E. Podjarny

Effect of Tetrahydrobiopterin on Blood Pressure in Rats after Subtotal Nephrectomy

Background: Previous studies have shown that endothelial dysfunction after 5/6 nephrectomy (5/6 Nx) in rats is associated with decreased nitric oxide (NO) bioavailability and increased vascular superoxide production. Blood pressure is significantly increased by day 10 after surgery. Tetrahydrobiopterin (BH4) is a key cofactor of NO synthase. Suboptimal levels of BH4 result in uncoupling of NO synthase, low NO synthesis and augmented production of superoxide anions. The aim of this study was to evaluate whether BH4 supplementation may improve NO production and prevent the increase of blood pressure after 5/6 Nx. Methods: Three groups were evaluated: 5/6 Nx (untreated rats), BH4 (5/6 Nx rats treated with BH4, 10 mg/day i.p. for 10 days) and L-ARG (5/6 Nx rats treated with L-arginine, 260 mg/kg BW, p.o for 10 days). Systolic blood pressure (SBP), urinary nitrate excretion (UNO3) and creatinine clearance (CCR) were measured before surgery and on days 3 and 10 after surgery. Endothelial NO synthase (eNOS) protein content of mesenteric resistance vessels was measured at the end of the study. Results: SBP increased from 107 ± 2 to 127 ± 4 mm Hg in untreated 5/6 Nx rats (p < 0.01). By contrast, rats treated with BH4 or L-ARG remained normotensive. Ten days after 5/6 Nx, creatinine clearance decreased similarly in all groups. Both BH4 and L-ARG supplementation markedly increased UNO3 excretion. The mesenteric vascular expression of eNOs protein was significantly higher in BH4 but not in L-ARG, compared with Nx rats. Conclusions: BH4 supplementation prevents the earlier increase in blood pressure observed in rats after 5/6 Nx, possibly by upregulating eNOS in resistance vessels.

Captopril, but not diltiazem, favorably affects the course of early chronic renal disease in rats

The concepts that increased intracellular Ca2+ content and increased glomerular capillary pressure play an important role in the progression of chronic renal diseases has led to the suggestion that treatment with calcium-blocking agents (diltiazem; CBB) or converting enzyme inhibitors (captopril; CEI) may be indicated to prevent renal failure. We studied the effects of CCB and CEI on the early course of adriamycin (ADR) nephropathy, where glomerular pressure has been shown to be unchanged, blood pressure was only mildly elevated and renal failure incipient. Animals were studied 2, 7, 12, 16 and 20 weeks after the second injection of ADR, 2 mg/kg. In treated rats, blood pressure remained normal. At the end of the study, proteinuria and serum creatine were lower in ADR-CEI than in ADR rats (149 +/- 42 vs. 616 +/- 90 mg/day, p less than 0.01 and 0.36 +/- 0.04 vs. 0.58 +/- 0.02 mg%, p less than 0.01, respectively). ADR-CCB had values similar to those of untreated ADR rats. Mesangial expansion and focal glomerulosclerosis were present only in ADR and ADR-CCB rats, whereas in ADR-CEI rats the glomeruli were virtually normal. Glomerular 45Ca uptake was increased in ADR, decreased in ADR-CCB rats, and normal in ADR-CEI. Glomerular 6-keto PGF1 alpha and TxB2 were significantly increased in ADR rats, and both treatments decreased TxB2. The results suggest that endogenous angiotensin II is important for the early progression of glomerular injury toward renal insufficiency, while tissue Ca2+ accumulation may play an important role in more advanced phases.

Effect of Methyldopa on Renal Function in Rats with L-NAME-Induced Hypertension in Pregnancy

Background: Pregnancy-induced hypertension is characterized by an increased sympathetic activity and probably by a decreased synthesis/activity of nitric oxide. The aim of the present study is to evaluate whether the beneficial action of the sympathetic antagonist methyldopa (a first-choice hypotensive agent in the treatment of PIH) may be associated to changes in nitric-oxide synthesis. Methods: Forty pregnant Wistar rats received L-NAME (NO synthase inhibitor, 9–10 mg/kg/day) from mid-pregnancy (day 11) through to term. Some rats were treated with daltroban (TxA receptor antagonist, 60 mg/kg/day), diltiazem (calcium channel blocker, 30 mg/kg/day), methyldopa (central adrenergic antagonist, 400 mg/kg/day) or L-arginine (260 mg/kg/day) from mid-pregnancy. The effect of the different treatments on systolic blood pressure (SBP), creatinine clearance (CCR), urine protein excretion (UP) and urinary nitrate excretion (UNO3, representing urine NO metabolite) were evaluated and the results compared with those found in normal pregnancy. Normal pregnant rats receiving similar treatment were used as controls. Results: In normal pregnant (P) rats, SBP values decreased from 94 ± 2 to 83 ± 3 mm Hg at the end of pregnancy (p < 0.01) and CCR augmented significantly. Drug treatment had no significant effect. In NAME-treated rats, at the same period, the SBP augmented from 92 ± 1 to 129 ± 1.8 mm Hg (p < 0.01). At the end of pregnancy, NAME rats had significantly lower CCR values and higher UP excretion when compared with P rats. UNO3 increased significantly in P and in P rats treated with methyldopa. As expected, in NAME rats UNO3 excretion was significantly reduced. Treatment with methyldopa normalized SBP, improved CCR and proteinuria and was associated with an increase in UNO3. Similar results were obtained with L-arginine treatment. Diltiazem lowered SBP significantly but had no effect on renal function or UNO3 and daltroban had no effect. Conclusion: The increased UNO3 found in NAME rats treated with methyldopa suggests that the vasoconstriction secondary to chronic NO inhibition may be partially related to an increased sympathetic activity. The efficient action of the sympathetic antagonist methyldopa may be due not only to its antihypertensive effects but also by its stimulating effect on NO synthesis leading also to an improvement of renal function.

