Collecting duct (Na+/K+)-ATPase activity is correlated with urinary sodium excretion in rat nephrotic syndromes

In puromycin aminonucleoside (PAN)-treated nephrotic rats, sodium retention is associated with increased (Na+/K+)-ATPase activity in the cortical collecting ducts (CCD). This study was undertaken to determine whether stimulation of (Na+/K+)-ATPase in the CCD is a feature of other experimental nephrotic syndromes, whether it might be responsible for renal sodium retention, and whether it is mediated by increased plasma vasopressin levels or activation of calcineurin. For this purpose, the time courses of urinary excretion of sodium and protein, sodium balance, ascites, and (Na+/K+)-ATPase activities in microdissected CCD were studied in rats with PAN or adriamycin nephrosis or HgCl2 nephropathy. The roles of vasopressin and calcineurin in PAN nephrosis were evaluated by measuring these parameters in Brattleboro rats and in rats treated with cyclosporin or tacrolimus. Despite different patterns of changes in urinary sodium and protein excretion in the three nephrotic syndrome models, there was a linear relationship between CCD (Na+/K+)-ATPase activities and sodium excretion in all three cases. The results also indicated that there was no correlation between proteinuria and sodium retention, but ascites was present only when proteinuria was associated with marked reduction of sodium excretion. Finally, the lack of vasopressin in Brattleboro rats or the inhibition of calcineurin by administration of either cyclosporin or tacrolimus did not prevent development of the nephrotic syndrome in PAN-treated rats or stimulation of CCD (Na+/K+)-ATPase. It is concluded that stimulation of Na(+/K+)-ATPase in the CCD of nephrotic rats might be responsible for sodium retention and that this phenomenon is independent of proteinuria and vasopressin and calcineurin activities.     

Regulation of the proximal tubular sodium/proton exchanger NHE3 in rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome

Excessive proteinuria due to loss of glomerular permselectivity in nephrotic syndrome can cause disturbances in renal salt and water handling with edema formation. Apart from oncotic and hydrostatic mechanisms associated with hypoalbuminemia, primary derangements in renal tubular sodium transport may contribute to the pathogenesis of nephrotic edema. Whereas there is evidence for an increase of cortical collecting duct sodium reabsorption in nephrotic rats, it remains controversial whether proximal tubule sodium transport may also be activated in this condition. The regulation of the cortical Na/H exchanger NHE3, the main pathway for Na reabsorption in the proximal tubule (PT), was investigated in rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. PAN rats developed reduced GFR, severe proteinuria, and sodium retention within 3 d. After 10 d, immunoblots of brush border vesicles revealed a decreased abundance of NHE3 in nephrotic animals. However, the Na/H antiporter activity in the same vesicle preparations was not significantly altered. Antiporter activity normalized for NHE3 protein was increased by 88% in nephrotic animals (P = 0.025). Immunohistochemistry with the same polyclonal antibody as for immunoblots revealed a decrease of NHE3 abundance in PT. In contrast, immunoreactivity for the monoclonal antibody 2B9, which specifically recognizes the non-megalin-associated, transport-competent pool of NHE3, was higher in PAN-treated rats than in controls. In conclusion, increased sodium reabsorption might be associated with a shift of NHE3 from an inactive pool to an active pool, thus contributing to sodium retention in a state of proteinuria.     

Effects of uroguanylin on natriuresis in experimental nephrotic rats

Aim:   Uroguanylin, isolated from human and opossum urine, is a candidate intestinal natriuretic hormone that controls the sodium and water balance between the intestine and the kidneys. Levels of immunoreactive (ir)-uroguanylin in the plasma and urine are increased in rats and humans with nephrotic syndrome, which is physiologically characterized by sodium retention with massive proteinuria. The present study evaluates the effect of natriuresis induced by uroguanylin on nephrotic rats.
Methods:   Normal rats and rats rendered nephrotic by injections of puromycin aminonucleoside (PAN) were treated with uroguanylin (0.5 nmol/h, delivered by an osmotic pump) or with vehicle during the sodium retention phase. All rats consumed the same quantity of sodium.
Results:   Uroguanylin did not increase urinary excretion of sodium and water in normal rats, but significantly increased urinary sodium excretion during the sodium retention phase in nephrotic rats (untreated vs uroguanylin-treated nephrotic rats in mmol/mmol creatinine; 2.92 ± 0.65 vs 8.93 ± 2.53 on day 6, P  < 0.05; 3.55 ± 0.47 vs 10.37 ± 1.73 on day 7, P  < 0.01; 14.88 ± 2.32 vs 24.47 ± 2.86 on day 8, P  < 0.05). Plasma levels of ir-uroguanylin in uroguanylin-treated nephrotic rats on day 6 were significantly increased compared with those in uroguanylin-treated control and untreated nephrotic rats.
Conclusion:   Uroguanylin increased urinary sodium excretion in rats with PAN-induced nephrosis, and might be useful for treating sodium retention in patients with nephrotic syndrome.     

