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Calcium-dependent diuretic system in preascitic liver cirrhosis
Research Article
Calcium-dependent diuretic system in preascitic liver cirrhosis q,qq G. Sansoè1,*, M. Aragno2, C.E. Tomasinelli2, L. Valfrè di Bonzo2, F. Wong3, M. Parola2 1
Division of Gastroenterology, Gradenigo Hospital, Torino, Italy; 2Department of Experimental Medicine and Oncology, University of Torino, Torino, Italy; 3Department of Medicine, Toronto General Hospital, Toronto, Ontario, Canada See Editorial, pages 790–792
Background & Aims: Extracellular Ca++ activates cell membrane calcium-sensing receptors (CaRs), leading to renal tubule production of prostaglandins E2 (PGE2), which decrease both sodium reabsorption in the thick ascending limb of Henle’s loop and free-water reabsorption in collecting ducts. Aims & Methods: To assess the activity of this diuretic system in experimental cirrhosis, we evaluated renal function, hormonal status, PGE2 urinary excretion, and renal tissue concentrations of Na+–K+–2Cl co-transporters (BSC-1) and CaRs in three groups of rats: one group of controls receiving 5% glucose solution (vehicle) intravenously and two groups of rats with CCl4-induced preascitic cirrhosis receiving either vehicle or 0.5 mg i.v. PolyL-Arginine (PolyAg), a CaR-selective agonist. Results: Compared to controls, cirrhotic rats showed reduced urine volume and sodium excretion (p <0.05). Western blot analysis revealed reduced CaRs and increased BSC-1 protein content in kidneys of cirrhotic rats compared with controls (all p <0.01). PolyAg-treated cirrhotic rats had their urine and sodium excretion returned to normal; PolyAg also increased renal plasma flow, PGE2 urinary excretion, and free-water clearance in cirrhotic rats (all p <0.01 v. untreated cirrhotic animals).
Keywords: Experimental liver cirrhosis; Ascites; Sodium retention; Calcium; Calcium-sensing receptor; Poly-L-Arginine. Received 13 December 2009; received in revised form 9 May 2010; accepted 14 May 2010; available online 24 July 2010 q These data were presented orally in part at the 2006 annual meeting of the American Association for the Study of Liver Diseases (AASLD), Boston, MA, USA (November 2006), in part at the 2007 annual meeting of the European Association for the Study of the Liver (EASL), Barcelona, Spain (April 2007), and in part at the Digestive Disease Week (DDW), Chicago, IL, USA (May 2009). qq This is an experimental animal study: all animals received humane care and experiments in rats were performed in compliance with the procedures outlined in the Italian Ministry of Health guidelines (No. 86/609/EEC) and according to the Principles of Laboratory Animal Care (NIH No. 85-23, revised in 1985). * Corresponding author. Address: Division of Gastroenterology, Gradenigo Hospital, Corso Regina Margherita 10, 10153 Torino, Italy. Tel.: +39 011 8151250; fax: +39 011 8974222. E-mail address: [email protected] (G. Sansoè). Abbreviations: A, aldosterone; AVP, vasopressin; BSA, bovine serum albumin; BSC-1, Na+–K+–2Cl cotransporter; CaRs, extracellular calcium/polyvalent cation-sensing receptors; CIN, inulin clearance; CK, potassium clearance; CNA, sodium clearance; CPAH, para-aminohippurate clearance; FEK, fractional excretion of potassium; FENa, fractional excretion of sodium; FlNa, filtered sodium load; F-WCl, free-water clearance; GFR, glomerular filtration rate; IN, inulin; N, norepinephrine; NO, nitric oxide; NO2/NO3, nitrites and nitrates; PAH, paraaminohippurate; PGE2, prostaglandin E2; PolyAg, Poly-L-Arginine; PRA, plasma renin activity; RPF, renal plasma flow; TAL, thick ascending limb.
Conclusions: In preascitic cirrhosis, sodium retention may be linked to down-regulation of renal CaRs and up-regulation of tubular sodium-retaining channels. Calcimimetic drugs normalize preascitic sodium retention. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Calcium is an established intracellular second messenger. Recently, the molecular identification of sensors for extracellular Ca++ – extracellular calcium/polyvalent cation-sensing receptors (CaRs) – suggests that Ca++ might also work as a messenger outside cells [1]. In the kidney, CaRs are expressed in the thick ascending limb (TAL) of Henle’s loop and in collecting ducts [2]. Parathyroid glands detect changes in the blood levels of calcium by means of CaRs [3]. CaRs have also been described in the mammary gland, the brain, and the bowel [4]. In these organs, stimulation of CaRs by extracellular Ca++ is coupled to G protein-linked intracellular signaling pathways. Key elements in these pathways include stimulation of various kinases and phospholipases leading to the synthesis of prostaglandin E2. In addition, the stimulation of CaRs by elevated levels of extracellular Ca++ leads to the inhibition of adenylate cyclase [5]. In the kidney, stimulation of tubular CaRs suppresses expression of water channels and vasopressin-dependent water reabsorption in the collecting duct, a putative defense mechanism against the formation of calcium-containing stones [6]. Moreover, CaRs stimulation in the TAL of Henle’s loop reduces the amount of sodium–potassium–chloride co-transporters and leads to an increased natriuresis (Fig. 1) [7]. Retention of sodium and water is a frequent complication of liver cirrhosis. In rats with preascitic liver cirrhosis, Jonassen and colleagues demonstrated hypertrophy of the TAL of Henle’s loop crossing the outer medulla of kidney [8,9]. Those rats retained sodium before ascites development and showed normal GFR, proximal tubular sodium handling, and plasma aldosterone levels. The natriuretic response to furosemide and the medullary interstitial sodium concentration were also increased [10,11], suggesting primary sodium retention in the medullary TAL of Henle’s loop. In addition, in rats with CCl4-induced liver cirrhosis, significantly increased amount of Na+–K+–2Cl co-transporters was found in the TAL of Henle’s loop [12].
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JOURNAL OF HEPATOLOGY was induced by CCl4 (Riedel de Haen, Sigma–Aldrich, Seelze, Germany) administered by gavage twice weekly. Cirrhotic rats were studied 9 weeks after starting the cirrhosis induction program, when cirrhosis was fully developed. Control rats were studied following a similar period of standardized diet. Experiments in rats were performed in compliance with the procedures outlined in the Italian Ministry of Health guidelines (No. 86/609/EEC) and according to the Principles of Laboratory Animal Care (NIH No. 85-23, revised in 1985). PolyAg (molecular weight: 5000–15,000) a specific CaRs agonist, was provided by Sigma–Aldrich, Seelze, Germany. Animal groups PolyAg (0.5 mg) was dissolved in 1 ml 5% glucose solution as vehicle to obtain the solution (F0.5) to be administered intravenously to the rats. The rats were divided into three groups: eight control rats receiving 1 ml vehicle alone (G1), 10 cirrhotic rats receiving 1 ml vehicle (G2), and 10 cirrhotic rats receiving F0.5 (G3). Study protocol
Fig. 1. Mechanism of calcium-dependent inhibition of Na+–K+–2Cl cotransport in the thick ascending limb of Henle’s loop. CaRs, Calcium-sensing receptor; COX2, cyclo-oxygenase; PGE2, prostaglandin E2; 20-HETE, 20-hydroxyeicosatetraenoic acid; P450, cytochrome P450; TAL, thick ascending limb, , positive charge; , negative charge; X, blockage of action. Reprinted with permission from Gut 2007; 56: 1117–1123, with modifications.
Recently, it has been shown that intravenous calcium loading significantly increases natriuresis and aquaresis in patients with compensated cirrhosis, suggesting selective sodium and water retention in Ca++-sensitive segments of the nephron, that is in tubular segments endowed with CaRs (TAL of Henle’s loop and collecting ducts) [13]. Poly-L-Arginine (PolyAg) and Poly-L-Lysine are synthetic polycationic polypeptides and specific agonists of CaRs. These compounds are called calcimimetic agents since they are able to elicit the effects of intravenous calcium loading, avoiding the metabolic effects of hypercalcemia (i.e., without metabolic acidosis) [14,15]. We hypothesize that in an animal model of preascitic cirrhosis the Henle’s loop contributes to subtle sodium retention. We further hypothesize that i.v. administration of PolyAg, by decreasing sodium reabsorption at the loop of Henle, should significantly improve sodium excretion in preascitic cirrhotic rats. Therefore, a major aim of the present study is to evaluate the diuretic and hormonal effects of i.v. PolyAg in rats with CCl4-induced preascitic cirrhosis and sodium retention. As CaRs content and function in the kidney of cirrhotic rats has never been studied, the secondary aim of this study is the assessment of the expression of CaRs and Na+–K+–2Cl co-transporters (BSC-1) in the tubular nephron of rats with experimental cirrhosis.
