Various peripheral tissues show circadian rhythmicity, which is generated at the cellular level by their own core oscillators that are composed of transcriptional/translational feedback loops involving a set of clock genes. Although the circulating levels of some adipocytokines, i.e. bioactive substances secreted by adipocytes, are on a 24-h rhythmic cycle, it remains to be elucidated whether the clock gene system works in adipose tissue. To address this issue, we investigated the daily mRNA expression profiles of the clock genes and adipocytokines in mouse perigonadal adipose tissues. In C57BL/6J mice, all transcript levels of the clock genes (Bmal1, Per1, Per2, Cry1, Cry2, and Dbp) and adipocytokines (adiponectin, resistin, and visfatin) clearly showed 24-h rhythms. On the other hand, the rhythmic expression of these genes was mildly attenuated in obese KK mice and greatly attenuated in more obese, diabetic KK-A y mice. Obese diabetes also diminished the rhythmic expression of the clock genes in the liver. Interestingly, a 2-wk treatment of KK and KK-A y mice with pioglitazone impaired the 24-h rhythmicity of the mRNA expression of the clock genes and adipocytokines despite the antidiabetic effect of the drug. In contrast, pioglitazone improved the attenuated rhythmicity in the liver. These findings suggest that the intracellular clock gene system acts in visceral adipose tissues as well as liver and is influenced by the conditions of obesity/type 2 diabetes and pioglitazone treatment.

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2 −/− ). The Cldn2 −/− mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2 −/− kidney. Ultrathin section and freezefracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2 −/− mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na + , Cl -, and water was significantly decreased in Cldn2 −/− mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na + ) selectivity and therefore relative anion (Cl -) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na + and Cl -excretions in Cldn2 −/− mice were similar to those in wild types, but both were greater in Cldn2 −/− mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na + )-selective paracellular channels within tight junctions of mouse proximal tubules. mouse proximal tubule | tight junction | paracellular transport | Na/Cl transport | water transport T ight junctions (TJs) are circumferential seals around cells that selectively modulate paracellular permeability between extracellular compartments (1-3). On ultrathin-section electron microscopy, TJs appear as foci where the plasma membranes of neighboring cells make complete contact (4). On freeze-fracture electron microscopy, TJs appear as a continuous and anastomosing network of intramembranous particle strands (TJ strands) (5). These strands are mainly composed of linearly polymerized integral membrane proteins called claudins with molecular masses of ∼23 kDa (2, 3, 6). The claudin gene family contains more than 20 members in humans and in mice (2, 3, 7). The expression pattern of claudins varies considerably; most cell types express more than two claudins in various combinations to constitute mosaic TJ strands.Through the formation of TJ strands, claudins are directly involved in creating a primary barrier to the paracellular diffusion of solutes and water across epithelia (8). However, TJs are not a simple barrier: the barrier varies in tightness, measured by the transepithelial electrical resistance (R T ), and charge selectivity. Furuse et al. (9) reported that, when canine claudin-2 cDNA was transfected into high-resistance Madin-Darby canine kidney (MDCK) I cells primarily expressing claudins-1 and -4, the R T decreas...

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Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of arteriosclerosis. Clinical studies show that uremic toxins deteriorate the arteriosclerosis in renal failure patients. Indoxyl sulfate (IS) is a strong protein-bound uremic toxin, but the effect of IS on VSMC proliferation has not been studied. We examined the effect of IS on rat VSMC proliferation, assessed by a cell counting kit (4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] assay) and by [(3)H]thymidine incorporation in vitro. We further evaluated a contribution of mitogen-activated protein kinase (MAPK; p44/42 MAPK) to VSMC proliferation by IS. Immunohistochemical staining was performed for VSMCs using antirat organic anion transporter (OAT)3 antibody. The mRNA expressions of platelet-derived growth factor (PDGF)-A and -C chains, and PDGF-beta receptor were evaluated by real-time PCR. IS stimulated the proliferation of VSMCs in a concentration-dependent manner and activated p44/42 MAPK. Concentration of IS needed to stimulate the proliferation of rat VSMC was about 250 microM, which is compatible with that in the serum of end-stage renal failure patients. PD98059 (10 microM), a selective inhibitor of MAPK/extracellular signal-regulated kinase, inhibited the IS-induced (250 microM) VSMC proliferation and phosphorylation of MAPK. Probenecid (0.5 mM), an inhibitor and substrate of OAT, inhibited the IS-induced (250 microM) VSMC proliferation. Rat OAT3 was detected in VSMCs. The mRNA expressions of PDGF-C chain and PDGF-beta receptor were significantly increased by IS. We conclude that IS directly stimulates rat VSMC proliferation and activates MAPK in vitro. This might be one of the mechanisms underlying the progression of atherosclerotic lesions in end-stage renal disease patients.