Selective albuminuria via podocyte albumin transport in puromycin nephrotic rats is attenuated by an inhibitor of NADPH oxidase

The mechanism of selective albuminuria in minimal change nephrotic syndrome, in which glomerular capillaries are diffusely covered by effaced podocyte foot processes with reduced slit diaphragms, is unknown. Podocyte injury is due, in part, to NADPH-induced oxidative stress. Here we studied mechanism of selective albuminuria in puromycin aminonucleoside (PAN) nephrotic rats, a model of minimal change nephrotic syndrome. In these rats, Evans Blue-labeled human albumin was taken up by podocytes and its urinary excretion markedly increased, with retained selectivity for albumin. Immunogold scanning electron micrographic images found increased human albumin in podocyte vesicles and on the apical membrane in nephrotic compared with control rats. Apocynin, an inhibitor of NADPH oxidase, decreased superoxide production in podocytes, and inhibited endocytosis and urinary albumin excretion. Real-time confocal microscopy found an initial delay in the appearance of Evans Blue-labeled human albumin in the tubular lumen, reflecting the time needed for transcellular transport. Immunoprecipitation analysis indicated that FcRn, a receptor for albumin transport, mediated podocyte albumin transport, and treatment with anti-FcRn antibody reduced proteinuria in these nephrotic rats. Thus, podocyte albumin transport was enhanced in PAN nephrotic rats by means of FcRn, which may explain the mechanism of selective proteinuria. This was blocked by apocynin, suggesting a new therapeutic approach.     

Hyperaldosteronemia and activation of the epithelial sodium channel are not required for sodium retention in puromycin-induced nephrosis

Edema and ascites in nephrotic syndrome mainly result from increased Na+ reabsorption along connecting tubules and cortical collecting ducts (CCD). In puromycin aminonucleoside (PAN)-induced nephrosis, increased Na+ reabsorption is associated with increased activity of the epithelial sodium channel (ENaC) and Na+,K+-ATPase, two targets of aldosterone. Because plasma aldosterone increases in PAN-nephrotic rats, the aldosterone dependence of ENaC activation in PAN nephrosis was investigated. For this purpose, (1) the mechanism of ENaC activation was compared in nephrotic and sodium-depleted rats, and (2) ENaC activity in PAN-nephrotic rats was evaluated in the absence of hyperaldosteronemia. The mechanism of ENaC activation was similar in CCD from nephrotic and sodium-depleted rats, as demonstrated by (1) increased number of active ENaC evaluated by patch clamp, (2) recruitment of ENaC to the apical membrane determined by immunohistochemistry, (3) shift in the electrophoretic profile of gamma-ENaC, and (4) increased abundance of beta-ENaC mRNA. Corticosteroid clamp fully prevented all PAN-induced changes in ENaC but did not alter the development of a full-blown nephrotic syndrome with massive albuminuria, amiloride-sensitive sodium retention, induction of CCD Na+,K+-ATPase, and ascites. It is concluded that in PAN-nephrosis, (1) ENaC activation in CCD is secondary to hyperaldosteronemia, (2) sodium retention and induction of Na+,K+-ATPase in CCD are independent of hyperaldosteronemia, and (3) ENaC is not necessarily limiting for sodium reabsorption in the distal nephron.     