Materials and methods Studies were performed on anaesthetized male adult Wistar rats (Harlan Italy, Udine, Italy) with preascitic cirrhosis and anaesthetized male adult Wistar control rats. Both groups were fed ad libitum with standard chow and water. Cirrhosis
Rats were anaesthetized as described elsewhere [16]. Blood was sampled (time 0) by cardiac puncture (0.3 cc) and inulin (IN) 10% (w/v) (Laevosan-Gesellschaft, Linz/Donau, Austria) plus para-aminohippurate (PAH) 20% (w/v) (Nephrotest, BAG Gmbh, Munich, Germany) were administered into the caudal vein as a priming bolus followed by a continuous infusion for 150 min, in order to assess glomerular filtration rate (GFR) and renal plasma flow (RPF) at different times by means of their respective steady-state plasma clearances (CIN and CPAH) [17– 19]. The steady-state technique has the following advantages over the urine collection clearance method: catheterization of the bladder can be omitted, less analytical work is needed, exact timing of serum and urine samples is unnecessary, and more urine is available for other tests [18]. After 90 min (i.e., once IN and PAH steady-state plasma concentrations were reached), laparotomy was performed and the urinary bladder was emptied; a clamp was positioned on the urethral orifice. Cardiac blood was then sampled (time 1) to assess basal values of CIN and CPAH, and then either vehicle or F0.5 was injected as a single bolus (1 ml) into the right femoral vein. Cardiac blood was then sampled (0.3 cc) at precise intervals (20 min) for 1 h (times 2, 3, and 4) to measure plasma osmolality and concentrations of inulin, PAH, sodium, potassium, and calcium. This technical procedure (cardiac puncture to withdraw 0.3 ml of blood, followed by replacement of the amount of blood withdrawn with an equal volume of i.v. saline) has been employed in our laboratory for years. We never experienced any hemorrhagic shock in our rats and uniformly applied in control and cirrhotic animals, this sampling of small amounts of blood never induced significant hypotension or impairment of cardiac function [16,19]. Blood samples withdrawn 60 min after the injection of vehicle or F0.5 were also used to measure plasma concentrations of vasopressin (AVP), plasma renin activity (PRA), aldosterone (A), norepinephrine (N), and plasma concentration of nitrites and nitrates (NO2/NO3). The latter determination was performed in order to ascertain that PolyAg, injected as a single i.v. bolus dose, had not worked as a nitric oxide-donor, influencing the results of this protocol. At time four, upon collecting from the bladder the urine produced during the 60-min period after the administration of PolyAg or vehicle, the rats were killed. This urine was used to determine its osmolality and the excretion of sodium, potassium, and PGE2. In a further group of five anaesthetized cirrhotic rats, mean arterial pressure was evaluated by means of tail sphygmomanometry [16], before and 40 min after administration of 0.5 mg i.v. PolyAg in the caudal vein, without performing any laparotomy. Three out of these five rats were submitted to the measurement of 1-h urinary excretion of nitrites and nitrates [20] before and after i.v. PolyAg. CaRs and BSC-1 protein concentrations in rat kidneys For Western blot analysis, membrane fractions were prepared from kidneys removed from five rats in each experimental group (G1–G3) [19]. Blots were incubated with a rabbit polyclonal CaRs antibody (ABR Affinity BioReagents, Golden, CO, USA) and an antibody against b-actin (Sigma, St. Louis, Missouri, USA). Washing and incubation times, along with detection, were performed as standardized [16,19]. Densitometric quantification was performed using b-actin as an internal standard: before any comparison the net intensity of CaR bands in each experiment was normalized to the intensity of the corresponding b-actin band, used as an internal standard in order to evaluate the degree of non-specific protein content in the homogenate. The same procedure was performed for determination of BSC-1 protein concentrations in rat kidneys, with the exception that blots were incubated with a rabbit polyclonal BSC-1 antibody (Alpha Diagnostic International, San Antonio, TX, USA) and an antibody against b-actin [21].
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Research Article Kidney CaRs and BSC-1 indirect immunofluorescence
Renal CaRs and BSC-1 content and immunostaining
Indirect immunofluorescence was performed as described elsewhere [22]. Mouse monoclonal anti-CD31, an endothelial marker (BD-Pharmingen, Erembodegem, Belgium), and rabbit polyclonal anti-CaRs and anti-BSC-1 primary antibodies (see above) were used. As secondary antibodies, anti-mouse C3y-conjugated antibodies (Amersham Biosciences, Braunschweig, Germany), as well as FITC-conjugated anti-rabbit antibodies (Sigma–Aldrich, Milan, Italy), were used. At the end of the conventional immunofluorescence staining procedure, kidney sections were counterstained with the DNA fluorescent dye DAPI to visualize kidney cell nuclei [23].
CaRs appeared significantly reduced and, conversely, BSC-1 was significantly increased in the membrane fraction of renal tissue homogenate from cirrhotic animals (all p <0.03) (Fig. 2), without any difference in the expression between PolyAg-treated and untreated animals. CaRs- and BSC-1-positive tubular cells (TAL of Henle’s loop) were clearly identified in normal and in cirrhotic animal kidney sections (Fig. 3). Intense CaRs indirect immunofluorescence staining was detected in the sub-endothelial layers of renal arterioles located in the kidney medulla of cirrhotic rats (Fig. 4). In control and cirrhotic rats, CaRs immunostaining was also confirmed in medullary nephron tubular structures that did not contain BSC-1 or the endothelial marker CD31, i.e., in collecting ducts (Fig. 5).
Plasma and urine analyses Plasma and urinary concentrations of electrolytes and IN, and PAH plasma concentrations were measured as described elsewhere [19,24,25]. Plasma AVP, aldosterone, norepinephirne, and PRA were determined as described elsewhere [13,16,19]. Plasma and urine NO2/NO3 concentrations were determined through standard HPLC methods [20,26]. Urine samples were assayed for PGE2 concentrations by ELISA (Neogen, Lexington, KY, USA). Calculations Sodium and potassium clearances [CNa and CK] were calculated through the usual formula [13]. Inulin clearance (CIN) and para-aminohippurate clearance (CPAH) were calculated through the steady-state plasma clearance formula as: Cx ¼ Infusion rateðxÞ=ssP-x where ssP-x is the steady-state plasma concentration of x. CIN and CPAH were taken as measures of GFR and RPF, respectively [17,18]. Filtered sodium load (FlNa) was derived following Boer et al. [27]. Fractional sodium excretion (FENa) and fractional potassium excretion (FEK) were also calculated. Free-water clearance (F-WCl) was calculated, following Rose and Post [28], through the formula: F-WCl ¼ V Cosm where V is the urinary output (ml/min); Cosm is the osmolar clearance. All renal function parameters measured after vehicle or PolyAg administration were derived by computing the mean of three determinations of osmolality, inulin, PAH, sodium, potassium, and calcium in plasma during the 60 min urine collection period (blood sampling times 2, 3, and 4). Mean arterial pressure (MAP) was calculated from the formula: 1=3ðsystolic blood pressure diastolic blood pressureÞ
Hormonal status (Table 1) and mean arterial pressure Infusion of PolyAg caused a significant increase in plasma concentrations of the neurotransmitter N (p <0.03), as a consequence of the diuretic effect itself (see over), but had no effect on PRA, A, and AVP plasma levels in cirrhotic animals. Plasma renin did not rise in relation to a reduction in the extracellular fluid volume which was secondary to the diuretic effects of PolyAg. This most likely is related to renal overproduction of PGE2 (see over) and ensuing kidney vasodilatation. To put it differently, CaRs agonists, due to their effects on PGE2 production, are the only known diuretics increasing renal perfusion. Therefore, the difference between the mechanisms of adrenergic activation (reduced volume in systemic circulation) and renin activation (decreased delivery of Na+ to the distal tubule due to intra-renal hemodynamic changes) accounts for the reason why noradrenaline was activated while renin was not, in the cirrhotic animals receiving PolyAg. This drug significantly raised urinary excretion rates of PGE2 in cirrhotic animals, as expected when CaRs are specifically stimulated. Cirrhotic rats injected with PolyAg had no significant changes in plasma levels or urinary excretion of NO2/NO3. This
þ diastolic blood pressure:
CaRs
Morphological liver studies
BSC-1
Livers were removed from all rats submitted to CCl4-intoxication, and hepatic tissue samples for light microscopy were placed in buffered 4% formaldehyde solution (pH 7.4). The sections were stained with hematoxylin and eosin to assess fibrosis. Silver-impregnated liver sections were used to observe portal–central or central–central bridging fibrosis.
β-actin C
Micronodular cirrhosis with hepatocellular necrosis and micro/ macro-vacuolar steatosis was found in all the livers removed from CCl4-treated rats (data not shown). 858
Cir
% of controls
% of controls
C CaRs
100 80 60 40 20 0
C
Liver morphological studies
Cir 120
140 120 100 80 60 40 20 0
Results
Cir
BSC-1
Statistical analysis The main comparisons were between renal or hormonal parameters measured after administration of PolyAg or after vehicle alone. Results are expressed as means ± SD. All comparisons between groups of rats were made by one-tailed Wilcoxon Rank Sum Test for unpaired data. Significance is accepted at the 5% probability level.
