Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e., distal convoluted tubule (DCT) and connecting tubule (CNT) and the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf, and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, ␣ENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms, and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function.circadian rhythm ͉ homeostasis ͉ renal function R ecent evidence suggests that many if not all specific physiological functions are under the control of the circadian timing system. The mammalian circadian timing system is a hierarchically organized network of molecular oscillators driven by a central pacemaker located in the suprachiasmatic nucleus (SCN) of hypothalamus. This central pacemaker functions in a self-sustained fashion, but is reset each day by exposure to environmental synchronizers, mainly the light/dark cycle. The SCN masterclock drives the rest-activity cycle, which in turn imposes the feeding pattern [reviewed in (1, 2)]. The feeding time seems to be the dominant cue for circadian rhythms in the peripheral tissues (3, 4). Central and peripheral oscillators share a similar molecular core clock based on a set of self-autonomous transcriptional/ translational feedback loops. The key molecular components of these loops are the PAS domain transcriptional factors CLOCK, BMAL1, and NPAS2 and the feedback repressors PER1, PER2, CRY1, and CRY2. The orphan nuclear receptors NR1D1 and, probably, NR1D2 form an accessory feedback loop. The core oscillators confer circadian rhythmicity on a set of output genes underlying the tissue-specific functional rhythms. Current estimates indicate that up to 10% of the cellular transcriptome may follow a circadian expression pattern (5-7). Several recent studies have also demonstrated that the transcription of only a minority of these circadian genes is driven by systemic humoral or neurona...

The circadian clock contributes to the control of BP, but the underlying mechanisms remain unclear. We analyzed circadian rhythms in kidneys of wild-type mice and mice lacking the circadian transcriptional activator clock gene. Mice deficient in clock exhibited dramatic changes in the circadian rhythm of renal sodium excretion. In parallel, these mice lost the normal circadian rhythm of plasma aldosterone levels. Analysis of renal circadian transcriptomes demonstrated changes in multiple mechanisms involved in maintaining sodium balance. Pathway analysis revealed the strongest effect on the enzymatic system involved in the formation of 20-HETE, a powerful regulator of renal sodium excretion, renal vascular tone, and BP. This correlated with a significant decrease in the renal and urinary content of 20-HETE in clockdeficient mice. In summary, this study demonstrates that the circadian clock modulates renal function and identifies the 20-HETE synthesis pathway as one of its principal renal targets. It also suggests that the circadian clock affects BP, at least in part, by exerting dynamic control over renal sodium handling. Recent evidence indicates that the circadian clock is involved in BP control. In mice, suppression or decrease of the circadian clock activity via deletion of the circadian transcriptional activators Bmal1, Clock, or Npas2 leads to low BP, whereas its constitutive activation via deletion of the circadian repressors Cry1 and Cry2 results in salt-sensitive hypertension. 1-4 Wang et al. have recently shown that mice simultaneously devoid of three prolineand acidic amino acid-rich basic leucine zipper circadian transcriptional factors Dbp, Hlf, and Tef exhibit a significant reduction in BP. 5 Maintaining BP within the normal range strongly depends on the capacity of the kidney to precisely regulate sodium content in the extracellular space. Thus, dysregulation of molecular mechanisms involved in renal sodium handling could be partially responsible for the elevated or decreased BP observed in mice with genetically altered clocks. This hypothesis is supported by evidence in humans suggesting that alteration of circadian rhythms of urinary sodium excretion is the primary cause of disease in several forms of hyper-or hypotension. For instance, a decreased renal capacity to excrete sodium during the daytime correlates with nocturnal hypertension, whereas increased sodium excretion during the nighttime contributes to the maintenance of orthostatic hypotension. 6,7 Of note, important changes in the amplitude or the circadian phase of urinary excretion of sodium can be provoked not only by a pathologic process but also by a misalignment between the endogenous circadian clock and the imposed rest-activity or feeding cycles, or by sleep disturbance. For instance, Kamperis et al. have shown that acute sleep deprivation in humans leads to excessive natriuresis and kaliuresis during the subjective night and attenuation of the

The circadian clock controls a wide variety of metabolic and homeostatic processes in a number of tissues, including the kidney. However, the role of the renal circadian clocks remains largely unknown. To address this question, we performed a combined functional, transcriptomic, and metabolomic analysis in mice with inducible conditional knockout (cKO) of BMAL1, which is critically involved in the circadian clock system, in renal tubular cells (Bmal1/Pax8-rtTA/LC1 mice). Induction of cKO in adult mice did not produce obvious abnormalities in renal sodium, potassium, or water handling. Deep sequencing of the renal transcriptome revealed significant changes in the expression of genes related to metabolic pathways and organic anion transport in cKO mice compared with control littermates. Furthermore, kidneys from cKO mice exhibited a significant decrease in the NAD-to-NADH ratio, which reflects the oxidative phosphorylation-to-glycolysis ratio and/or the status of mitochondrial function. Metabolome profiling showed significant changes in plasma levels of amino acids, biogenic amines, acylcarnitines, and lipids. In-depth analysis of two selected pathways revealed a significant increase in plasma urea level correlating with increased renal Arginase II activity, hyperargininemia, and increased kidney arginine content as well as a significant increase in plasma creatinine concentration and a reduced capacity of the kidney to secrete anionic drugs (furosemide) paralleled by an approximate 80% decrease in the expression level of organic anion transporter 3 (SLC22a8). Collectively, these results indicate that the renal circadian clocks control a variety of metabolic/homeostatic processes at the intrarenal and systemic levels and are involved in drug disposition.

