Showing papers in "American Journal of Physiology-renal Physiology in 2004"
TL;DR: The upregulation of expression of Kim-1 and its presence in the urine in response to exposure to various types of nephrotoxicants suggest that this protein may serve as a general biomarker for tubular injury and repair processes.
Abstract: Nephrotoxicity is a common side effect of therapeutic interventions, environmental insults, and exposure to toxicants in the workplace. Although biomarkers for nephrotoxicity are available, they of...
608 citations
TL;DR: The studies demonstrate the feasibility of repeated nonisotopic measurement of inulin clearance in conscious mice and found that diabetes mellitus induced by streptozotocin was associated with increased GFR.
Abstract: Two nonradioactive methods for determining glomerular filtration rate (GFR) in conscious mice using FITC-labeled inulin (FITC-inulin) were evaluated. The first method measured GFR using clearance kinetics of plasma FITC-inulin after a single bolus injection. Based on a two-compartment model, estimated GFR was 236.69 ± 16.55 and 140.20 ± 22.27 μl/min in male and female C57BL/6J mice, respectively. Total or ⅚ nephrectomy reduced inulin clearance to 0 or 32.80 ± 9.32 μl/min, respectively. Conversely, diabetes mellitus induced by streptozotocin was associated with increased GFR. The other approach measured urinary inulin clearance using intraperitoneal microosmotic pumps to deliver FITC-inulin and metabolic cages to collect timed urine samples. This approach yielded similar GFR values of 211.11 ± 26.56 and 157.36 ± 20.02 μl/min in male and female mice, respectively. These studies demonstrate the feasibility of repeated nonisotopic measurement of inulin clearance in conscious mice.
328 citations
TL;DR: It is shown that hypoxia response elements present upstream of Ctgf enable direct interaction of Hif-1 transcription factor with the CtgF promoter, resulting in increased transcription of CTGf mRNA.
Abstract: CTGF plays a significant role in the development of renal fibrosis by mediating the fibrotic effects of transforming growth factor (TGF)-β1 and has been shown to be hypoxia inducible in human breas...
273 citations
TL;DR: The mechanisms involved in the hypertensive effects of oxidant stress and tubulointerstitial inflammation, in particular intrarenal ANG II activity, are discussed, focusing on their potential for sodium retention.
Abstract: Recent evidence indicates that interstitial infiltration of T cells and macrophages plays a role in the pathogenesis of salt-sensitive hypertension. The present review examines this evidence and summarizes the investigations linking the renal accumulation of immune cells and oxidative stress in the development of hypertension. The mechanisms involved in the hypertensive effects of oxidant stress and tubulointerstitial inflammation, in particular intrarenal ANG II activity, are discussed, focusing on their potential for sodium retention. The possibility of autoimmune reactivity in hypertension is raised in the light of the proinflammatory and immunogenic pathways stimulated by the interrelationship between oxidant stress and inflammatory response. Finally, we present some clinical considerations derived from the recognition of this interrelationship.
272 citations
TL;DR: Recent advances in the understanding of the cellular and molecular mechanisms underlying HGF inhibition of renal fibrosis are reviewed.
Abstract: Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an imperative role in tubular repair and regeneration after acute renal injury. Growing evidence indicates that HGF is also an endo...
255 citations
TL;DR: This review will focus on the molecular regulation of the HO-1 gene in renal injury and will highlight the interspecies differences, predominantly between the rodent and humanHO-1 genes.
Abstract: Heme oxygenases (HOs) catalyze the rate-limiting step in heme degradation, resulting in the formation of iron, carbon monoxide, and biliverdin, the latter of which is subsequently converted to bilirubin by biliverdin reductase. Recent attention has focused on the biological effects of product(s) of this enzymatic reaction, which have important antioxidant, anti-inflammatory, and cytoprotective functions. Two major isoforms of the HO enzyme have been described: an inducible isoform, HO-1, and a constitutively expressed isoform, HO-2. A third isoform, HO-3, closely related to HO-2, has also been described. Several stimuli implicated in the pathogenesis of renal injury, such as heme, nitric oxide, growth factors, angiotensin II, cytokines, and nephrotoxins, induce HO-1. Induction of HO-1 occurs as an adaptive and beneficial response to these stimuli, as demonstrated by studies in renal and non-renal disease states. This review will focus on the molecular regulation of the HO-1 gene in renal injury and will highlight the interspecies differences, predominantly between the rodent and human HO-1 genes.