Net HCO transport in the rabbit kidney cortical collecting duct (CCD) is mediated by simultaneous H ϩ secretion and HCO secretion, most likely occurring in ␣ -and ␤ -intercalated cells (ICs), respectively. The polarity of net HCO transport is shifted from secretion to absorption after metabolic acidosis or acid incubation of the CCD. We investigated this adaptation by measuring net HCO flux before and after incubating CCDs 1 h at pH 6.8 followed by 2 h at pH 7.4. Acid incubation always reversed HCO flux from net secretion to absorption, whereas incubation for 3 h at pH 7.4 did not. Inhibition of ␣ -IC function (bath Cl Ϫ removal or DIDS, luminal bafilomycin) stimulated net HCO secretion by ‫ف‬ 2 pmol ր min per mm before acid incubation, whereas after incubation these agents inhibited net HCO absorption by ‫ف‬ 5 pmol ր min per mm. Inhibition of ␤ -IC function (luminal Cl Ϫ removal) inhibited HCO secretion by ‫ف‬ 9 pmol ր min per mm before incubation, whereas after incubation HCO absorption was stimulated by only ‫ف‬ 3 pmol ր min per mm. After acid incubation, luminal SCH28080 inhibited HCO absorption by only 5-15% vs the ‫ف‬ 90% inhibitory effect of bafilomycin. In outer CCDs, which contain fewer ␣ -ICs than midcortical segments, the reversal in polarity of HCO flux was blunted after acid incubation. We conclude that the CCD adapts to low pH in vitro by downregulating HCO secretion in ␤ -ICs via decreased apical Cl Ϫ ր base exchange activity and upregulating HCO absorption in ␣ -ICs via increased apical H ϩ -ATPase and basolateral Cl Ϫ ր base exchange activities. Whether or not there is a reversal of IC polarity or recruitment of ␥ -ICs in this adaptation remains to be established. (

Idiopathic hypercalciuria is a frequent cause of calcium (Ca) containing kidney stones. We have previously shown that there is increased intestinal Ca absorption in selectively inbred genetic hypercalciuric stone forming (GHS) rats; however, excess Ca excretion persists when the rats are fed a low Ca diet indicating a defect in renal Ca reabsorption and/or increased bone resorption. To determine if GHS rats have a defect in renal Ca reabsorption we performed 14C-inulin clearance studies on parathyroidectomized female GHS and control (Ctl) rats. After three baseline collections, chlorothiazide (CTZ) or furosemide (FUR) was infused and three more collections were obtained. Both GFR and filtered load of Ca did not differ among the groups; however, fractional and absolute excretion (UcaV) of Ca was three times higher in GHS rats. The increased Ca excretion was not diminished by a low Ca diet. Urine flow rate nearly tripled in all rats after either FUR or CTZ. After CTZ, UcaV was decreased to a greater extent in GHS compared to Ctl rats. After FUR, UcaV was increased to a greater extent in Ctl rats compared to GHS rats. These data indicate that GHS rats have a defect in renal Ca reabsorption, in addition to increased intestinal Ca absorption. The effect of CTZ was greater, and that of FUR was smaller, in GHS compared with Ctl rats, suggesting that the defect in renal Ca handling might be at the level of the thick ascending limb.

The transient receptor vanilloid-4 (TRPV4) is a mechanosensitive, swell-activated cation channel that is abundant in the renal distal tubules. Immunolocalization studies, however, present conflicting data as to whether TRPV4 is expressed along the apical and/or basolateral membranes. To disclose the role of TRPV4 in flow-dependent K(+) secretion in distal tubules in vivo, urinary K(+) excretion and net transports of K(+) and Na(+) in the cortical collecting duct (CCD) were measured with an in vitro microperfusion technique in TRPV4(+/+) and TRPV4(-/-) mice. Both net K(+) secretion and Na(+) reabsorption were flow dependently increased in the CCDs isolated from TRPV4(+/+)mice, which were significantly enhanced by a luminal application of 50 microM 4alpha-phorbol-12,13-didecanoate (4alphaPDD), an agonist of TRPV4. No flow dependence of net K(+) and Na(+) transports or effects of 4alphaPDD on CCDs were observed in TRPV4(-/-) mice. A basolateral application of 4alphaPDD had little effect on these ion transports in the TRPV4(+/+) CCDs, while the luminal application did. Urinary K(+) excretion was significantly smaller in TRPV4(-/-) than in TRPV4(+/+) mice when urine production was stimulated by a venous application of furosemide. These observations suggested an essential role of the TRPV4 channels in the luminal or basolateral membrane as flow sensors in the mechanism underlying the flow-dependent K(+) secretion in mouse CCDs.

Medial arterial calcification (MAC) and renal osteodystrophy are complications of mineral bone disease (MBD) associated with chronic kidney disease (CKD). Our aim was to develop a novel mouse model to investigate the clinical course of CKD-MBD. Eight-week-old C57BL/6 J male mice were assigned to the following groups: the control group, fed a standard chow for 6 or 12 weeks; the CKD-normal phosphorus (NP) group, fed a chow containing 0.2% adenine, with normal (0.8%) phosphorus, for 6 or 12 weeks; and the CKD-high phosphorus (HP) group, fed 6 weeks with the 0.2% adenine/0.8% phosphorus diet, followed by a chow with 1.8% phosphorus for 2 weeks, 4 weeks or 6 weeks. Serum phosphorus was significantly increased in the CKD-HP group, and associated with MAC formation; the volume of calcification increased with longer exposure to the high phosphorus feed. MAC was associated with upregulated expression of runt-related transcription factor 2, alkaline phosphatase, and osteopontin, indicative of osteoblastic trans-differentiation of vascular smooth muscle cells. A significant mineral density depletion of cortical bone was observed. We describe the feasibility of developing a model of CKD-MBD and provide findings of a direct association between elevated serum phosphorus and the formation of MAC and renal osteodystrophy.