Low Molecular Weight Heparin Reduces Proteinuria and Modulates Glomerular TNF-α Production in the Early Phase of Adriamycin Nephropathy

Background/Aim: Heparin has been shown to be renoprotective in a number of experimental nephropathies. The inflammatory component in the early phase of Adriamycin (ADR) induced nephropathy has been established. A microdose of low molecular weight heparin (Fragmin; F) has been noted to exert immunomodulatory effects independent of its anticoagulant activity. We assessed the effects of microdoses of F on daily proteinuria and glomerular production of tumor necrosis factor alpha (TNF-α) and prostaglandins 8 and 15 days after induction of ADR nephropathy. Methods: Following intravenous injection of ADR (7 mg/kg) to Wistar rats weighing 200 ± 20 g, F 20 µg/day/rat s.c. was administered for 8 and 15 days (groups F8 and F15). The respective control groups (C8 and C15) received normal saline subcutaneously. Proteinuria, serum albumin, and creatinine clearance were evaluated on days 8 and 15. The production of TNF-α and prostaglandins from glomerular supernatants was measured by radioimmunoassay on days 8 and 15. Results: F significantly reduced proteinuria (mg/day) on day 8: 13.6 ± 1.2 in F8 versus 40.3 ± 2.7 in C8 (p = 0.008). The glomerular production of TNF-α (pi/ml) was significantly lower on day 8 in rats treated with F: 356 ± 33 in F8 versus 764 ± 81 in C8 (p = 0.006). A decrease in the prostaglandin E2/thromboxane B2 ratio was noted in the F group between 8 and 15 days (1.1 in F8 vs. 0.9 in F15, p = 0.005) which principally reflects an increase of thromboxane B2. The antiproteinuric effect of F shown after 8 days was no longer present after 15 days (354 ± 91 mg/day in F15 vs. 499 ± 69 mg/day in C15, p = 0.33). The same trend was seen for the glomerular production of TNF-α. Light microscopy and immunohistochemistry for interstitial and glomerular macrophages were negative. Conclusion: The lowering effect of microdoses of F on the proteinuria seen during the early phase of ADR nephropathy may be mediated by a decreased production of glomerular TNF-α, supporting the anti-inflammatory action of low molecular weight heparin.

Sodium loading and renal prostaglandins in old rats

1. Previous studies have shown that altered synthesis of prostaglandins (PGs) in the kidney of ageing rats contributes to impaired Na conservation during sodium deprivation. In the present study, we wished to assess whether the disturbance of prostaglandin synthesis also affects the response to sodium loading in old rats. 2. We measured the urinary excretion of thromboxane B2 (TXB2), 6-keto PGF1 alpha (6KPGF1 alpha) and PGE2 in young (3-4 months) and old (20-21 months) rats after 24, 48 and 72 h of Na loading. In a separate protocol, we measured prostanoid synthesis by isolated glomeruli, cortical homogenates, medullary and papillary slices from young and old rats in basal conditions and after 15 days of dietary Na loading. 3. Young and old rats excreted similarly the Na load. The urinary excretion (U) of TXB2 and 6KPGF1 alpha were unchanged during Na load in young rats. U6KPGF1 alpha, which was significantly higher in old rats and UTXB2 which also tended to be elevated, decreased in old rats with Na loading. Sodium loading was associated with a transient increase of UPGE2 in young, but not in old rats. 4. TXB2 synthesis was increased in all portions of the kidneys of old rats. 6KPGF1 alpha production was elevated in glomeruli and cortex and that of PGE2 in cortex. In medulla and papilla only TXB2 synthesis was enhanced. 5. Sodium loading did not significantly change prostanoid synthesis in the kidneys of young rats. In old rats, glomerular and cortical TXB2 decreased whereas medullary and papillary 6KPGF1 alpha increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in Renal Prostanoid Synthesis Induced by Potassium Loading in Rats