245 citations
TL;DR: Results indicate that claudin-4, -8, and -12 are expressed in umbrella cells, where they may impart the high-resistance phenotype associated with this cell type, and that in some instances tight junction proteins are also associated at the sites of cell contact of the underlying cell layers, perhaps playing some role in cell-cell adhesion.
Abstract: In mammals, the bladder stores urine without permitting the passage of urine contents into the bloodstream, a function, in part, of the uroepithelial-associated tight junction complex. The protein constituents that make up this high-resistance barrier in the bladder are currently unknown, although the claudins, a multigene family, are thought to govern paracellular transport in other epithelia. Reverse transcriptase-polymerase chain reaction analysis was used to define that mRNA for claudin-2, -4, -8, -12, and -13 was expressed in mouse bladder tissue. The localization of these claudins, as well as the tight junction-associated proteins zonula occludens-1 (ZO-1) and occludin, within the bladder epithelium was determined by immunofluorescence microscopy. As expected, occludin and ZO-1 were localized to the tight junctions of rat, mouse, and rabbit umbrella cells. Intriguingly, ZO-1 in mouse epithelium, ZO-1 in the dome region of rabbit bladders and occludin in rat and mouse bladders were also expressed in the underlying intermediate and basal cell layers. Claudin-4, -8, and -12 were found in the umbrella cell tight junction; however, additional staining of claudin-4 was observed along the sites of cell-cell contact in the underlying cell layers of rat, mouse, and rabbit tissue. No claudin-2 staining was associated with tight junctions in the uroepithelium. Our results indicate that claudin-4, -8, and -12 are expressed in umbrella cells, where they may impart the high-resistance phenotype associated with this cell type, and that in some instances tight junction proteins are also associated at the sites of cell contact of the underlying cell layers, perhaps playing some role in cell-cell adhesion.
230 citations
TL;DR: The podocyte is identified as an important target cell for the renoprotective action of 1,25(OH)(2)D(3), suggested by less evidence of podocyte injury, decreased podocytes loss, and abrogation of podocytes hypertrophy, findings that may also explain less pronounced albuminuria and glomerulosclerosis.
Abstract: 1,25(OH)2D3 has antiproliferative effects and promotes cell differentiation. This consideration has provided the rationale for studies in subtotally nephrectomized rats showing that 1,25(OH)2D3 int...
226 citations
TL;DR: An archetype is presented that explains multiple nuances of kidney function in early diabetes, including diabetic hyperfiltration, a paradoxical effect of dietary salt on glomerular filtration rate in diabetes, and the renal response to dietary protein and amino acid infusion in diabetes.
Abstract: At the onset of diabetes mellitus, the glomerular filtration rate becomes supranormal. Discovery science has identified many abnormalities in the early diabetic kidney that apparently contribute to...
225 citations
TL;DR: It is suggested that caspase-3 mediates cisplatin-induced cell death in TKPTS cells via an EGFR/src/ERK-dependent pathway and it is proposed that injury-specific outcome diverges downstream from ERK in cis platin- or H(2)O(2)-mediated cell survival and death.
Abstract: Cisplatin treatment induces extensive death of the proximal tubules in mice. We also demonstrated that treatment of immortalized mouse proximal tubule cells (TKPTS) with 25 μM cisplatin induces apo...
212 citations
TL;DR: The results suggest that the HG-induced increase in ANG II generation in MC results from an increase in intracellular renin activity mediated by at least three factors: a time-dependent stimulation of (pro)renin gene transcription, a reduction in prorenin enzyme secretion, and an increased rate of conversion of proren in to active renin, probably mediated by cathepsin B.
