT also increased urine flow and urinary excretion of sodium. These increases were reversed by pretreatment with ketanserin and abolished by methysergide. We propose that 5-HT may exert its antidiuretic action via a 5-HT1-like receptor in the tubules but that the renal hemodynamic changes induced by 5-HT may overcome its antidiuretic action. The present results suggest the existence of a 5-HT1-like and 5-HT2 receptor in the renal vasculature and a 5-HT1-like receptor in the renal tubules.

Our recent findings on the subcellular localization of exogenously administered 125I-labeled mouse submaxillary renin in the mouse kidney are summarized herein. 125I-labeled renin was given intravenously to anesthetized male ICR mice (6 weeks) in a dose of 20 ng (2 muCi)/40 g body weight and the subcellular distribution was identified by the reaction with a specific antiserum of the mouse submaxillary renin. 125I-labeled renin was identified mainly by differential centrifugation in the heavy mitochondrial fraction. With discontinuous sucrose density gradient centrifugation, the radioactivity was most readily detected with 1.2 M and 1.5 M sucrose. Over 90% of the radioactivity was identified in the form of 125I-labeled renin with a molecular weight of 38,000 daltons in each fraction. Light and electron microscopic autoradiography revealed that the 125I-labeled renin had accumulated in the apical region of the proximal convoluted tubular cells and was mainly located in the lysosomal granules. All these data taken together indicate that the exogenous renin filtered through the glomerular capillaries is reabsorbed and localized in the lysosomes of the proximal convoluted tubular cells.

The expression of messenger (m)RNA for renin, angiotensinogen and atrial natriuretic factor (ANF) was investigated in rats on different sodium intakes. Messenger RNA was measured by a radiodensitometric hybridization assay. In the high-sodium state, renal renin mRNA decreased, but it increased in the low-sodium state. A further increase in renin mRNA was seen in the low-sodium state after captopril administration. Angiotensinogen mRNA levels in the liver, kidney and brain were altered by varying sodium intake. In the high-sodium state angiotensinogen mRNA decreased, but in the low-sodium state it increased. After treatment with captopril, angiotensinogen mRNA levels decreased in the liver and kidney. Angiotensinogen mRNA showed tissue specificity for expression, especially in the brain. Atrial ANF mRNA levels changed slightly with different levels of sodium intake.

The effect of alpha- and beta-adrenergic agonists on renal and submaxillary renin of different molecular weights was studied using male albino mice as experimental animals. Phenylephrine or isoproterenol was administered intravenously after removal of the submaxillary glands and/or kidneys. Renin was isolated from plasma by column chromatography and then measured by a direct radioimmunoassay. Phenylephrine increased both 68,500-dalton renin (big renin) and 38,000-dalton renin (small renin) in the plasma of nephrectomized mice. Isoproterenol increased big and small renin in the plasma of mice whose submaxillary glands were removed. In both cases, the increase of small renin was significantly greater than that of big renin. The results suggest that the alpha-adrenergic agonist phenylephrine affects the submaxillary gland, leading to the increase of both big and small plasma renin. In contrast, the beta-adrenergic agonist isoproterenol affects the kidney, leading to the increase of both big and small plasma renin.