Previous works have demonstrated changes in the urinary excretion of prostaglandins (PG) in response to changes in potassium (K) or sodium (Na) intake. In the present study, the production of PGE2, PGF2 alpha, 6-keto-PGF1 alpha and thromboxane B2 (TXB2) by isolated glomeruli, cortical homogenates, medullary and papillary slices was measured in K-loaded rats on either a normal or a low Na intake. In glomeruli, K loading increased selectively PGE2 synthesis. In Na-depleted animals, all prostanoids were elevated and K loading did not induce a further increase. In cortical and medullary preparations, PGE2 was decreased by K loading irrespective of the state of Na balance. In papilla, PGE2 decreased (in all K-loaded rats) and PGF2 alpha increased (only in rats with normal Na intake). 6-Keto-PGF1 alpha and TXB2 did not change significantly. No correlation was present between changes of PG synthesis and urinary kallikrein excretion. The results demonstrate a specific effect of K on PGE2 and PGF2 alpha, and suggest a role for these substances in K homeOstasis.

Thrombin Inhibits the Synthesis of Prostanoids by Isolated Glomeruli and Peritoneal Macrophages in Rats

Activation of macrophages and release of mediators that activate the coagulation system characterize proliferative glomerulonephritis. To evaluate the possible role of prostanoids in this process, isolated rat glomeruli (G) and peritoneal macrophages (M) or a combination of the two (G + M) were incubated in the presence of thrombin (2 U/ml). In G, thrombin inhibited only the synthesis of thromboxane B2. In M and G + M incubations, the synthesis of prostaglandin I2 and thromboxane A2 was inhibited by thrombin. This effect was abolished by the addition of arachidonic acid. As prostanoids may play a modulatory role in the interaction between macrophages and glomerular cells, inhibition of their synthesis by thrombin might enhance macrophage activity.

Effect of sodium and chloride depletion on urinary prostaglandin F2α excretion in potassium loaded rats

Previous studies have shown that the urinary excretion of prostaglandin (PG) F2 alpha is stimulated by potassium (K) loading. Because changes of sodium chloride (NaCl) intake also affect renal PG production, in this study we investigated the interaction between the effect of K and that of concomitant reduction of Na and Cl intake. The urinary excretion of PGF2 alpha and PGE2 was measured in 12 groups of female rats on normal, high or low K intake. Na and Cl intake were adjusted so that rats had normal intake (controls, C), were selectively Cl depleted (CD), selectively Na depleted (ND) or Na and Cl depleted (NCD). In rats with normal K intake, urinary PGF2 alpha was not modified by changes of Na or Cl intake, whereas PGE2 was increased in by Cl depletion (in both NCD or CD groups). Potassium chloride loading increased urinary PGF2 alpha and selective Na depletion (ND group) induced a further increase. On the other hand, PGF2 alpha was not stimulated when K load was associated with Cl depletion. Urine PGF2 alpha was directly correlated with plasma aldosterone and urinary kallikrein. Urinary PGE2 did not change with K-loading. The results suggest that PGF2 alpha participates in the renal adaptation to KCl-loading but not when K is accompanied by non-Cl anions.

NADH-dependent prostaglandin E2-9-ketoreductase activity and prostaglandin synthesis in the Brattleboro rat kidney: effects of the antidiuretic hormone

The activity of prostaglandin (PG)E2-9-ketoreductase (9KR), an enzyme catalyzing the conversion of PGE2 to PGF2 alpha, was significantly increased in glomerular and cortical homogenates of diabetes insipidus (DI) rats, as compared to normal Long Evans (LE) rats, and did not change with ADH treatment. Medullary 9KR was similar in the three groups and papillary 9KR was increased, but not significantly, in both groups of DI rats. Km values for PGE2 and NADH were compared in the various compartments of the kidney. Levels of 9KR were not correlated with the PGE/PGF ratio in urine or supernatants. The synthesis of PGE2 and PGF2 alpha by isolated glomeruli was increased in DI rats. This was not reversed by ADH treatment, PGE2 synthesis increasing even further, especially in the presence of arachidonic acid. In contrast, medullary slices produced significantly less PGs in DI than in LE rats and returned to normal with ADH treatment. Papillary slices produced similar quantities of prostaglandins in all groups. The results do not support the concept that the alterations in PG synthesis observed in DI rat are related only to changes in 9KR activity, but do not exclude the possibility that the enzyme participates in the regulation of PG biosynthesis.

Effect of the prostacyclin analogue iloprost in sodium-depleted rats pretreated with captopril

Previous studies have shown that administration of captopril to sodium-depleted rats decreases the glomerular filtration rate (GFR) and blunts the increase in glomerular prostacyclin synthesis normally occurring in response to sodium depletion. To clarify the relationship between these two responses, iloprost, a stable analogue of prostacyclin, was administered to Na-depleted, captopril-treated (LNC) rats. At a dosage not affecting systemic blood pressure (12.5 ng/kg/min), iloprost increased GFR in LNC rats by 25% (from 0.26 +/- 0.03 to 0.35 +/- 0.03 ml/min/100 g body wt, P less than 0.01), without significant effects on renal plasma flow. No effect was observed in control rats. The results suggest that altered prostacyclin synthesis could contribute to the decrease of GFR in this model.