Abstract: Increased intrarenal renin-angiotensin system activity contributes to diabetic nephropathy. ANG II generation in mesangial cells (MC) is increased by high-glucose (HG) exposure. This study assessed the mechanisms involved in the glucose-induced ANG II generation in rat MC. Under basal conditions, MC mainly secreted prorenin. HG decreased prorenin secretion and induced a striking 30-fold increase in intracellular renin activity. After 72 h of HG exposure, only the mRNA levels for angiotensinogen and angiotensin-converting enzyme (ACE) were significantly elevated. However, after shorter periods of 24 h of HG stimulation the mRNA levels of the enzymes prorenin and cathepsin B, besides that for ACE, were significantly increased. The results suggest that the HG-induced increase in ANG II generation in MC results from an increase in intracellular renin activity mediated by at least three factors: a time-dependent stimulation of (pro)renin gene transcription, a reduction in prorenin enzyme secretion, and an increased rate of conversion of prorenin to active renin, probably mediated by cathepsin B. The increase in angiotensinogen mRNA in parallel to increased renin activity indicates that HG also increased the availability of the renin substrate. The consistent upregulation of ACE mRNA suggests that, besides renin, ACE is directly involved in the increased mesangial ANG II generation induced by HG.
TL;DR: This review focuses on TRPM6, an ion channel of the "transient receptor potential (TRP) gene family, which, when mutated, causes a combined defect of intestinal magnesium absorption and renal magnesium conservation as observed in primary hypomagnesemia with secondary hypocalcemia.
Abstract: Magnesium is an important cofactor for many biological processes, such as protein synthesis, nucleic acid stability, or neuromuscular excitability. Extracellular magnesium concentration is tightly ...
TL;DR: It is demonstrated that exposure of kidney graft recipients to CO at a low concentration can impart significant protective effects against renal I/R injury and improve function of renal grafts.
Abstract: Carbon monoxide (CO), a product of heme metabolism by heme oxygenases, is known to impart protection against oxidative stress. We hypothesized that CO would protect ischemia-reperfusion (I/R) injur...
TL;DR: It is likely that the homeostatic role of the distal convoluted and connecting tubules, which are technically difficult to study, has been underestimated, whereas the more easily accessible collecting duct may have been overemphasized.
Abstract: Sodium reabsorption and potassium secretion in the distal convoluted tubule and in the connecting tubule can maintain the homeostasis of the body, especially when dietary sodium intake is high and potassium intake is low. Under these conditions, a large proportion of the aldosterone-regulated sodium and potassium transport would occur in these nephron segments before the tubular fluid reaches the collecting duct. The differences between these two segments and the collecting duct would be more quantitative than qualitative. The collecting duct would come into play when the upstream segments are overloaded by a primary genetic defect that affects sodium and/or potassium transport or by a diet that is exceedingly poor in sodium and rich in potassium. It is likely that the homeostatic role of the distal convoluted and connecting tubules, which are technically difficult to study, has been underestimated, whereas the role of the more easily accessible collecting duct may have been overemphasized.
TL;DR: To determine the nephron segment localization of claudin-7 and -8, immunofluorescence staining of mouse kidney sections was performed using isoform-specific antibodies and found Claudin-8 was found to be expressed primarily at the tight junction along the entire aldosterone-sensitive distal nephRON and in the late segments of the thin descending limbs of long-looped nephrons.
Abstract: Claudins are integral membrane proteins of the tight junction that determine the magnitude and selectivity of paracellular permeability in epithelial tissues. The mammalian renal tubule exhibits considerable heterogeneity in the permeability properties of its different segments. To determine the nephron segment localization of claudin-7 and -8, immunofluorescence staining of mouse kidney sections was performed using isoform-specific antibodies. Claudin-8 was found to be expressed primarily at the tight junction along the entire aldosterone-sensitive distal nephron and in the late segments of the thin descending limbs of long-looped nephrons. This pattern of expression is consistent with the putative role of claudin-8 as a paracellular cation barrier. By contrast, claudin-7 was found in the same nephron segments as claudin-8, but it was expressed primarily at the basolateral membrane.
TL;DR: It is demonstrated that intestinal Na(+)-dependent P(i) cotransport adaptation to a low-P( i) diet occurs independently of vitamin D.
