The extracellular calcium-sensing receptor promotes porcine egg activation via calcium/calmodulin-dependent protein kinase II.

Extracellular calcium is required for intracellular Ca2+ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium-sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R-568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R-568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation-dependent [Ca2+ ]i rise. When egg activation was conducted in intracellular Ca2+ chelator BAPTA-AM and NPS R-568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca2+ ]i effector p-CAMKII, and the presence of KN-93, an inhibitor of CAMKII, significantly reduced the CASR-mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca2+ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII.

Identification of a Calcium-sensing Receptor in Human Dental Pulp Cells That Regulates Mineral Trioxide Aggregate-induced Mineralization.

INTRODUCTION: The purpose of this study was to verify the expression of the calcium-sensing receptor (CaSR) and its role in mineral trioxide aggregate (MTA)-induced odontoblastic differentiation and mineralization in human dental pulp cells (hDPCs). METHODS: The expression of CaSR in human dental pulp tissue and hDPCs was detected using immunohistochemical and immunofluorescent assays. Then, hDPCs were cultured in specific medium supplemented with defined concentrations of MTA dilute alone or in combination with calcimimetic R-568 (a positive allosteric modulator of CaSR [Tocris Bioscience, Bristol, UK]), and cell viability was monitored by Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) analysis. Alkaline phosphatase activity, alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot were used to investigate the gene/protein expression of odontoblastic-associated markers and CaSR in medium supplemented with different combinations of diluted MTA, R-568, and calcilytic Calhex 231 (a negative allosteric modulator of CaSR [Sigma-Aldrich, St Louis, MO]). RESULTS: CaSR was slightly expressed in the central pulp tissue, whereas it was strongly expressed in the odontoblast layer, plasma membrane, and cytoplasm of hDPCs. Cell Counting Kit-8 assay indicated maximum cell viability in cultures treated with 1:8 diluted MTA additives. Compared with undifferentiated controls, the cells at the early stage of odontoblastic differentiation exhibited lower CaSR protein expression. The combination of 1:8 diluted MTA with 0.1 and 1.0 µmol/L R-568 led to significantly increased cell vitality but decreased alkaline phosphatase activity and mineralized deposit formation, and this negative effect could be attenuated by 1.0 µmol/L Calhex 231 supplementation. Quantitative polymerase chain reaction results showed a significant up-regulation of RUNX2, DSPP, DMP-1, and OCN gene expression in the 1 µmol/L R-568-treated hDPCs. Western blot analysis indicated that the treatment by MTA and R-568 alone or their combination gave no clear trend on the protein levels of CaSR and dentin sialophosphoprotein, whereas Calhex 231 can increase their expressions. In addition, the up-regulation of Akt phosphorylation was observed in R-568- and Calhex 231-treated hDPCs. CONCLUSIONS: Our data indicated that CaSR is expressed in human dental pulp and hDPCs and that it can negatively or positively regulate MTA-induced mineralization of hDPCs via the phosphoinositide 3-kinase/Akt pathway in a ligand-dependent manner, suggesting a therapeutic target for modulating reparative dentin formation.

Astragalosides increase the cardiac diastolic function and regulate the "Calcium sensing receptor-protein kinase C-protein phosphatase 1" pathway in rats with heart failure.

This study was designed to investigate the effects of astragalosides on cardiac diastolic function, and an emphasis was placed on the variation of the upstream molecular regulators of phospholamban. Chronic heart failure (CHF) rats were induced by ligaturing the left anterior coronary artery, and rats in the therapeutic groups were treated with either a 50 mg/kg dose of captopril, 10 mg/kg dose of astragalosides or 20 mg/kg dose of astragalosides. Four weeks after treatment, the ratio of the early and atrial peak filling velocities (E/A) and maximal slope diastolic pressure decrement (-dp/dt) both decreased in CHF rats (by 30.3% and 25.5%, respectively) and significantly increased in 20 mg/kg astragalosides and captopril-treated rats. The protein phosphatase-1 activity was lower in the 20 mg/kg astragalosides group than in the CHF group (0.22 vs 0.44, P < 0.01), and the inhibitor-1 levels in the astragalosides and captopril-treated groups were increased. Chronic heart failure increased expression of protein kinase C-α and calcium-sensing receptor, and these changes were attenuated by astragalosides therapy. Astragalosides restored the diastolic dysfunction of chronic heart failure rats, possibly by downregulation of calcium-sensing receptor and protein kinase C-α, which in turn augmented inhibitor-1 expression, reduced protein phosphatase-1 activity and increased phospholamban phosphorylation.