Cir
Cir
C
Cir
Fig. 2. Western blots of representative experiments showing CaRs and Na+– K+–Cl cotransporter (BSC-1) levels in the membrane fractions of kidneys of control and cirrhotic rats receiving vehicle alone; c, control; cir, cirrhosis. bActin is an internal standard used to evaluate the degree of non-specific protein expression in tissue homogenate. CaRs are significantly reduced and BSC-1 significantly increased in the membrane fraction of renal tissue homogenate from cirrhotic animals (all p <0.03).
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JOURNAL OF HEPATOLOGY
Fig. 3. Left hand side: thick ascending limb of Henle’s loop intensely positive for BSC-1 protein in the kidney medulla of a cirrhotic rat. Right hand side: the same tubular structures that are positive for BSC-1 in red (TAL of Henle’s loop) are also positive for CaRs in green, in the outer layers of kidney medulla in cirrhotic rats. Magnification 200. Fig. 5. CaRs immunostaining in a medullary collecting duct (cirrhotic rat). Magnification 200.
Discussion
Fig. 4. Intense CaRs indirect immunofluorescence staining in the subendothelial layers of renal arterioles located in the kidney medulla of cirrhotic rats. CD31 is an endothelial marker. Magnification 200.
confirms that renal and hormonal effects described herein were not dependent on nitric oxide synthesis stimulation by PolyAg. PolyAg had no effect on blood pressure (89 ± 27 vs. 86 ± 37 mm Hg, p > 0.05) in cirrhotic rats. Renal function (Table 2) Cirrhotic rats receiving vehicle alone (G2) showed significantly reduced values of urine volume, absolute and fractional excretion of sodium with respect to controls (G1). Free-water clearance was also lower, and GFR and RPF were both higher, in cirrhotic animals with respect to controls, at baseline. In cirrhotic rats, 0.5 mg i.v. PolyAg significantly increased urine volume, absolute and fractional excretion of sodium, kaliuresis, free-water clearance, and renal plasma flow; while also significantly reducing plasma Ca++ levels. On the other hand, GFR was not significantly affected by PolyAg.
In patients or animal models with liver cirrhosis, sodium retention occurs before the development of ascites despite normal or increased glomerular filtration rate (GFR) [29–35]. The renal tubular site of early sodium retention is still ill-defined. Micropuncture studies showed that preascitic sodium-retaining dogs had normal delivery of tubular fluid to the Henle’s loop [36]. Accordingly, lithium clearance (an established index of distal tubular delivery of fluid) was found to be significantly reduced only in standing preascitic cirrhotic patients, whereas no significant changes were detected in supine compensated patients [37– 39] and in animals with experimental preascitic cirrhosis [8,9,40]. Indeed, in supine compensated patients with liver cirrhosis, retention of sodium was found in unidentified nephron segments that follow the proximal convoluted tubule [35]. Plasma renin activity and aldosterone levels are slightly decreased in early cirrhosis, suggesting that increased sodium reabsorption in aldosterone-sensitive nephron segments (i.e., distal convoluted tubule and collecting ducts) is unlikely to occur [35,41–43]. Nevertheless, treatment with mineralocorticoidreceptor antagonists can delay ascites development in experimental cirrhosis, suggesting that the distal nephron sensitivity to aldosterone might be increased in preascitic cirrhosis [44,45]. The greatest merit of studying a model of preascitic sodiumretaining rats with cirrhosis lies in the fact that interference and concomitant effects of different activated hormones and neurotransmitters do not occur at this stage of the disease, at variance with what happens in the ascitic stage. This study confirms the occurrence of renal tubular sodium retention (Table 2) in animals with experimental preascitic cirrhosis [8,9,36,40]. A further finding is the occurrence of reduced osmolar and free-water clearance, leading to reduced urine volume, in our cirrhotic rats with respect to healthy ones; this was detected when both groups were administered intravenously just 5% glucose solution, i.e., electrolyte-free water (Table 2). The main finding of the present study is that sodium and fluid retention is associated with decreased protein content of CaRs
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Research Article Table 1. Hormonal plasma, serum, and urine determinations in different groups of rats.
G1 (n = 8)
G2 (n = 10)
G3 (n = 10)
Plasma AVP (pg/ml)
108 ± 68
98 ± 12
95 ± 35
PRA (ng/ml/h)
24 ± 1
20 ± 2
21 ± 8
Plasma A (ng/ml)
2.5 ± 1.8
2.9 ± 1.7
2.7 ± 1.8
Plasma N (pg/ml)
125 ± 82
132 ± 84
245 ± 125
Serum NO2/NO3 (µM/L)
31 ± 6
67 ± 22b
48 ± 14
15 ± 3.2
12 ± 6.2
1.5 ± 0.1
6.4 ± 1.1d
Urinary NO2/NO3 (µM/h) Urinary PGE2 (ng/h)
2.1 ± 0.3
Data are means ± SD. G1: control rats receiving vehicle alone; G2: cirrhotic rats receiving vehicle alone; G3: cirrhotic rats receiving 0.5 mg PolyAg. a p <0.03 versus G2; b p <0.03 versus G1; c p <0.01 versus G1; d p <0.01 versus G2 (Wilcoxon rank sum test).
Table 2. Renal function data.
G1 ( n = 8) Urine volume (ml/h)
0.8 ± 0.1
G2 (n = 10) a
0.5 ± 0.1 a
G3 (n = 10) 0.9 ± 0.1b 141 ± 7c
Kaliuresis (µM/h)
142 ± 9 2.2 ± 0.3 137 ± 15
124 ± 8 1.4 ± 0.07a 102 ± 8a
FEK (%) Cosm (ml/h) F - WCl (ml/h) Uosm (mOsm/Kg H2O)
8.1 ± 2.2 0.21 ± 0.03 0.61 ± 0.07 1142 ± 226
3.1 ± 0.9a 0.12 ± 0.03d 0.40 ± 0.05d 1340 ± 399a
11.1 ± 3.1b 0.22 ± 0.04b 0.73 ± 0.08b 1254 ± 23c
GFR (ml/min)
Natriuresis (µM/h) FENa (%)
2.1 ± 0.06c 181 ± 9c
0.63 ± 0.21
1.02 ± 0.31a
0.79 ± 0.54
RPF (ml/min) Plasma Na+ (mEq/L)
0.96 ± 0.21 142.8 ± 6.5
1.26 ± 0.23a 143.3 ± 2.1
2.08 ± 0.6b 148.7 ± 8.1c
Plasma K+ (mEq/L) Plasma Ca2+ (mM/L)
4.5 ± 1 2.7 ± 0.6
4.7 ± 0.8 2.5 ± 0.3
4.2 ± 0.5c 2 ± 0.5c
Data are means ± SD. G1, control rats receiving vehicle alone; G2, cirrhotic rats receiving vehicle alone; G3, cirrhotic rats receiving 0.5 mg PolyAg. a p <0.05 versus G1; b p <0.01 versus G2; c p <0.05 versus G2; d p <0.03 versus G1 (Wilcoxon rank sum test).