Abstract: Recent studies suggest that vitamin D may play a role in intestinal Na(+)-dependent phosphate transport adaptation to variable levels of dietary P(i). Therefore, the goal of the current study was to assess Na(+)-dependent P(i) cotransport activity in transgenic mice to determine whether vitamin D is an essential mediator of this process. Intestinal brush-border membrane (BBM), Na(+)-dependent P(i) cotransport activity was significantly decreased in vitamin D receptor (VDR) null [VDR (-/-)] mice compared with wild-type (VDR+/+) mice. While intestinal Na-P(i) cotransporter (type IIb) mRNA levels were similar in VDR (-/-) and VDR (+/+) mice, type IIb Na-P(i) cotransporter protein expression was markedly suppressed in VDR (-/-) mice compared with VDR (+/+) mice. Furthermore, Na-P(i) cotransport activity in renal BBM was similar in VDR (-/-) and VDR (+/+) mice, but type IIa Na-P(i) cotransporter protein expression was decreased in VDR (-/-) mice. After administration of a low-P(i) diet, type IIb protein expression was significantly increased in VDR (+/+) and VDR (-/-) mice, and type IIb protein expression was present in the intestinal BBM of VDR (-/-) mice. These data demonstrate that intestinal Na-P(i) cotransport adaptation to a low-P(i) diet occurs independently of vitamin D.
TL;DR: Investigation of the distribution of purinoceptors in the urothelium, smooth muscle, and nerves of the normal cat urinary bladder as well as possible changes in the expression of these receptors in cats with a chronic painful bladder condition termed feline interstitial cystitis (FIC) raises the possibility that purinergic mechanisms in the UROthelium and bladder smooth muscle are altered in FIC cats.
Abstract: Purinergic mechanisms appear to be involved in motor as well as sensory functions in the urinary bladder. ATP released from efferent nerves excites bladder smooth muscle, whereas ATP released from ...
TL;DR: It is found that THP deficiency predisposes mice to bladder infections by type 1-fimbriated E. coli, and the results suggest that potential THP defects, either quantitative or qualitative, could predispose the urinary bladder to bacterial infections.
Abstract: The adhesion of uropathogenic Escherichia coli to the urothelial surface of the bladder is a prerequisite for the establishment of bladder infections. This adhesion process relies on E. coli adhesi...
TL;DR: Gender differences in the rat renal cortical OAT1 and OAT3 (M > F) appear after puberty and are determined by both a stimulatory effect of androgens (and progesterone in the case of OAT2) and an inhibitory effect of estrogens.
Abstract: In rats, the secretion of p-aminohippurate (PAH) by the kidney is higher in males (M) than in females (F). The role of the major renal PAH transporters, OAT1 and OAT3, in the generation of these ge...
TL;DR: An important role is found for p53 in cisplatin-induced apoptosis in renal tubular cells and the results suggest that p53 activation might be an early signal for apoptosis during cisPlatin treatment.
Abstract: Tubular damage by cisplatin leads to acute renal failure, which limits its use in cancer therapy. In tubular cells, a primary target for cisplatin is presumably the genomic DNA. However, the pathway relaying the signals of DNA damage to tubular cell death is unclear. In response to DNA damage, the tumor suppressor gene p53 is induced and is implicated in subsequent DNA repair and cell death by apoptosis. The current study was designed to examine the role of p53 in cisplatin-induced apoptosis in cultured rat kidney proximal tubular cells. Cisplatin at 20 microM induced apoptosis in approximately 70% of cells, which was partially suppressed by carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone (VAD), a general caspase inhibitor. Of interest, cisplatin-induced apoptosis was also suppressed by pifithrin-alpha, a pharmacological inhibitor of p53. Cisplatin-induced caspase activation was completely inhibited by VAD, but only partially by pifithrin-alpha. Early during cisplatin treatment, p53 was phosphorylated and upregulated. The p53 activation was blocked by pifithrin-alpha, but not by VAD. Bcl-2 expression abolished cisplatin-induced apoptosis without blocking p53 phosphorylation or induction. The results suggest that p53 activation might be an early signal for apoptosis during cisplatin treatment. To further determine the role of p53, tubular cells were stably transfected with a dominant-negative mutant of p53 with diminished transcriptional activity. Expression of the mutant attenuated cisplatin-induced apoptosis and caspase activation. In conclusion, the results support an important role for p53 in cisplatin-induced apoptosis in renal tubular cells. p53 May regulate apoptosis through the transcription of apoptotic genes.