A Forward Genetic Screen in Zebrafish Identifies the G-Protein-Coupled Receptor CaSR as a Modulator of Sensorimotor Decision Making.

Animals continuously integrate sensory information and select contextually appropriate responses. Here, we show that zebrafish larvae select a behavioral response to acoustic stimuli from a pre-existing choice repertoire in a context-dependent manner. We demonstrate that this sensorimotor choice is modulated by stimulus quality and history, as well as by neuromodulatory systems-all hallmarks of more complex decision making. Moreover, from a genetic screen coupled with whole-genome sequencing, we identified eight mutants with deficits in this sensorimotor choice, including mutants of the vertebrate-specific G-protein-coupled extracellular calcium-sensing receptor (CaSR), whose function in the nervous system is not well understood. We demonstrate that CaSR promotes sensorimotor decision making acutely through Gαi/o and Gαq/11 signaling, modulated by clathrin-mediated endocytosis. Combined, our results identify the first set of genes critical for behavioral choice modulation in a vertebrate and reveal an unexpected critical role for CaSR in sensorimotor decision making.

PTH-independent regulation of blood calcium concentration by the calcium-sensing receptor.

Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.

The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A.

Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of ß-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

Novel activating mutations of the calcium-sensing receptor: the calcilytic NPS-2143 mitigates excessive signal transduction of mutant receptors.

CONTEXT AND OBJECTIVE: Activating mutations in the calcium-sensing receptor (CaSR) gene cause autosomal dominant hypocalcemia (ADH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcilytic NPS-2143 on the signaling of mutant receptors as a potential new treatment for ADH patients. METHODS: Wild-type and mutant CaSR (T151R, P221L, E767Q, G830S, and A844T) were expressed in human embryonic kidney cells (HEK 293T). Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca(2+)](o)) in the presence or absence of NPS-2143. RESULTS: All ADH patients had lowered serum calcium ranging from 1.7 to 2.0 mm and inadequate intact PTH and urinary calcium excretion. In vitro testing of CaSR mutations from these patients revealed exaggerated [Ca(2+)](o)-induced cytosolic Ca(2+) responses with EC(50) values for [Ca(2+)](o) ranging from 1.56 to 3.15 mM, which was lower than for the wild-type receptor (4.27 mM). The calcilytic NPS-2143 diminished the responsiveness to [Ca(2+)](o) in the CaSR mutants T151R, E767Q, G830S, and A844T. The mutant P221L, however, was only responsive when coexpressed with the wild-type CaSR. CONCLUSION: Calcilytics might offer medical treatment for patients with autosomal dominant hypocalcemia caused by calcilytic-sensitive CaSR mutants.

[The role of calcium, calcitriol and their receptors in parathyroid regulation]. / Papel de calcio, calcitriol y sus receptores en la regulación de la paratiroides.

The mechanism of regulation of Parathyroid hormone (PTH) is complex, and diverse factors are involved: the fundamental ones are calcium, calcitriol and phosphorus. Calcium and calcitriol's mechanism of action takes place through its specific receptors, the calcium-sensing receptor (CaR) and the Vitamin D Receptor (VDR). These two factors have an effect not only on its specific receptors, but also they can modify the other receptor in a positive manner, promoting its actions and demonstrating a cooperative effect between the two. Along with calcium and calcitriol, drugs used in the treatment of Chronic Kidney Disease Mineral Bone Disorders (CKD-MBD) also act directly or indirectly on CaR and VDR and therefore are also responsible for the regulation of the parathyroid gland.

Enterotoxin preconditioning restores calcium-sensing receptor-mediated cytostasis in colon cancer cells.

Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca(2+) as the intracellular messenger. Similarly, extracellular Ca(2+) (Ca(2+)(o)) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca(2+)(o). Here, preconditioning with ST, mediated by GCC signaling through cyclic nucleotide-gated channels, restored Ca(2+)(o)-dependent cytostasis, reflecting posttranscriptional regulation of calcium-sensing receptors (CaRs). ST-induced GCC signaling deployed CaRs to the surface of human colon cancer cells, whereas elimination of GCC signaling in mice nearly abolished CaR expression in enterocytes. Moreover, ST-induced Ca(2+)(o)-dependent cytostasis was abrogated by CaR-specific antisense oligonucleotides. Importantly, following ST preconditioning, newly expressed CaRs at the cell surface represented tumor cell receptor targets for antiproliferative signaling by CaR agonists. Since expression of the endogenous paracrine hormones for GCC is uniformly lost early in carcinogenesis, these observations offer a mechanistic explanation for the Ca(2+)(o)-resistant phenotype of colon cancer cells. Restoration of antitumorigenic CaR signaling by GCC ligand replacement therapy represents a previously unrecognized paradigm for the prevention and treatment of human colorectal cancer employing dietary Ca(2+) supplementation.

[Hyperparathyroidism--new aspects]. / Neue Aspekte bei Hyperparathyreoidismus.

Hyperparathyroidism is generally classified into a primary and secondary form. The primary form is caused by an autonomous adenomatous hypertrophy and/or hyperplasia of parythyroideal glands without known cause in most of the patients. Resulting elevated levels of parathyroid hormone cause elevation of serum calcium, subsequently followed by cerebral symptoms, fatigue and calcinosis of vessels and kidneys. The mainstay of secondary HPT is the initial vitamin D deficiency such as associated with kidney failure. Via an increased PTH secretion, calcium homeostasis will be maintained together with ongoing hyperplasia of the parathyroidea. Therapeutic approaches are related to pathophysiological mechanisms. While surgical removal of adenomatous glands is the mainstay of therapy in primary and late secondary forms, during the still regulated initial period of secondary HPT supplementation of vitamin D and/or sensitation of parathyroideal Calcium-sensing-receptors are therapy of choice.

[Calcium-sensing receptor and hypoparathyroidism].

Serum level of parathyroid hormone (PTH) is strictly regulated by serum calcium concentration, which is sensed by the calcium-sensing receptor (CaSR) , a member of G-protein-coupled-receptor superfamily. Activating CaSR mutations result in the impaired PTH secretion and hypocalcemia, and the increased sensitivity of the receptor in kidney leads to relative hypercalciuria despite hypocalcemia. Recognizing the patients with activating mutations in CaSR is quite important, because these patients can exhibit unusual sensitivity to treatment of their hypocalcemia with calcium and vitamin D supplementation, with toxic effects including nephrocalcinosis, renal stones, and diminished renal function.

Calcium-sensing receptor stimulates secretion of an interferon-gamma-induced monokine (CXCL10) and monocyte chemoattractant protein-3 in immortalized GnRH neurons.

Biology of GnRH neurons is critically dependent on extracellular Ca(2+) (Ca(2+) (o)). We evaluated differences in gene expression patterns with low and high Ca(2+) (o) in an immortalized GnRH neuron line, GT1-7 cells. Mouse global oligonucleotide microarray was used to evaluate transcriptional differences among the genes regulated by elevated Ca(2+) (o). Our result identified two interferon-gamma (IFNgamma)-inducible chemokines, CXCL9 and CXCL10, and a beta chemokine, monocyte chemoattractant protein-3 (MCP-3/CCL7), being up-regulated in GT1-7 cells treated with high Ca(2+) (o) (3.0 mM) compared with low Ca(2+) (o) (0.5 mM). Up-regulation of these mRNAs by elevated Ca(2+) (o) was confirmed by quantitative PCR. Elevated Ca(2+) (o) stimulated secretion of CXCL10 and MCP-3 but not CXCL9 in GT1-7 cells, and this effect was mediated by an extracellular calcium-sensing receptor (CaR) as the dominant negative CaR attenuated secretion of CXCL10 and MCP-3. CXCL10 and MCP-3 were localized in mouse GnRH neurons in the preoptic hypothalamus. Suppression of K(+) channels (BK channels) with 25 nM charybdotoxin inhibited high-Ca(2+) (o)-stimulated CXCL10 release. Accordingly, CaR activation by a specific CaR agonist, NPS-467, resulted in the activation of a Ca(2+)-activated K(+) channel in these cells. CaR-mediated MCP-3 secretion involves the PI3 kinase pathway in GT1-7 cells. MCP-3 stimulated chemotaxis of astrocytes treated with transforming growth factor-beta (TGFbeta). With TGFbeta-treated astrocytes, we next observed that conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of astrocytes, and this effect was attenuated by a neutralizing antibody to MCP-3. These results implicate CaR as an important regulator of GnRH neuron function in vivo by stimulating secretion of heretofore unsuspected cytokines, i.e., CXCL10 and MCP-3.