(the mediators of diuretic effects of extracellular Ca++) in Henle’s loop and collecting ducts and increased amount of Na+–K+–2Cl co-transporters (BSC-1) in the TAL of Henle’s loop. The latter observation confirms previous findings in the literature [12]. When downregulated CaRs were stimulated with a specific polycationic agonist (PolyAg), we achieved a significant increase in urinary sodium and water excretion in cirrhotic rats, which led to parameters of renal function equal to those measured in controls (Table 2). At least in the present model of preascitic cirrhosis, this finding should be sufficient to rule out relevant sodium retention in aldosterone-dependent segments of the nephron, which are devoid of CaRs. The latter statements deserve further explanations. Decreased content of CaRs in renal tissue homogenate (Fig. 2) means that these receptors are downregulated just in the TAL of Henle’s loop and medullary collecting ducts but not in other portions of the nephron, since we found positive immunostaining for these proteins only in those tubular segments of control and cirrhotic rats
860
(Figs. 3 and 5), exactly as described in the literature [6,7]. Of course, upregulated BSC-1 protein was strictly located in the TAL of Henle’s loop (Fig. 3). By means of PolyAg injections, we were able to target CaRs: PolyAg caused a large increase in tubular production and urinary excretion of PGE2, the second messenger produced by CaRs-positive cells when these receptors are stimulated by increased extracellular Ca++ [5]. PolyAg also significantly decreased the plasma levels of Ca++, as occurs when parathyroid gland CaRs are targeted by calcimimetics [3]. Moreover, despite the possibility that arginine may represent a nitric oxide (NO) precursor, the synthetic polymer we used (PolyAg, molecular weight 5000–15,000) did not cause any increase in NO2/NO3 plasma levels or urinary excretion. This confirms that renal and hormonal effects described herein were not influenced by the stimulation of nitric oxide synthesis by PolyAg, at least when this compound was administered as a single bolus injection. PolyAg exerted its natriuretic effects in the TAL of Henle’s loop, where this compound reduced sodium–potassium–chloride co-transport by means of CaRs stimulation, as unequivocally indicated by the associated increase in sodium and potassium urinary excretion, eventually leading to decreased plasma potassium levels in cirrhotic rats (Table 2). Unlike other diuretics that exert their action in the TAL of Henle’s loop, such as furosemide, [46,47], PolyAg increased free-water clearance because CaRs agonists blunt vasopressin-dependent water reabsorption in the distal nephron [6,48]. A further finding of this study is the considerable renal vasodilatation, without systemic hypotension, that we obtained by means of PolyAg injection in cirrhotic rats (Table 2). This was due to significant expression of CaRs in the sub-endothelial layers of renal arterioles located in the outer medulla of kidneys of cirrhotic rats (Fig. 4), where these receptors may mediate PGE2 synthesis when stimulated by calcimimetic agents. In conclusion, as Jonassen and colleagues did with furosemide [8,11], and our group did with intravenous calcium loading [13], we showed remarkable natriuretic efficiency of a further diuretic agent targeting the TAL of Henle’s loop in preascitic cirrhosis: the CaRs agonist PolyAg. The clinical perspectives of the present study are, therefore, twofold. On the one hand, the information that calcimimetics normalize sodium excretion in preascitic cirrhosis by decreasing sodium reabsorption in the Henle’s loop means that a significant component of sodium retention does occur in the Henle’s loop through up-regulation of Na–K–2Cl co-transporters, prior to ascites development. This statement has been recently reinforced by our preliminary findings that pharmacological hyperparathyroidism, by physiologically reducing the overexpression of Na–K–2Cl cotransporters in the Henle’s loop [21], completely normalized urinary sodium excretion in rats with preascitic cirrhosis [49]. On the other hand, calcimimetic agents, especially orally active drugs of this kind [50] or oral calcium itself [13], due to their diuretic, aquaretic, and renal vasodilating properties might represent a promising tool to delay ascites development in patients with compensated cirrhosis, when administered chronically. Unfortunately, to date the experience with calcimimetic agents in humans is confined to treatment of secondary hyperparathyroidism and bone disease in patients with chronic renal failure. Nothing has been published about the use of these drugs in models or patients with cirrhosis.
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JOURNAL OF HEPATOLOGY Study highlights What is current knowledge s Tubular segmental contribution to sodium retention in preascitic cirrhosis is still ill-defined. s Intravenous calcium infusion does induce diuresis by reducing sodium reabsorption at the loop of Henle in preascitic human cirrhosis. s The mechanism of calcium-dependent natriuresis in liver cirrhosis is unknown. What is new here s The use of a calcimimetic drug, i.e., a specific agonist of membrane-bound calcium-sensing receptors (CaRs), can induce diuresis in an animal model of preascitic cirrhosis. s The biomolecular content of CaRs is decreased in the nephron of sodium-retaining rats with preascitic cirrhosis. s The biomolecular content of Na+–K+–2Cl co-transporters, normally down-regulated by CaRs stimulation, is increased in the Henle’s loop of preascitic cirrhotic rats, leading to primary sodium retention in this segment of the nephron. Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgement Supported by grants from the Italian Ministry of University and of Scientific Research (60%), 2006. References [1] Brown EM, Pollack MD, Seidman CE, Seidman JG, Chou YHW, Riccardi D, et al. Calcium-ion-sensing cell-surface receptors. N Engl J Med 1995;333: 234–240. [2] Riccardi D, Park J, Lee WS, Gamba G, Brown EM, Hebert SC. Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci USA 1995;92:131–135. [3] Okabe M, Graham A. The origin of the parathyroid gland. Proc Natl Acad Sci USA 2004;101:17716–17719. [4] Conigrave AD, Brown EM. Taste receptors in the gastrointestinal tract II. LAmino acid sensing by calcium sensing receptors: implications for GI physiology. Am J Physiol Gastrointest Liver Physiol 2006;291:G753–G761. [5] Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001;81:239–297. [6] Procino G, Carmosino M, Tamma G, Gouraud S, Laera A, Riccardi D, et al. Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells. Kidney Int 2004;66:2245–2255. [7] Wang WH, Lu M, Hebert SC. Cytochrome P-450 metabolites mediate extracellular Ca++-induced inhibition of apical Na+–K+–Cl channels in the thick ascending limb of Henle. Am J Physiol Cell Physiol 1996;271: C103–C111. [8] Jonassen TE, Marcussen N, Haugan K, Skyum H, Christensen S, Andreasen F, et al. Functional and structural changes in the thick ascending limb of Henle’s loop in rats with liver cirrhosis. Am J Physiol Regul Integr Comp Physiol 1997;273:R568–R577. [9] Jonassen TE, Christensen S, Sorensen AM, Marcussen N, Flyvbjerg A, Andreasen F, et al. Effects of chronic octreotide treatment on renal changes during compensated liver cirrhosis in rats. Hepatology 1999;29:1387–1395. [10] Jonassen TE, Nielsen S, Christensen S, Petersen JS. Decreased vasopressinmediated renal water reabsorption in rats with compensated liver cirrhosis. Am J Physiol Renal Physiol 1998;275:F216–F225.
[11] Jonassen TE, Sorensen AM, Petersen JS, Andreasen F, Christensen S. Increased natriuretic efficiency of furosemide in rats with carbon tetrachlorideinduced cirrhosis. Hepatology 2000;31:1224–1230. [12] Fernandez-Llama P, Ageloff S, Fernandez-Varo G, Ros J, Wang X, Garra N, et al. Sodium retention in cirrhotic rats is associated with increased renal abundance of sodium transporter proteins. Kidney Int 2005;67:622– 630. [13] Sansoè G, Wong F. Natriuretic and aquaretic effects of intravenously infused calcium in preascitic human cirrhosis: physiopathological and clinical implications. Gut 2007;56:1117–1123. [14] Wang D, Pedraza PL, Abdullah HI, McGiff JC, Ferreri R. Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells. Am J Physiol Renal Physiol 2002;283:F963–F970. [15] Nemeth EF, Fox J. Calcimimetic compounds: a direct approach to controlling plasma levels of parathyroid hormone in hyperparathyroidism. Trends Endocrinol Metab 1999;10:66–71. [16] Sansoè G, Aragno M, Mastrocola R, Restivo F, Mengozzi G, Smedile A, et al. Neutral endopeptidase (EC 3.4.24.11) in cirrhotic liver: a new target to treat portal hypertension? J Hepatol 2005;43:791–798. [17] Cole BR, Giangiacomo J, Ingelfinger JR, Robson AM. Measurement of renal function without urine collection. A critical evaluation of the constantinfusion technique for determination of inulin and para-aminohippurate. N Engl J Med 1972;287:1109–1114. [18] Schnurr E, Lahme W, Kuppers H. Measurement of renal clearance of inulin and PAH in the steady state without urine collection. Clin Nephrol 1980;13:26–29. [19] Sansoè G, Aragno M, Mastrocola R, Cutrìn JC, Silvano S, Mengozzi G, et al. Overexpression of kidney neutral endopeptidase (EC 3.4.24.11) and renal function in experimental cirrhosis. Am J Physiol Renal Physiol 2006;290: F1337–F1343. [20] Radisavljevic Z, George M, Dries DJ, Gamelli RL. Determination of intracellular and extracellular nitrite and nitrate by anion chromatography. J Liq Chromatogr 1996;19:1061–1079. [21] Wang W, Li C, Kwon TH, Miller T, Knepper MA, Frokiaer J, et al. Reduced expression of renal Na+ transporters in rats with PTH-induced hypercalcemia. Am J Physiol Renal Physiol 2004;286:F534–F545. [22] Parola M, Leonarduzzi G, Robino G, Albano E, Poli G, Dianzani MU. On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis. Free Radic Biol Med 1996;20:351–359. [23] Zamara E, Novo E, Marra F, Gentilini A, Romanelli RG, Caligiuri A, et al. 4Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells. J Hepatol 2004;40:60–68. [24] Roe JH, Epstein JH, Goldstein NP. A photometric method for the determination of inulin in plasma and urine. J Biol Chem 1949;178:839–845. [25] Smith HW, Finkelstein N, Alminosa L. The renal clearance of substituted hippuric acid derivatives and other aromatic acids in dog and men. J Clin Invest 1945;24:388–403. [26] Jedlickova V, Paluch Z, Alusik S. Determination of nitrate and nitrite by highperformance liquid chromatography in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2002;780:193–197. [27] Boer WH, Koomans HA, Dorhout-Mees EJ. Lithium clearance during paradoxical natriuresis of hypotonic expansion in man. Kidney Int 1987;32: 376–381. [28] Rose BD, Post TW, editors. Clinical physiology of acid–base and electrolyte disorders. Berlin: McGraw-Hill; 2001. p. 285–298. [29] Wong F, Massie D, Hsu P, Dudley F. Renal response to a saline load in wellcompensated alcoholic cirrhosis. Hepatology 1994;20:873–881. [30] Wong F, Liu P, Allidina Y, Blendis LM. Pattern of sodium handling and its consequences in pre-ascitic cirrhosis. Gastroenterology 1995;108: 1820–1827. [31] Wood LJ, Massie D, McLean AJ, Dudley F. Renal sodium retention in cirrhosis: tubular site and relation to hepatic disfunction. Hepatology 1988;8: 831–836. [32] Bernardi M, Di Marco C, Trevisani F, Fornalè L, Andreone P, Cursaro C, et al. Renal sodium retention during upright posture in preascitic cirrhosis. Gastroenterology 1993;105:188–193. [33] Wong F, Liu P, Tobe S, Morali G, Blendis L. Central blood volume in cirrhosis: measurement with radionuclide angiography. Hepatology 1994;19: 312–321. [34] Lewis FW, Adair O, Rector Jr WG. Arterial vasodilatation is not the cause of increased cardiac output in cirrhosis. Gastroenterology 1992;102: 1024–1029. [35] Sansoè G, Ferrari A, Baraldi E, Castellana CN, De Santis MC, Manenti F. Renal distal tubular handling of sodium in central fluid volume homoeostasis in preascitic cirrhosis. Gut 1999;45:750–755.