Role of uroguanylin, a Peptide with natriuretic activity, in rats with experimental nephrotic syndrome

Uroguanylin induces natriuresis and diuresis in vivo as well as in vitro and is found mainly in the intestine and the kidney. However, the roles of uroguanylin in nephrotic syndrome, which is associated with sodium and water retention, have not been determined. Therefore, changes in the urine and plasma concentration of immunoreactive uroguanylin (ir-uroguanylin) and its mRNA expression in the kidney and intestine were examined using rats with puromycin aminonucleoside (PAN)-induced nephrosis. Male Sprague-Dawley rats were separated into control and nephrotic groups, and then the urinary excretion of sodium, protein, and ir-uroguanylin was examined over time. The plasma levels and renal and intestinal mRNA expression of uroguanylin at the periods of sodium retention and remarkable natriuresis also were evaluated. The sequential changes of urinary ir-uroguanylin excretion in the nephrotic group were similar to those of urinary sodium excretion. When the urinary excretion of ir-uroguanylin and sodium peaked, the plasma level of ir-uroguanylin also increased compared with that of the control group. Uroguanylin mRNA expression in the kidney increased during the period of sodium retention and then decreased during the period of remarkable natriuresis. Uroguanylin mRNA expression in the small intestines of control and nephrotic rats were identical. However, in a unilateral PAN-induced proteinuria, uroguanylin expression significantly increased in the PAN-perfused kidney compared with that in the opposite kidney. Considering the natriuretic effect of uroguanylin, these results suggested that uroguanylin plays an important role as a natriuretic factor in nephrotic syndrome via both the circulation and the kidney itself.     

Collecting duct is a site of sodium retention in PAN nephrosis: a rationale for amiloride therapy

Micropuncture studies of the distal nephron and measurements of Na,K-ATPase activity in microdissected collecting tubules have suggested that renal retention of sodium in puromycin aminonucleoside (PAN) nephrotic rats originates in the collecting duct. The present study demonstrated this hypothesis by in vitro microperfusion and showed that amiloride was able to restore sodium balance. Indeed, isolated perfused cortical collecting ducts from PAN-treated rats exhibited an abnormally high transepithelial sodium reabsorption that was abolished by amiloride, and in vivo administration of amiloride fully prevented decreased urinary sodium excretion and positive sodium balance in nephrotic rats. As expected from the aldosterone independence of Na(+) retention in PAN nephrotic rats, blockade of aldosterone receptor by potassium canrenoate did not alter urinary Na(+) excretion, Na(+) balance, or ascites formation in PAN nephrotic rats.     

Blunted renal dopaminergic system activity in puromycin aminonucleoside-induced nephrotic syndrome.

BACKGROUND: A primary tubular sodium handling abnormality has been implicated in the edema formation of nephrotic syndrome. Dopamine synthesized by renal proximal tubules behaves as an endogenous natriuretic hormone by activating D(1)-like receptors as a paracrine/autocrine substance. METHODS: We examined the time courses of the urinary excretion of sodium, protein and dopamine in puromycin aminonucleoside (PAN)-treated and control rats. The rats were sacrificed during greatest sodium retention (day 7) as well as during negative sodium balance (day 14) for the evaluation of renal aromatic l-amino acid decarboxylase (AADC) activity, the enzyme responsible for the synthesis of renal dopamine. Also, the influence of volume expansion (VE) and the effects of the D(1)-like agonist fenoldopam (10 microg/kg bw/min) on natriuresis and on proximal tubular Na(+),K(+)-ATPase activity were examined on day 7. RESULTS: The daily urinary excretion of dopamine was decreased in PAN-treated rats, from day 5 and beyond. This was accompanied by a marked decrease in the renal AADC activity, on days 7 and 14. During VE, the fenoldopam-induced decrease in proximal tubular Na(+),K(+)-ATPase activity was more pronounced in PAN-treated rats than in controls. However, the urinary sodium excretion during fenoldopam infusion was markedly increased in control rats but was not altered in PAN-treated animals. CONCLUSION: PAN nephrosis is associated with a blunted renal dopaminergic system activity which may contribute to enhance the proximal tubular Na(+),K(+)-ATPase activity. However, the lack of renal dopamine appears not to be related with the overall renal sodium retention in a state of proteinuria.     