TL;DR: The current knowledge about the signaling pathways that regulate the adaptive response to osmotic stress are summarized and new insights from yeast that could be relevant to the osmostress response in mammals are discussed.
Abstract: The adaptation to hypertonicity in mammalian cells is driven by multiple signaling pathways that include p38 kinase, Fyn, the catalytic subunit of PKA, ATM, and JNK2. In addition to the well-characterized tonicity enhancer (TonE)-TonE binding protein interaction, other transcription factors (and their respective cis elements) can potentially respond to hypertonicity. This review summarizes the current knowledge about the signaling pathways that regulate the adaptive response to osmotic stress and discusses new insights from yeast that could be relevant to the osmostress response in mammals.
TL;DR: The evidence consistent with and for Cl secretion in, respectively, proximal and distal tubules of the mammalian kidney calls for a reexamination of basic assumptions in renal physiology that may lead to new opportunities for managing some forms of renal disease.
Abstract: The evolution of the vertebrate kidney records three occasions, each separated by about 50 million years, when fish have abandoned glomeruli to produce urine by tubular mechanisms. The recurring dismissal of glomeruli suggests a mechanism of aglomerular urine formation intrinsic to renal tubules. Indeed, the transepithelial secretion of organic solutes and of inorganic solutes such as sulfate, phosphate, and magnesium can all drive secretory water flow in renal proximal tubules of fish. However, the secretion of NaCl via secondary active transport of Cl is the primary mover of secretory water flow in, surprisingly, proximal tubules of both glomerular and aglomerular fish. In filtering kidneys, the tubular secretion of solute and water is overshadowed by reabsorptive transport activities, but secretion progressively comes to light as glomerular filtration decreases. Thus the difference between glomerular and aglomerular urine formation is more a difference of degree than of kind. At low rates of glomerular filtration in seawater fish, NaCl-coupled water secretion serves to increase the renal excretory capacity by increasing the luminal volume into which waste, excess, and toxic solutes can be secreted. The reabsorption of NaCl and water in the distal nephron and urinary bladder concentrates unwanted solutes for excretion while minimizing renal water loss. In aglomerular fish, NaCl-coupled water secretion across proximal tubules replaces glomerular filtration to increase renal excretory capacity. A review of the literature suggests that tubular secretion of NaCl and water is an early function of the vertebrate proximal tubule that has been retained throughout evolution. Active transepithelial Cl secretion takes place in gall bladders studied as models of the mammalian proximal tubule and in proximal tubules of amphibians and apparently also of mammals. The tubular secretion of Cl is also observed in mammalian distal tubules. The evidence consistent with and for Cl secretion in, respectively, proximal and distal tubules of the mammalian kidney calls for a reexamination of basic assumptions in renal physiology that may lead to new opportunities for managing some forms of renal disease.
TL;DR: Experimental studies identified the release of iron and free radicals, activation of calpain, and formation of F(2)-isoprostanes as important components of cold ischemic injury, as are the swelling of mitochondria and activation of mitochondrial apoptotic pathways, and other new experimental data that have implications for reducing cold isChemic transplant injury.
Abstract: Kidney transplantation is the preferred and definitive treatment for end-stage renal disease (ESRD), and kidneys from deceased donors are a major source for it. These kidneys are routinely cold sto...
TL;DR: The results show that prenatal programming of hypertension is associated with an abnormal pattern of intrarenal RAS ontogeny that may play a pathogenetic role, for instance, by constitutively altering renal hemodynamics or Na reabsorption.
Abstract: Adult hypertension may be programmed by the prenatal environment in humans and in experimental animals. The potential role of the intrarenal renin-angiotensin system (RAS) in prenatally programmed ...
TL;DR: The current review summarizes recent developments that enhance the understanding of the CaSR and its fundamental importance in parathyroid gland physiology.