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[44] Jimènez W, Martìnez-Pardo A, Arroyo V, Bruix J, Rimola A, Gaya J, et al. Temporal relationship between hyperaldosteronism, sodium retention and ascites formation in rats with experimental cirrhosis. Hepatology 1985;5:245–250. [45] Jonassen TEN, Petersen JS, Sorensen AM, Andreasen F, Christensen S. Aldosterone receptor blockade inhibits increased furosemide-sensitive sodium reabsorption in rats with liver cirrhosis. J Pharmacol Exp Ther 1998;287:931–936. [46] Szatalowicz VL, Miller PD, Lacher JW, Gordon JA, Schrier RW. Comparative effects of diuretics on renal water excretion in hyponatremic oedematous disorders. Clin Sci 1982;62:235–242. [47] Anderson RJ, Chung HM, Kluge R, Schrier RW. Hyponatremia: a prospective analysis of its epidemiology and the pathogenetic role of vasopressin. Ann Intern Med 1985;102:164–171. [48] Sands JM, Naruse M, Baum M, Jo I, Hebert SC, Brown EM, et al. Apical extracellular calcium/polyvalent cation-sensing receptor regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct. J Clin Invest 1997;99:1399–1405. [49] Sansoè G, Aragno M, Novo E, Mastrocola R, Parola M. Identification of the Henle’s loop as a major site of sodium retention in experimental preascitic cirrhosis: effects of parathyroid hormone. J Hepatol 2010;52:S8. [50] Colloton M, Shatzen E, Miller G, Stehman-Breen G, Wada M, Lacey D, et al. Cinacalcet HCl attenuates parathyroid hyperplasia in a rat model of secondary hyperparathyroidism. Kidney Int 2005;67:467–476.
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Calcium-dependent diuretic system in preascitic liver cirrhosis q,qq G. Sansoè1,*, M. Aragno2, C.E. Tomasinelli2, L. Valfrè di Bonzo2, F. Wong3, M. Parola2 1
Division of Gastroenterology, Gradenigo Hospital, Torino, Italy; 2Department of Experimental Medicine and Oncology, University of Torino, Torino, Italy; 3Department of Medicine, Toronto General Hospital, Toronto, Ontario, Canada See Editorial, pages 790–792
Background & Aims: Extracellular Ca++ activates cell membrane calcium-sensing receptors (CaRs), leading to renal tubule production of prostaglandins E2 (PGE2), which decrease both sodium reabsorption in the thick ascending limb of Henle’s loop and free-water reabsorption in collecting ducts. Aims & Methods: To assess the activity of this diuretic system in experimental cirrhosis, we evaluated renal function, hormonal status, PGE2 urinary excretion, and renal tissue concentrations of Na+–K+–2Cl co-transporters (BSC-1) and CaRs in three groups of rats: one group of controls receiving 5% glucose solution (vehicle) intravenously and two groups of rats with CCl4-induced preascitic cirrhosis receiving either vehicle or 0.5 mg i.v. PolyL-Arginine (PolyAg), a CaR-selective agonist. Results: Compared to controls, cirrhotic rats showed reduced urine volume and sodium excretion (p <0.05). Western blot analysis revealed reduced CaRs and increased BSC-1 protein content in kidneys of cirrhotic rats compared with controls (all p <0.01). PolyAg-treated cirrhotic rats had their urine and sodium excretion returned to normal; PolyAg also increased renal plasma flow, PGE2 urinary excretion, and free-water clearance in cirrhotic rats (all p <0.01 v. untreated cirrhotic animals).
Keywords: Experimental liver cirrhosis; Ascites; Sodium retention; Calcium; Calcium-sensing receptor; Poly-L-Arginine. Received 13 December 2009; received in revised form 9 May 2010; accepted 14 May 2010; available online 24 July 2010 q These data were presented orally in part at the 2006 annual meeting of the American Association for the Study of Liver Diseases (AASLD), Boston, MA, USA (November 2006), in part at the 2007 annual meeting of the European Association for the Study of the Liver (EASL), Barcelona, Spain (April 2007), and in part at the Digestive Disease Week (DDW), Chicago, IL, USA (May 2009). qq This is an experimental animal study: all animals received humane care and experiments in rats were performed in compliance with the procedures outlined in the Italian Ministry of Health guidelines (No. 86/609/EEC) and according to the Principles of Laboratory Animal Care (NIH No. 85-23, revised in 1985). * Corresponding author. Address: Division of Gastroenterology, Gradenigo Hospital, Corso Regina Margherita 10, 10153 Torino, Italy. Tel.: +39 011 8151250; fax: +39 011 8974222. E-mail address: [email protected] (G. Sansoè). Abbreviations: A, aldosterone; AVP, vasopressin; BSA, bovine serum albumin; BSC-1, Na+–K+–2Cl cotransporter; CaRs, extracellular calcium/polyvalent cation-sensing receptors; CIN, inulin clearance; CK, potassium clearance; CNA, sodium clearance; CPAH, para-aminohippurate clearance; FEK, fractional excretion of potassium; FENa, fractional excretion of sodium; FlNa, filtered sodium load; F-WCl, free-water clearance; GFR, glomerular filtration rate; IN, inulin; N, norepinephrine; NO, nitric oxide; NO2/NO3, nitrites and nitrates; PAH, paraaminohippurate; PGE2, prostaglandin E2; PolyAg, Poly-L-Arginine; PRA, plasma renin activity; RPF, renal plasma flow; TAL, thick ascending limb.
Conclusions: In preascitic cirrhosis, sodium retention may be linked to down-regulation of renal CaRs and up-regulation of tubular sodium-retaining channels. Calcimimetic drugs normalize preascitic sodium retention. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Calcium is an established intracellular second messenger. Recently, the molecular identification of sensors for extracellular Ca++ – extracellular calcium/polyvalent cation-sensing receptors (CaRs) – suggests that Ca++ might also work as a messenger outside cells [1]. In the kidney, CaRs are expressed in the thick ascending limb (TAL) of Henle’s loop and in collecting ducts [2]. Parathyroid glands detect changes in the blood levels of calcium by means of CaRs [3]. CaRs have also been described in the mammary gland, the brain, and the bowel [4]. In these organs, stimulation of CaRs by extracellular Ca++ is coupled to G protein-linked intracellular signaling pathways. Key elements in these pathways include stimulation of various kinases and phospholipases leading to the synthesis of prostaglandin E2. In addition, the stimulation of CaRs by elevated levels of extracellular Ca++ leads to the inhibition of adenylate cyclase [5]. In the kidney, stimulation of tubular CaRs suppresses expression of water channels and vasopressin-dependent water reabsorption in the collecting duct, a putative defense mechanism against the formation of calcium-containing stones [6]. Moreover, CaRs stimulation in the TAL of Henle’s loop reduces the amount of sodium–potassium–chloride co-transporters and leads to an increased natriuresis (Fig. 1) [7]. Retention of sodium and water is a frequent complication of liver cirrhosis. In rats with preascitic liver cirrhosis, Jonassen and colleagues demonstrated hypertrophy of the TAL of Henle’s loop crossing the outer medulla of kidney [8,9]. Those rats retained sodium before ascites development and showed normal GFR, proximal tubular sodium handling, and plasma aldosterone levels. The natriuretic response to furosemide and the medullary interstitial sodium concentration were also increased [10,11], suggesting primary sodium retention in the medullary TAL of Henle’s loop. In addition, in rats with CCl4-induced liver cirrhosis, significantly increased amount of Na+–K+–2Cl co-transporters was found in the TAL of Henle’s loop [12].