Protective effect of radical scavenger edaravone against puromycin nephrosis

AIM: Recent studies have indicated that reactive oxygen species (ROS) play a role in the pathogenesis of glomerular injury leading to proteinuria in nephrotic syndrome. In the present investigation, we examined the effects of the radical scavenger edaravone administered at various time points to rats with puromycin nephrosis. MATERIALS AND METHODS: 35 Wistar rats were divided into five groups: treatment with puromycin aminonucleoside (PAN) alone, treatment with PAN followed by edaravone in the early period, treatment with PAN followed by edaravone administration in the late period, treatment with PAN and administration of edaravone for the whole experimental period, and untreated controls. On Days 3, 6 and 9, urinary protein excretion was measured. The levels of glomerular thiobarbituric acid-reactive substance (TBArs) were determined in all animals on Day 10. RESULTS: On Day 9, rats that had been administered edaravone showed reduced urinary protein excretion and reduced glomerular TBArs. In particular, edaravone administration in the late period, during which proteinuria was most acute, had the effect of reducing the severity of proteinuria. Glomerular TBArs were suppressed to the control level. Our results indicate that edaravone exerts a protective effect in the acute phase of PAN nephrosis when administered as antioxidant therapy at the onset of proteinuria. CONCLUSIONS: Edaravone can ameliorate urinary protein excretion after the onset of proteinuria in nephrotic syndrome.     

Salt-sensitivity of blood pressure and decreased 11beta-hydroxysteroid dehydrogenase type 2 activity after renal transplantation

BACKGROUND: High blood pressure (BP) predicts a poor long-term kidney graft outcome. The mechanisms for hypertension in renal graft recipients are only partly understood. There is evidence that BP is salt dependent in renal transplant recipients. We hypothesize that renal transplantation induces salt sensitivity by decreasing 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) activity. METHODS: A syngenic uninephrectomized rat transplantation model (Lewis to Lewis) (n=7) was used to demonstrate salt sensitivity after transplantation. Sham-operated (n=5) and denervated rats (n=5) were used as controls. In all rats, BP was measured continuously by telemetry 24 hr a day, whereas the rats were set successively on a normal- (0.45% NaCl), high- (8% NaCl), low- (0.1% NaCl), and, again, normal-salt (0.45% NaCl) diet during a 6-day period to assess salt-related changes in mean arterial pressure (MAP). 11betaHSD2 activity was assessed by determining the ratio of corticosterone to dehydrocorticosterone metabolites (THB+5alphaTHB)/THA in urine. RESULTS: After uninephrectomy and implantation of the telemetry device, MAP was comparable in rats assigned to undergo sham operation (100+/-3 mmHg), denervation (105+/-5 mmHg), or transplantation (102+/-6 mmHg). When animals were switched from the normal- to high-salt diet, the increase in MAP was more pronounced in the transplanted group (13.9+/-5.1 mmHg) than in those undergoing sham operation (5.1+/-1.7 mmHg, P<0.004) or denervation (7.1+/-1.8 mmHg, P<0.021). Urinary (THB+5alphaTHB)/THA increased more than 2-fold in the transplanted rats but remained stable in the sham-operated and denervated animals (P<0.0001), indicating reduced activity of 11betaHSD2. CONCLUSION: Syngenic renal transplantation causes salt sensitivity with increased BP associated with a reduced activity of 11betaHSD2.     

Jejunal dopamine and Na,K+-ATPase activity in nephrotic syndrome

BACKGROUND: The occurrence of complementary functions in sodium transport between the intestine and the kidney was suggested to occur when the renal function is immature or compromised and jejunal dopamine has been implicated in this renal-intestinal cross-talk. The jejunal sodium transport was not previously evaluated in the nephrotic syndrome. METHODS: We examined the jejunal Na(+),K(+)-ATPase activity and the role of dopamine in puromycin aminonucleoside (PAN) and HgCl(2)-induced nephrotic syndrome rat models. RESULTS: In both nephrotic syndrome rat models, the jejunal Na(+),K(+)-ATPase activity was reduced during greatest sodium retention and ascites accumulation (PAN nephrosis, day 7; HgCl(2) nephrosis, day 14), whereas during enhanced sodium excretion and ascites mobilization the jejunal Na(+),K(+)-ATPase activity was increased in HgCl(2) nephrosis (day 21) and was similar to controls in PAN nephrosis (day 14). In both PAN- and HgCl(2)-induced nephrosis, the jejunal aromatic L-amino acid decarboxylase (AADC) activity, the enzyme responsible for the synthesis of jejunal dopamine, did not differ from controls. In addition, the jejunal Na(+),K(+)-ATPase activity was not sensitive to inhibition by dopamine (1 microM) in both experimental groups throughout the study. CONCLUSIONS: In the nephrotic syndrome the jejunal Na(+),K(+)-ATPase activity may respond in a compensatory way to changes in extracellular volume, through dopamine-independent mechanisms.     