The circadian timing system is critically involved in the maintenance of fluid and electrolyte balance and BP control. However, the role of peripheral circadian clocks in these homeostatic mechanisms remains unknown. We addressed this question in a mouse model carrying a conditional allele of the circadian clock gene Bmal1 and expressing Cre recombinase under the endogenous Renin promoter (Bmal1 Cre mice exhibited multiple abnormalities, including increased urine volume, changes in the circadian rhythm of urinary sodium excretion, increased GFR, and significantly reduced plasma aldosterone levels. These changes were accompanied by a reduction in BP. These results show that local renal circadian clocks control body fluid and BP homeostasis. Circadian rhythmicity is a feature of a wide variety of physiologic functions. Many of the functional rhythms are driven by the circadian timing system, a complex mechanism that coordinates all major cellular processes with geophysical time. It is thought that this coordination allows for the anticipatory adaptation of cells and tissues to the circadian changes in functional requirements. The circadian timing system is organized in a hierarchical manner. The master clock of the system is located in the suprachiasmatic nucleus of the hypothalamus and synchronized to the daily light/dark cycle through the retinohypothalamic tract. The peripheral circadian clocks, which are present in virtually all peripheral tissues, are synchronized to geophysical time through a wide range of master clock-dependent stimuli, many of which remain unknown (reviewed in ref. 1 ). It is important to note, however, that, despite the hierarchical structure of the circadian timing system and its continuous resetting by environmental time cues, the intrinsic activity of both central and peripheral circadian clocks is largely self-sustained. On the molecular level, the master clock and peripheral clocks share similar machinery based on transcriptional/translational feedback loops involving transcriptional activators BMAL1, CLOCK, and NPAS2 and their own repressors PER1 and PER2 as well as CRY1 and CRY2 (reviewed in ref. 2 ).The circadian timing system is critically involved in the maintenance of fluid and electrolyte balance

No abstract

Single-Point Incremental Forming (SPIF) is an emerging manufacturing process especially suitable to produce small batches of metal parts. Moreover, the enhanced formability of metal sheets deformed by SPIF makes this technology useful to those industrial applications requiring high deformation levels. In this sense, the precise setting of limit strains in SPIF in relation to the conventional formability limits of the material, as well as the influence of the process parameters on these strains, are essential variables to understand how and how much can be deformed the metal sheets in real production. On the other hand, the forming force in SPIF is an essential variable, especially for the design of dedicated equipment or for the safe use of adapted machinery. This paper revisits failure in SPIF by means of an experimental analysis of the influence of process parameters, such as the tool diameter, the spindle speed and the step down, on the formability in SPIF (spifability) of AISI 304 metal sheets, studied in the light of circle grid analysis. The work also involves the independent determination of conventional formability limits by necking and fracture under laboratory conditions by using stretching tests (Nakazima tests), in conjunction with stretch-bending tests performed in order to quantify the influence of the bending induced by the tool radius. Failure strains are experimentally obtained and compared in stretch-bending and SPIF tests, being the failure mode discussed in each case. Finally, the axial forming force evolution was recorded with the aim of analyzing the range of process parameters that would guarantee the safely utilization of the non-dedicated process equipmentThe authors wish to thank the Spanish Government for its financial support through the research project DPI2012-32913 and DPI2012-36042. The work of M.Sc. Carlos Suntaxi within this investigation is also greatly acknowledged. The second author would also like to thank the support of the Spanish scholarship FPU12/0540

No abstract

Abstract. Aldosterone exerts its effects through interactions with two types of binding sites, the mineralocorticoid (MR) and the glucocorticoid (GR) receptors. Although both receptors are known to be involved in the anti-natriuretic response to aldosterone, the mechanisms of signal transduction leading to modulation of electrolyte transport are not yet fully understood. This study measured the Na ϩ and K ϩ urinary excretion and the mRNA levels of three known aldosterone-induced transcripts, the serum and glucocorticoid-induced kinase (Sgk-1), the ␣ subunit of the epithelial Na ϩ channel (␣ENaC), and the glucocorticoid-induced-leucine-zipper protein (GILZ) in the whole kidney and in isolated cortical collecting tubules of adrenalectomized rats treated with low doses of aldosterone and/or dexamethasone. The resulting plasma concentrations of both steroids were close to 1 nmol/L. Aldosterone, given with or without dexamethasone, induced anti-natriuresis and kaliuresis, whereas dexamethasone alone did not. GILZ and ␣ENaC transcripts were higher after treatment with either or both hormones, whereas the mRNA abundance of Sgk-1 was increased in the cortical collecting tubule by aldosterone but not by dexamethasone. We conclude the increased expression of Sgk-1 in the cortical collecting tubules is a primary event in the early antinatriuretic and kaliuretic responses to physiologic concentrations of aldosterone. Induction of ␣ENaC and/or GILZ mRNAs may play a permissive role in the enhancement of the early and/or late responses; these effects may be necessary for a full response but do not by themselves promote early changes in urinary Na ϩ and K ϩ excretion.