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JOURNAL OF HEPATOLOGY was induced by CCl4 (Riedel de Haen, Sigma–Aldrich, Seelze, Germany) administered by gavage twice weekly. Cirrhotic rats were studied 9 weeks after starting the cirrhosis induction program, when cirrhosis was fully developed. Control rats were studied following a similar period of standardized diet. Experiments in rats were performed in compliance with the procedures outlined in the Italian Ministry of Health guidelines (No. 86/609/EEC) and according to the Principles of Laboratory Animal Care (NIH No. 85-23, revised in 1985). PolyAg (molecular weight: 5000–15,000) a specific CaRs agonist, was provided by Sigma–Aldrich, Seelze, Germany. Animal groups PolyAg (0.5 mg) was dissolved in 1 ml 5% glucose solution as vehicle to obtain the solution (F0.5) to be administered intravenously to the rats. The rats were divided into three groups: eight control rats receiving 1 ml vehicle alone (G1), 10 cirrhotic rats receiving 1 ml vehicle (G2), and 10 cirrhotic rats receiving F0.5 (G3). Study protocol
Fig. 1. Mechanism of calcium-dependent inhibition of Na+–K+–2Cl cotransport in the thick ascending limb of Henle’s loop. CaRs, Calcium-sensing receptor; COX2, cyclo-oxygenase; PGE2, prostaglandin E2; 20-HETE, 20-hydroxyeicosatetraenoic acid; P450, cytochrome P450; TAL, thick ascending limb, , positive charge; , negative charge; X, blockage of action. Reprinted with permission from Gut 2007; 56: 1117–1123, with modifications.
Recently, it has been shown that intravenous calcium loading significantly increases natriuresis and aquaresis in patients with compensated cirrhosis, suggesting selective sodium and water retention in Ca++-sensitive segments of the nephron, that is in tubular segments endowed with CaRs (TAL of Henle’s loop and collecting ducts) [13]. Poly-L-Arginine (PolyAg) and Poly-L-Lysine are synthetic polycationic polypeptides and specific agonists of CaRs. These compounds are called calcimimetic agents since they are able to elicit the effects of intravenous calcium loading, avoiding the metabolic effects of hypercalcemia (i.e., without metabolic acidosis) [14,15]. We hypothesize that in an animal model of preascitic cirrhosis the Henle’s loop contributes to subtle sodium retention. We further hypothesize that i.v. administration of PolyAg, by decreasing sodium reabsorption at the loop of Henle, should significantly improve sodium excretion in preascitic cirrhotic rats. Therefore, a major aim of the present study is to evaluate the diuretic and hormonal effects of i.v. PolyAg in rats with CCl4-induced preascitic cirrhosis and sodium retention. As CaRs content and function in the kidney of cirrhotic rats has never been studied, the secondary aim of this study is the assessment of the expression of CaRs and Na+–K+–2Cl co-transporters (BSC-1) in the tubular nephron of rats with experimental cirrhosis.
Materials and methods Studies were performed on anaesthetized male adult Wistar rats (Harlan Italy, Udine, Italy) with preascitic cirrhosis and anaesthetized male adult Wistar control rats. Both groups were fed ad libitum with standard chow and water. Cirrhosis
Rats were anaesthetized as described elsewhere [16]. Blood was sampled (time 0) by cardiac puncture (0.3 cc) and inulin (IN) 10% (w/v) (Laevosan-Gesellschaft, Linz/Donau, Austria) plus para-aminohippurate (PAH) 20% (w/v) (Nephrotest, BAG Gmbh, Munich, Germany) were administered into the caudal vein as a priming bolus followed by a continuous infusion for 150 min, in order to assess glomerular filtration rate (GFR) and renal plasma flow (RPF) at different times by means of their respective steady-state plasma clearances (CIN and CPAH) [17– 19]. The steady-state technique has the following advantages over the urine collection clearance method: catheterization of the bladder can be omitted, less analytical work is needed, exact timing of serum and urine samples is unnecessary, and more urine is available for other tests [18]. After 90 min (i.e., once IN and PAH steady-state plasma concentrations were reached), laparotomy was performed and the urinary bladder was emptied; a clamp was positioned on the urethral orifice. Cardiac blood was then sampled (time 1) to assess basal values of CIN and CPAH, and then either vehicle or F0.5 was injected as a single bolus (1 ml) into the right femoral vein. Cardiac blood was then sampled (0.3 cc) at precise intervals (20 min) for 1 h (times 2, 3, and 4) to measure plasma osmolality and concentrations of inulin, PAH, sodium, potassium, and calcium. This technical procedure (cardiac puncture to withdraw 0.3 ml of blood, followed by replacement of the amount of blood withdrawn with an equal volume of i.v. saline) has been employed in our laboratory for years. We never experienced any hemorrhagic shock in our rats and uniformly applied in control and cirrhotic animals, this sampling of small amounts of blood never induced significant hypotension or impairment of cardiac function [16,19]. Blood samples withdrawn 60 min after the injection of vehicle or F0.5 were also used to measure plasma concentrations of vasopressin (AVP), plasma renin activity (PRA), aldosterone (A), norepinephrine (N), and plasma concentration of nitrites and nitrates (NO2/NO3). The latter determination was performed in order to ascertain that PolyAg, injected as a single i.v. bolus dose, had not worked as a nitric oxide-donor, influencing the results of this protocol. At time four, upon collecting from the bladder the urine produced during the 60-min period after the administration of PolyAg or vehicle, the rats were killed. This urine was used to determine its osmolality and the excretion of sodium, potassium, and PGE2. In a further group of five anaesthetized cirrhotic rats, mean arterial pressure was evaluated by means of tail sphygmomanometry [16], before and 40 min after administration of 0.5 mg i.v. PolyAg in the caudal vein, without performing any laparotomy. Three out of these five rats were submitted to the measurement of 1-h urinary excretion of nitrites and nitrates [20] before and after i.v. PolyAg. CaRs and BSC-1 protein concentrations in rat kidneys For Western blot analysis, membrane fractions were prepared from kidneys removed from five rats in each experimental group (G1–G3) [19]. Blots were incubated with a rabbit polyclonal CaRs antibody (ABR Affinity BioReagents, Golden, CO, USA) and an antibody against b-actin (Sigma, St. Louis, Missouri, USA). Washing and incubation times, along with detection, were performed as standardized [16,19]. Densitometric quantification was performed using b-actin as an internal standard: before any comparison the net intensity of CaR bands in each experiment was normalized to the intensity of the corresponding b-actin band, used as an internal standard in order to evaluate the degree of non-specific protein content in the homogenate. The same procedure was performed for determination of BSC-1 protein concentrations in rat kidneys, with the exception that blots were incubated with a rabbit polyclonal BSC-1 antibody (Alpha Diagnostic International, San Antonio, TX, USA) and an antibody against b-actin [21].
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Research Article Kidney CaRs and BSC-1 indirect immunofluorescence
Renal CaRs and BSC-1 content and immunostaining
Indirect immunofluorescence was performed as described elsewhere [22]. Mouse monoclonal anti-CD31, an endothelial marker (BD-Pharmingen, Erembodegem, Belgium), and rabbit polyclonal anti-CaRs and anti-BSC-1 primary antibodies (see above) were used. As secondary antibodies, anti-mouse C3y-conjugated antibodies (Amersham Biosciences, Braunschweig, Germany), as well as FITC-conjugated anti-rabbit antibodies (Sigma–Aldrich, Milan, Italy), were used. At the end of the conventional immunofluorescence staining procedure, kidney sections were counterstained with the DNA fluorescent dye DAPI to visualize kidney cell nuclei [23].
CaRs appeared significantly reduced and, conversely, BSC-1 was significantly increased in the membrane fraction of renal tissue homogenate from cirrhotic animals (all p <0.03) (Fig. 2), without any difference in the expression between PolyAg-treated and untreated animals. CaRs- and BSC-1-positive tubular cells (TAL of Henle’s loop) were clearly identified in normal and in cirrhotic animal kidney sections (Fig. 3). Intense CaRs indirect immunofluorescence staining was detected in the sub-endothelial layers of renal arterioles located in the kidney medulla of cirrhotic rats (Fig. 4). In control and cirrhotic rats, CaRs immunostaining was also confirmed in medullary nephron tubular structures that did not contain BSC-1 or the endothelial marker CD31, i.e., in collecting ducts (Fig. 5).