In vivo microvascular clearance of albumin in renal and extrarenal tissues in puromycin aminonucleoside (PAN) induced nephrotic syndrome.

BACKGROUND: The nephrotic syndrome is characterized by generalized oedema considered to be due to the fall in serum albumin and to sodium retention. The aim of the present study was to investigate whether a generalized disturbance in vascular integrity contributes to the oedema formation. METHODS: We used the PAN-(puromycin aminonucleoside) nephritis model in order to induce the nephrotic syndrome in female Wistar rats. Eight rats were given PAN, 15 mg/100 g body weight, intraperitoneally 10 days prior to the study, whereas 21 rats served as controls. Albumin clearance to tissues was measured using a dual isotope technique. Repeated blood samples as well as samples from various muscles, kidney, liver, lung, heart, abdominal wall and from ascites fluid were taken to determine radioactivity and tissue dry-to-wet weights. Clearance of albumin (Clalb) from plasma to interstitium was calculated from the (linear) increment in 'plasma equivalent tissue albumin space' as a function of time, corrected for intravascular volume and oedema. The plasma and urine concentrations of albumin were determined in a parallel study by single radial diffusion using monospecific rabbit anti-rat antiserum in seven PAN animals and 13 controls. RESULTS: A marked fall in dry-to-wet weight ratios together with pronounced proteinuria, oedema and ascites were found in the PAN animals. Haematocrit decreased from 45% (32-51) to 30% (28-38) and serum albumin from 22.0 g/l (16.3-25.2) to 4.94 g/l (3.20-6.72) in control and PAN animals, respectively. However, Clalb apparently remained unchanged in the PAN animals in comparison to controls in most tissues examined. Thus, in these in vivo experiments there was no direct evidence of an increased extravasation of albumin in extrarenal tissues. CONCLUSIONS: There was no strong support for the contention that a generalized disturbance of capillary integrity outside the renal vasculature would contribute to the oedema formation in the PAN nephrotic syndrome.     

Differences in puromycin aminonucleoside nephrosis in two rat strains

Administration of puromycin aminonucleoside (PAN) to Wistar rats induces proteinuria and enhanced mesangial deposition of circulating macromolecules. After proteinuria of longer duration focal and segmental glomerular hyalinosis and sclerosis (FSGHS) develops. The present report analyzes these aspects of PAN nephrosis in PVG/c rats, a strain previously shown to be remarkably resistant to proteinuria and FSGHS with aging or after uninephrectomy. In Wistar rats multiple injections of PAN during five months resulted in sustained severe proteinuria and FSGHS lesions in 8.1 +/- 1.0% (mean +/- 1 SEM) of their glomeruli (N = 6). In PVG/c rats a 1.3-fold higher dose of PAN was needed to induce chronic proteinuria similar to the Wistar rats. After five months 3.3 +/- 0.9% of their glomeruli showed FSGHS (N = 6, P less than 0.01) and the glomerular lesions were considerably less advanced. In acute PAN nephrosis induced by a single intravenous injection of PAN the mesangium of Wistar rats contained large amounts of lipid in contrast to a few small mesangial lipid droplets in nephrotic PVG/c rats. After injection of colloidal carbon in nephrotic PVG/c rats no enhanced carbon accumulation was found in the mesangium when compared to nonproteinuric controls. This result clearly differs from the increased mesangial sequestration of circulating material in nephrotic Wistar, and most other rat strains. The unchanged mesangial traficking of macromolecules in nephrotic PVG/c rats and the low incidence of FSGHS lesions in the presence of sustained glomerular proteinuria may reflect a relative resistance to PAN-induced glomerular damage in this particular rat strain.     