Abstract: The calcium-sensing receptor (CaSR) represents the molecular mechanism by which parathyroid cells detect changes in blood ionized calcium concentration and modulate parathyroid hormone (PTH) secretion to maintain serum calcium levels within a narrow physiological range. Much has been learned in recent years about the diversity of signal transduction through the CaSR and the various factors that affect receptor expression. Beyond its classic role as a determinant of calcium-regulated PTH secretion, signaling through the CaSR also influences both gene transcription and cell proliferation in parathyroid cells. The CaSR thus serves a broad physiological role by integrating several distinct aspects of parathyroid gland function. The current review summarizes recent developments that enhance our understanding of the CaSR and its fundamental importance in parathyroid gland physiology.
TL;DR: It is concluded that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.
Abstract: Aquaporin-4 (AQP4) water channels exist as heterotetramers of M1 and M23 splice variants and appear to be present in orthogonal arrays of intramembraneous particles (OAPs) visualized by freeze-fracture microscopy. We report that AQP4 forms OAPs in rat gastric parietal cells but not in parietal cells from the mouse or kangaroo rat. Furthermore, the organization of principal cell OAPs in Brattleboro rat kidney is perturbed by vasopressin (arginine vasopressin). Membranes of LLC-PK(1) cells expressing M23-AQP4 showed large, abundant OAPs, but none were detectable in cells expressing M1-AQP4. Measurements of osmotic swelling of transfected LLC-PK(1) cells using videomicroscopy, gave osmotic water permeability coefficient (P(f)) values (in cm/s) of 0.018 (M1-AQP4), 0.019 (M23-AQP4), and 0.003 (control). Quantitative immunoblot and immunofluorescence showed an eightfold greater expression of M1- over M23-AQP4 in the cell lines, suggesting that single-channel p(f) (cm(3)/s) is much greater for the M23 variant. Somatic fusion of M1- and M23-AQP4 cells (P(f) = 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios ( approximately 1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel p(f) for the M23 variant. Expression of an M23-AQP4-Ser(111E) mutant produced approximately 1.5-fold greater single-channel p(f) and OAPs that were up to 2.5-fold larger than wild-type M23-AQP4 OAPs, suggesting that a putative PKA phosphorylation site Ser(111) is involved in OAP formation. We conclude that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.
TL;DR: ANG II induces secretion and activation of transforming growth factor-β (TGF-β) by glomerular mesangial cells, but the mechanisms that operate this are unclear.
Abstract: ANG II induces secretion and activation of transforming growth factor-β (TGF-β) by glomerular mesangial cells. However, the mechanisms that operate this are unclear. Thrombospondin-1 (TSP-1), which...
TL;DR: An overview of recent advances in LX physiology is provided, with particular emphasis on the cellular and molecular processes involved, to suggest that LX bioactions may be amenable to pharmacological mimicry for therapeutic gain.
Abstract: Over the past decade, compelling in vivo and in vitro studies have highlighted lipoxins (LXs) and aspirin-triggered LXs (ATLs) as endogenously produced anti-inflammatory eicosanoids. LXs and ATLs elicit distinct anti-inflammatory and proresolution bioactions that include inhibition of leukocyte-mediated injury, stimulation of macrophage clearance of apoptotic neutrophils, repression of proinflammatory cytokine production, modulation of cytokine-stimulated metalloproteinase activity, and inhibition of cell proliferation and migration. An overview of recent advances in LX physiology is provided, with particular emphasis on the cellular and molecular processes involved. These data coupled with in vivo models of inflammatory diseases suggest that LX bioactions may be amenable to pharmacological mimicry for therapeutic gain.
TL;DR: The data show that rapid and extensive plasma membrane accumulation of AQP2 can occur in a vasopressin receptor (V2R)- and phosphorylation-independent manner, pointing to a potential means of bypassing the mutated V2R in X-linked nephrogenic diabetes insipidus to achieve cell surface expression of AQp2.
Abstract: Inhibition of clathrin-mediated endocytosis by expression of a GTPase-deficient dynamin mutant (dynamin-2/K44A) for 16 h results in an accumulation of plasma membrane aquaporin-2 (AQP2) in epitheli...
TL;DR: A model of an autocrine/paracrine RAS in polycystic kidney disease, whereby overactivity of the intrarenal system results in sustained increases in intratubular ANG II concentrations is proposed.
Abstract: Hypertension is a common complication of autosomal dominant polycystic kidney disease (ADPKD), often present before the onset of renal failure. A role for the renin-angiotensin system (RAS) has bee...