Plasma and urine analyses Plasma and urinary concentrations of electrolytes and IN, and PAH plasma concentrations were measured as described elsewhere [19,24,25]. Plasma AVP, aldosterone, norepinephirne, and PRA were determined as described elsewhere [13,16,19]. Plasma and urine NO2/NO3 concentrations were determined through standard HPLC methods [20,26]. Urine samples were assayed for PGE2 concentrations by ELISA (Neogen, Lexington, KY, USA). Calculations Sodium and potassium clearances [CNa and CK] were calculated through the usual formula [13]. Inulin clearance (CIN) and para-aminohippurate clearance (CPAH) were calculated through the steady-state plasma clearance formula as: Cx ¼ Infusion rateðxÞ=ssP-x where ssP-x is the steady-state plasma concentration of x. CIN and CPAH were taken as measures of GFR and RPF, respectively [17,18]. Filtered sodium load (FlNa) was derived following Boer et al. [27]. Fractional sodium excretion (FENa) and fractional potassium excretion (FEK) were also calculated. Free-water clearance (F-WCl) was calculated, following Rose and Post [28], through the formula: F-WCl ¼ V Cosm where V is the urinary output (ml/min); Cosm is the osmolar clearance. All renal function parameters measured after vehicle or PolyAg administration were derived by computing the mean of three determinations of osmolality, inulin, PAH, sodium, potassium, and calcium in plasma during the 60 min urine collection period (blood sampling times 2, 3, and 4). Mean arterial pressure (MAP) was calculated from the formula: 1=3ðsystolic blood pressure diastolic blood pressureÞ
Hormonal status (Table 1) and mean arterial pressure Infusion of PolyAg caused a significant increase in plasma concentrations of the neurotransmitter N (p <0.03), as a consequence of the diuretic effect itself (see over), but had no effect on PRA, A, and AVP plasma levels in cirrhotic animals. Plasma renin did not rise in relation to a reduction in the extracellular fluid volume which was secondary to the diuretic effects of PolyAg. This most likely is related to renal overproduction of PGE2 (see over) and ensuing kidney vasodilatation. To put it differently, CaRs agonists, due to their effects on PGE2 production, are the only known diuretics increasing renal perfusion. Therefore, the difference between the mechanisms of adrenergic activation (reduced volume in systemic circulation) and renin activation (decreased delivery of Na+ to the distal tubule due to intra-renal hemodynamic changes) accounts for the reason why noradrenaline was activated while renin was not, in the cirrhotic animals receiving PolyAg. This drug significantly raised urinary excretion rates of PGE2 in cirrhotic animals, as expected when CaRs are specifically stimulated. Cirrhotic rats injected with PolyAg had no significant changes in plasma levels or urinary excretion of NO2/NO3. This
þ diastolic blood pressure:
CaRs
Morphological liver studies
BSC-1
Livers were removed from all rats submitted to CCl4-intoxication, and hepatic tissue samples for light microscopy were placed in buffered 4% formaldehyde solution (pH 7.4). The sections were stained with hematoxylin and eosin to assess fibrosis. Silver-impregnated liver sections were used to observe portal–central or central–central bridging fibrosis.
β-actin C
Micronodular cirrhosis with hepatocellular necrosis and micro/ macro-vacuolar steatosis was found in all the livers removed from CCl4-treated rats (data not shown). 858
Cir
% of controls
% of controls
C CaRs
100 80 60 40 20 0
C
Liver morphological studies
Cir 120
140 120 100 80 60 40 20 0
Results
Cir
BSC-1
Statistical analysis The main comparisons were between renal or hormonal parameters measured after administration of PolyAg or after vehicle alone. Results are expressed as means ± SD. All comparisons between groups of rats were made by one-tailed Wilcoxon Rank Sum Test for unpaired data. Significance is accepted at the 5% probability level.
Cir
Cir
C
Cir
Fig. 2. Western blots of representative experiments showing CaRs and Na+– K+–Cl cotransporter (BSC-1) levels in the membrane fractions of kidneys of control and cirrhotic rats receiving vehicle alone; c, control; cir, cirrhosis. bActin is an internal standard used to evaluate the degree of non-specific protein expression in tissue homogenate. CaRs are significantly reduced and BSC-1 significantly increased in the membrane fraction of renal tissue homogenate from cirrhotic animals (all p <0.03).
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Fig. 3. Left hand side: thick ascending limb of Henle’s loop intensely positive for BSC-1 protein in the kidney medulla of a cirrhotic rat. Right hand side: the same tubular structures that are positive for BSC-1 in red (TAL of Henle’s loop) are also positive for CaRs in green, in the outer layers of kidney medulla in cirrhotic rats. Magnification 200. Fig. 5. CaRs immunostaining in a medullary collecting duct (cirrhotic rat). Magnification 200.
Discussion
Fig. 4. Intense CaRs indirect immunofluorescence staining in the subendothelial layers of renal arterioles located in the kidney medulla of cirrhotic rats. CD31 is an endothelial marker. Magnification 200.
confirms that renal and hormonal effects described herein were not dependent on nitric oxide synthesis stimulation by PolyAg. PolyAg had no effect on blood pressure (89 ± 27 vs. 86 ± 37 mm Hg, p > 0.05) in cirrhotic rats. Renal function (Table 2) Cirrhotic rats receiving vehicle alone (G2) showed significantly reduced values of urine volume, absolute and fractional excretion of sodium with respect to controls (G1). Free-water clearance was also lower, and GFR and RPF were both higher, in cirrhotic animals with respect to controls, at baseline. In cirrhotic rats, 0.5 mg i.v. PolyAg significantly increased urine volume, absolute and fractional excretion of sodium, kaliuresis, free-water clearance, and renal plasma flow; while also significantly reducing plasma Ca++ levels. On the other hand, GFR was not significantly affected by PolyAg.
In patients or animal models with liver cirrhosis, sodium retention occurs before the development of ascites despite normal or increased glomerular filtration rate (GFR) [29–35]. The renal tubular site of early sodium retention is still ill-defined. Micropuncture studies showed that preascitic sodium-retaining dogs had normal delivery of tubular fluid to the Henle’s loop [36]. Accordingly, lithium clearance (an established index of distal tubular delivery of fluid) was found to be significantly reduced only in standing preascitic cirrhotic patients, whereas no significant changes were detected in supine compensated patients [37– 39] and in animals with experimental preascitic cirrhosis [8,9,40]. Indeed, in supine compensated patients with liver cirrhosis, retention of sodium was found in unidentified nephron segments that follow the proximal convoluted tubule [35]. Plasma renin activity and aldosterone levels are slightly decreased in early cirrhosis, suggesting that increased sodium reabsorption in aldosterone-sensitive nephron segments (i.e., distal convoluted tubule and collecting ducts) is unlikely to occur [35,41–43]. Nevertheless, treatment with mineralocorticoidreceptor antagonists can delay ascites development in experimental cirrhosis, suggesting that the distal nephron sensitivity to aldosterone might be increased in preascitic cirrhosis [44,45]. The greatest merit of studying a model of preascitic sodiumretaining rats with cirrhosis lies in the fact that interference and concomitant effects of different activated hormones and neurotransmitters do not occur at this stage of the disease, at variance with what happens in the ascitic stage. This study confirms the occurrence of renal tubular sodium retention (Table 2) in animals with experimental preascitic cirrhosis [8,9,36,40]. A further finding is the occurrence of reduced osmolar and free-water clearance, leading to reduced urine volume, in our cirrhotic rats with respect to healthy ones; this was detected when both groups were administered intravenously just 5% glucose solution, i.e., electrolyte-free water (Table 2). The main finding of the present study is that sodium and fluid retention is associated with decreased protein content of CaRs
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Research Article Table 1. Hormonal plasma, serum, and urine determinations in different groups of rats.
G1 (n = 8)
G2 (n = 10)
G3 (n = 10)
Plasma AVP (pg/ml)
108 ± 68
98 ± 12
95 ± 35
PRA (ng/ml/h)
24 ± 1
20 ± 2
21 ± 8
Plasma A (ng/ml)
2.5 ± 1.8
2.9 ± 1.7
2.7 ± 1.8
Plasma N (pg/ml)
125 ± 82
132 ± 84
245 ± 125
Serum NO2/NO3 (µM/L)
31 ± 6
67 ± 22b
48 ± 14
15 ± 3.2
12 ± 6.2
1.5 ± 0.1
6.4 ± 1.1d
Urinary NO2/NO3 (µM/h) Urinary PGE2 (ng/h)
2.1 ± 0.3
Data are means ± SD. G1: control rats receiving vehicle alone; G2: cirrhotic rats receiving vehicle alone; G3: cirrhotic rats receiving 0.5 mg PolyAg. a p <0.03 versus G2; b p <0.03 versus G1; c p <0.01 versus G1; d p <0.01 versus G2 (Wilcoxon rank sum test).
Table 2. Renal function data.