Glycyrrhetinic acid decreases plasma potassium concentrations in patients with anuria.

ABSTRACT. Licorice-associated hypertension is thought to be due to increased renal sodium retention. The active compound of licorice, glycyrrhetinic acid (GA), inhibits renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) and by that mechanism increases access of cortisol to the mineralocorticoid receptor that causes renal sodium retention and potassium loss. In addition, a direct vascular effect of 11beta-HSD activity has recently been incriminated to promote hypertension, a contention based on in vitro observations. This investigation was designed to establish whether this extrarenal effect of 11beta-HSD is relevant for BP regulation and potassium concentrations in plasma. In a prospective, double-blind, cross-over study, seven patients with anuria on chronic hemodialysis were randomly assigned after a baseline period of 2 wk to placebo or GA (1 g/d) for 2 wk, separated by a washout phase of 3 wk. The ratio of plasma cortisol/cortisone, determined by gas chromatography-mass spectrometry, increased in all patients after GA intake (F = 9.705; P < 0.004), which indicates inhibition of 11beta-HSD. Twenty-four-hour BP values did not change throughout the study. The increase of the plasma cortisol/cortisone ratio was paralleled by a decline in the plasma potassium concentration in every patient. The mean +/- SD plasma potassium concentration decreased from 5.5 +/- 0.6 mM/L at baseline to 4.9 +/- 0.7 and 4.5 +/- 0.8 mM/L after 1 and 2 wk on GA, respectively (F = 9.934, P < 0.003). Extrarenal 11beta-HSD activity influences serum potassium concentrations but does not regulate BP independently of renal sodium retention.     

Decreased ATPase activity in adriamycin nephrosis is independent of proteinuria

In previous studies from this laboratory it has been shown that ATP-ase activity in situ in the glomerular basement membrane (GBM) is clearly reduced in rats rendered nephrotic after treatment with adriamycin (ADR). The question was raised whether this reduction of ATP-ase activity in the GBM is due to toxic activity of ADR or rather a result of the nephrotic condition per se. Therefore, we studied ATP-ase activity using the cerium-based method in kidneys from ADR-treated rats without proteinuria (48 hr after ADR injection), or with proteinuria (approximately 150 mg/24 hr) several weeks after ADR injection. Also kidneys from rats rendered nephrotic by surgical ablation and from non-nephrotic rats treated with local X-irradiation (2000 rads) as well as from normal control rats were studied. The results show that in the GBM of ADR-treated or irradiated rats, clear reduction of ATP-ase activity is observed irrespective of their proteinuria, whereas in the GBM of rats rendered nephrotic by renal ablation (approximately 156 mg/24 hr mean protein excretion) no reduction of enzyme activity is found. It is concluded that decreased ATP-ase activity of the glomerular filtration barrier in ADR-treated rats is due to an early toxic activity of this drug and not a result of the nephrotic state per se. In view of the identical results in X-irradiated rats, it is likely that ADR may act through production of toxic radicals leading to damage of this membrane-associated enzyme system.     

Interactions of mineralocorticoids and glucocorticoids in epithelial target tissues

BACKGROUND.: In Na+-transporting epithelial target tissues, such as mammalian kidney and the isolated toad bladder, glucocorticoids (GCs) do not normally elicit Na+ retention. In mammalian kidney, however, they do cause kaliuresis. The presence of 11beta-hydroxysteroid dehydrogenase isoform 2 (11beta-HSD2) in these target tissues inactivates the GCs, preventing them from accessing mineralocorticoid receptors (MRs) and stimulating Na+ transport. RESULTS.: The usually observed Na+ retention elicited by the mineralocorticoid aldosterone was blunted when the GC corticosterone was coadministered along with aldosterone. However, when corticosterone was administered along with a 11beta-HSD2 inhibitor, a strong Na+ transport was elicited by an MR-mediated mechanism. 11-Dehydrocorticosterone also blunted aldosterone-elicited Na+ transport in these target tissues. CONCLUSIONS.: 11beta-HSD2 appears to play two important roles in the epithelial target tissues, kidney and toad bladder. The first is to protect GC access to MR, and the second involves the product of the enzyme to regulate the magnitude of aldosterone-induced Na+ retention.