Requirement of the TRPC1 cation channel in the generation of transient Ca2+ oscillations by the calcium-sensing receptor.

The calcium-sensing receptor (CaR) is an allosteric protein that responds to extracellular Ca(2+) ([Ca(2+)](o)) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). An increase in [Ca(2+)](o) stimulates phospholipase C-mediated production of inositol 1,4,5-trisphosphate and causes sinusoidal oscillations in [Ca(2+)](i). Conversely, aromatic amino acid-induced CaR activation does not stimulate phospholipase C but engages an unidentified signaling mechanism that promotes transient oscillations in [Ca(2+)](i). We show here that the [Ca(2+)](i) oscillations stimulated by aromatic amino acids were selectively abolished by TRPC1 down-regulation using either a pool of small inhibitory RNAs (siRNAs) or two different individual siRNAs that targeted different coding regions of TRPC1. Furthermore, [Ca(2+)](i) oscillations stimulated by aromatic amino acids were also abolished by inhibition of TRPC1 function with an antibody that binds the pore region of the channel. We also show that aromatic amino acid-stimulated [Ca(2+)](i) oscillations can be prevented by protein kinase C (PKC) inhibitors or siRNA-mediated PKCalpha down-regulation and impaired by either calmodulin antagonists or by the expression of a dominant-negative calmodulin mutant. We propose a model for the generation of CaR-mediated transient [Ca(2+)](i) oscillations that integrates its stimulation by aromatic amino acids with TRPC1 regulation by PKC and calmodulin.

Autosomal dominant hypocalcemia with mild type 5 Bartter syndrome.

Type 5 Bartter syndrome has been recently defined as a Bartter syndrome due to the most activating mutations of the calcium-sensing receptor (CaSR). It has been attributed to the inhibition exerted by CaSR activity on sodium transport in the thick ascending limb of the loop of Henle (TALH). Two monozygotic twin sisters (T1 and T2) with autosomal dominant hypocalcemia (ADH) due to a nonconservative activating CaSR mutation in the extracellular domain (K29E) were studied. They developed a Bartter-like syndrome characterized by a mild phenotype: hypokalemia occurred only at the age of 22 years; it was corrected with small doses of oral potassium in one twin, while the other twin needed no potassium supplements to maintain borderline levels of plasma potassium; alkalosis was absent; plasma renin and aldosterone production were not markedly activated. Furthermore, the natriuretic response to furosemide, a inhibitor of sodium reabsorption in the TALH, was conserved in both twins. The K29E mutation was previously reported as one of the most activating mutations of the CaSR gene leading to a very marked increase in CaSR sensitivity to calcium ions. These findings confirm that Bartter syndrome is typically associated with ADH provided that the underlying mutation of CaSR is able to produce a conspicuous gain of function. However, the phenotype of type 5 Bartter syndrome may manifest with variable severity, not directly related with the in vitro potency of the CaSR activating mutation.

Parathyroid cell growth in patients with advanced secondary hyperparathyroidism: vitamin D receptor, calcium sensing receptor, and cell cycle regulating factors.