G1 ( n = 8) Urine volume (ml/h)
0.8 ± 0.1
G2 (n = 10) a
0.5 ± 0.1 a
G3 (n = 10) 0.9 ± 0.1b 141 ± 7c
Kaliuresis (µM/h)
142 ± 9 2.2 ± 0.3 137 ± 15
124 ± 8 1.4 ± 0.07a 102 ± 8a
FEK (%) Cosm (ml/h) F - WCl (ml/h) Uosm (mOsm/Kg H2O)
8.1 ± 2.2 0.21 ± 0.03 0.61 ± 0.07 1142 ± 226
3.1 ± 0.9a 0.12 ± 0.03d 0.40 ± 0.05d 1340 ± 399a
11.1 ± 3.1b 0.22 ± 0.04b 0.73 ± 0.08b 1254 ± 23c
GFR (ml/min)
Natriuresis (µM/h) FENa (%)
2.1 ± 0.06c 181 ± 9c
0.63 ± 0.21
1.02 ± 0.31a
0.79 ± 0.54
RPF (ml/min) Plasma Na+ (mEq/L)
0.96 ± 0.21 142.8 ± 6.5
1.26 ± 0.23a 143.3 ± 2.1
2.08 ± 0.6b 148.7 ± 8.1c
Plasma K+ (mEq/L) Plasma Ca2+ (mM/L)
4.5 ± 1 2.7 ± 0.6
4.7 ± 0.8 2.5 ± 0.3
4.2 ± 0.5c 2 ± 0.5c
Data are means ± SD. G1, control rats receiving vehicle alone; G2, cirrhotic rats receiving vehicle alone; G3, cirrhotic rats receiving 0.5 mg PolyAg. a p <0.05 versus G1; b p <0.01 versus G2; c p <0.05 versus G2; d p <0.03 versus G1 (Wilcoxon rank sum test).
(the mediators of diuretic effects of extracellular Ca++) in Henle’s loop and collecting ducts and increased amount of Na+–K+–2Cl co-transporters (BSC-1) in the TAL of Henle’s loop. The latter observation confirms previous findings in the literature [12]. When downregulated CaRs were stimulated with a specific polycationic agonist (PolyAg), we achieved a significant increase in urinary sodium and water excretion in cirrhotic rats, which led to parameters of renal function equal to those measured in controls (Table 2). At least in the present model of preascitic cirrhosis, this finding should be sufficient to rule out relevant sodium retention in aldosterone-dependent segments of the nephron, which are devoid of CaRs. The latter statements deserve further explanations. Decreased content of CaRs in renal tissue homogenate (Fig. 2) means that these receptors are downregulated just in the TAL of Henle’s loop and medullary collecting ducts but not in other portions of the nephron, since we found positive immunostaining for these proteins only in those tubular segments of control and cirrhotic rats
860
(Figs. 3 and 5), exactly as described in the literature [6,7]. Of course, upregulated BSC-1 protein was strictly located in the TAL of Henle’s loop (Fig. 3). By means of PolyAg injections, we were able to target CaRs: PolyAg caused a large increase in tubular production and urinary excretion of PGE2, the second messenger produced by CaRs-positive cells when these receptors are stimulated by increased extracellular Ca++ [5]. PolyAg also significantly decreased the plasma levels of Ca++, as occurs when parathyroid gland CaRs are targeted by calcimimetics [3]. Moreover, despite the possibility that arginine may represent a nitric oxide (NO) precursor, the synthetic polymer we used (PolyAg, molecular weight 5000–15,000) did not cause any increase in NO2/NO3 plasma levels or urinary excretion. This confirms that renal and hormonal effects described herein were not influenced by the stimulation of nitric oxide synthesis by PolyAg, at least when this compound was administered as a single bolus injection. PolyAg exerted its natriuretic effects in the TAL of Henle’s loop, where this compound reduced sodium–potassium–chloride co-transport by means of CaRs stimulation, as unequivocally indicated by the associated increase in sodium and potassium urinary excretion, eventually leading to decreased plasma potassium levels in cirrhotic rats (Table 2). Unlike other diuretics that exert their action in the TAL of Henle’s loop, such as furosemide, [46,47], PolyAg increased free-water clearance because CaRs agonists blunt vasopressin-dependent water reabsorption in the distal nephron [6,48]. A further finding of this study is the considerable renal vasodilatation, without systemic hypotension, that we obtained by means of PolyAg injection in cirrhotic rats (Table 2). This was due to significant expression of CaRs in the sub-endothelial layers of renal arterioles located in the outer medulla of kidneys of cirrhotic rats (Fig. 4), where these receptors may mediate PGE2 synthesis when stimulated by calcimimetic agents. In conclusion, as Jonassen and colleagues did with furosemide [8,11], and our group did with intravenous calcium loading [13], we showed remarkable natriuretic efficiency of a further diuretic agent targeting the TAL of Henle’s loop in preascitic cirrhosis: the CaRs agonist PolyAg. The clinical perspectives of the present study are, therefore, twofold. On the one hand, the information that calcimimetics normalize sodium excretion in preascitic cirrhosis by decreasing sodium reabsorption in the Henle’s loop means that a significant component of sodium retention does occur in the Henle’s loop through up-regulation of Na–K–2Cl co-transporters, prior to ascites development. This statement has been recently reinforced by our preliminary findings that pharmacological hyperparathyroidism, by physiologically reducing the overexpression of Na–K–2Cl cotransporters in the Henle’s loop [21], completely normalized urinary sodium excretion in rats with preascitic cirrhosis [49]. On the other hand, calcimimetic agents, especially orally active drugs of this kind [50] or oral calcium itself [13], due to their diuretic, aquaretic, and renal vasodilating properties might represent a promising tool to delay ascites development in patients with compensated cirrhosis, when administered chronically. Unfortunately, to date the experience with calcimimetic agents in humans is confined to treatment of secondary hyperparathyroidism and bone disease in patients with chronic renal failure. Nothing has been published about the use of these drugs in models or patients with cirrhosis.
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JOURNAL OF HEPATOLOGY Study highlights What is current knowledge s Tubular segmental contribution to sodium retention in preascitic cirrhosis is still ill-defined. s Intravenous calcium infusion does induce diuresis by reducing sodium reabsorption at the loop of Henle in preascitic human cirrhosis. s The mechanism of calcium-dependent natriuresis in liver cirrhosis is unknown. What is new here s The use of a calcimimetic drug, i.e., a specific agonist of membrane-bound calcium-sensing receptors (CaRs), can induce diuresis in an animal model of preascitic cirrhosis. s The biomolecular content of CaRs is decreased in the nephron of sodium-retaining rats with preascitic cirrhosis. s The biomolecular content of Na+–K+–2Cl co-transporters, normally down-regulated by CaRs stimulation, is increased in the Henle’s loop of preascitic cirrhotic rats, leading to primary sodium retention in this segment of the nephron. Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgement Supported by grants from the Italian Ministry of University and of Scientific Research (60%), 2006. References [1] Brown EM, Pollack MD, Seidman CE, Seidman JG, Chou YHW, Riccardi D, et al. Calcium-ion-sensing cell-surface receptors. N Engl J Med 1995;333: 234–240. [2] Riccardi D, Park J, Lee WS, Gamba G, Brown EM, Hebert SC. Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci USA 1995;92:131–135. [3] Okabe M, Graham A. The origin of the parathyroid gland. Proc Natl Acad Sci USA 2004;101:17716–17719. [4] Conigrave AD, Brown EM. Taste receptors in the gastrointestinal tract II. LAmino acid sensing by calcium sensing receptors: implications for GI physiology. Am J Physiol Gastrointest Liver Physiol 2006;291:G753–G761. [5] Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001;81:239–297. [6] Procino G, Carmosino M, Tamma G, Gouraud S, Laera A, Riccardi D, et al. Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells. Kidney Int 2004;66:2245–2255. [7] Wang WH, Lu M, Hebert SC. Cytochrome P-450 metabolites mediate extracellular Ca++-induced inhibition of apical Na+–K+–Cl channels in the thick ascending limb of Henle. Am J Physiol Cell Physiol 1996;271: C103–C111. [8] Jonassen TE, Marcussen N, Haugan K, Skyum H, Christensen S, Andreasen F, et al. Functional and structural changes in the thick ascending limb of Henle’s loop in rats with liver cirrhosis. Am J Physiol Regul Integr Comp Physiol 1997;273:R568–R577. [9] Jonassen TE, Christensen S, Sorensen AM, Marcussen N, Flyvbjerg A, Andreasen F, et al. Effects of chronic octreotide treatment on renal changes during compensated liver cirrhosis in rats. Hepatology 1999;29:1387–1395. [10] Jonassen TE, Nielsen S, Christensen S, Petersen JS. Decreased vasopressinmediated renal water reabsorption in rats with compensated liver cirrhosis. Am J Physiol Renal Physiol 1998;275:F216–F225.
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