The parathyroid gland (PTG) is a unique endocrine organ in which the quiescent glandular cells begin to proliferate in response to the demand for maintaining calcium (Ca) homeostasis in the progressive course of renal failure, leading to secondary hypereparathyroidism (SHPT). SHPT is characterized with continuous over-secretion of parathyroid hormone (PTH) and high turn-over bone disease, osteitis fibrosa, and the major factors include a deficiency of active vitamin D, hypocalcemia, and phosphate retention. With long-term end-stage renal failure, SHPT becomes resistant to conventional medical treatment such as phosphate binders and active vitamin D supplementation, and the growth of the PTG accelerates with the pattern of hyperplasia changing from diffuse to nodular type. In this process, the sigmoid curve between extracellular Ca concentration (exCa) and the plasma level of PTH shifts to the upper-rightward, indicating both an absolute increase in PTH secretion and the resistance of PT cells to exCa. Many experimental and human studies have revealed down-regulation of vitamin D receptor (VDR), calcium-sensing receptor (CaSR), and retinoid X receptor (RXR) in PT cells. The sustained proliferation of PT cells after obtaining autonomicity is another characteristic feature of SHPT. In this context, it has been demonstrated that the cell cycle is markedly progressed, where the expression of cyclin-dependent kinase inhibitor (CDKI), p21 and p27, is depressed in a VDR-dependent manner. These pathological features are most evident in nodular hyperplasia, in which monoclonal proliferation is obvious, indicating the phenotypic changes have occured in PT cells. It has been observed by Fukagawa and colleagues that pharmacologically high dose of active vitamin D administered orally can cause small-size PTG hyperplasia to regress in patients with advanced SHPT. Successful renal transplantation may also restore VDR and CaSR expressions in the diffuse type, in association with increasing TUNEL-positive cells. Thus, it is important to vigorously treat SHPT when the PT cell proliferation is in the reversible stage of diffuse hyperplasia.

cDNA cloning and functional expression of a Ca2+-sensing receptor with truncated C-terminal tail from the Mozambique tilapia (Oreochromis mossambicus).

The complete cDNA sequence of the tilapia extracellular Ca(2+)-sensing receptor (CaR) was determined. The transcript length of tilapia CaR (tCaR) is 3.4 kbp and encodes a 940-amino acid, 7-transmembrane domain protein that is consistent in its structural features with known mammalian and piscine CaRs. The tCaR extracellular domain includes a characteristic hydrophobic segment, conserved cysteine residues that are implicated in receptor dimerization (Cys(129) and Cys(131)) and in coupling to the transmembrane domain (nine conserved cysteine residues), and conserved serine residues (Ser(147) and Ser(169-171)) that are linked to receptor binding of Ca(2+) and L-amino acid-mediated potentiation of function. mRNA expression of tCaR was strong in kidney, brain, and gill. Weaker expression was observed in pituitary, stomach, intestine, urinary bladder, and heart. This distribution is consistent with possible physiological roles in endocrine cells, excitable tissues, and ion-transporting barrier epithelia. Expression of tCaR mRNA in kidney and intestine was salinity-dependent, suggesting a role for the receptor in iono-/osmoregulation in this euryhaline teleost species. Human embryonic kidney-293 cells transiently transfected with tCaR cDNA demonstrated dose-dependent phospholipase C activation in response to elevations in the extracellular Ca(2+) concentration ([Ca(2+)](o)). Functional activation of the mitogen-activated protein kinase cascade by high [Ca(2+)](o) was also confirmed in these cells despite the naturally occurring truncation of the receptor's intracellular tail, which removes segments variably linked in mammalian CaRs to filamin-coupled activation of mitogen-activated protein kinase cascades. Sensitivity of phospholipase C activation to [Ca(2+)](o) was dependent on the ionic strength of the bathing medium, supporting a role in salinity sensing.

Calcimimetics versus vitamin D: what are their relative roles?

A strict control of secondary hyperparathyroidism in patients with chronic kidney disease (CKD) is indicated to avoid serious complications linked to osteitis fibrosa and other parathyroid-hormone (PTH)-related bodily disturbances. However, such a control is often achieved only at the price of unacceptably high plasma calcium and phosphorus levels and the risk of soft tissue calcification, even when using the novel, so-called 'non-hypercalcemic' vitamin D analogs. The advent of a new class of drugs, the calcimimetics, should allow a more adequate control of the disturbed calcium-phosphorus metabolism in CKD patients. In my opinion, the calcimimetics will not replace currently used medications but will be a valuable supplement to presently available treatment options for this major complication in patients with renal failure.