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To clarify the characteristics of cellular ATP synthesis in individual nephron segments for assessing nephrotoxicity of chemicals, cellular ATP content was measured by the luciferin/luciferase system under various conditions using intact nephron segments isolated from male Sprague-Dawley rats. Increasing the duration of collagenase treatment of kidney slices significantly lowered the cellular level of ATP newly synthesized from 2 mm glutamine in PST at 37°C over 30 min (p < 0.01). The tubular incubation time significantly affected the cellular ATP content in the early and middle portions (S2) of the proximal tubule (p < 0.05 and p < 0.01, respectively) over 20 min and in the late proximal tubule over 10 min. Among numerous substrates tested, such as d-glucose, glutamine, pyruvate, dl-lactate, and β-hydroxybutyrate, the substrate utilization for maintaining cellular ATP content was entirely variable according to each nephron segment. Pyruvate and glutamine were the best substrates in the proximal tubule. On the other hand, ATP production from glutamine was less than that from the other substrates in the distally located nephron segments: medullary and cortical thick ascending limbs of Henle's loop (MAL and CAL, respectively), distal tubule, cortical and medullary collecting tubules (CCT and MCT, respectively). In general, glucose, pyruvate, and lactate appear to be equivalent in maintaining ATP content in the distal segments of renal tubules. A monovalent cation ionophore, monensin, at 10 μg/ml decreased the cellular ATP content in MAL, CAL, and MCT significantly. Mercuric chloride (HgCl2) was used as a model compound to study nephrotoxicity by investigating its effects on cellular ATP metabolism in microdissected nephron segments. HgCl2 at 1 × 10−6m significantly decreased ATP content only in S2 (p < 0.05), clearly demonstrating S2 to be the most sensitive segment within the nephron. These results indicate that measurement of cellular ATP content would be a useful method for forecasting the intrarenal toxic site and potency of possible nephrotoxic chemical compounds.

フルクトサミンはほぼ2週間の血糖を反映するとされ, 広く糖尿病の血糖管理に応用されつつある. 今回, 我々はフルクトサミン値への腎不全の影響について検討し, さらに, 糖尿病性腎症患者における有用性について検討した.対象は, 非糖尿病の保存期腎不全患者27名 (CRF群), 血液透析患者31名 (HD群), CAPD患者15名 (PD群), 糖尿病性腎症患者21名 (DM群), 糖尿病性腎症を原疾患とする血液透析患者4名 (DMHD群), 同CAPD患者2名 (DMPD群), および健常対照者12名 (C群) である. フルクトサミンの測定は, NBT比色法を用いたロッシュのキットにて施行した.各群のフルクトサミンは, CRF群2.59±0.46 (mean±SD), HD群2.96±0.34, PD群2.65±0.26, DM群3.29±0.46, DMHD群3.61±0.89, C群2.78±0.16mmol/lでCRF群, HD群, PD群はいずれもC群と有意差を認めなかった. 一方, DM群, DMHD群はC群, CRF群, HD群, PD群よりも有意に高値を示した. DMPD群2例は, 3.68±3.76mmol/lでいずれも高値を示した. フルクトサミンはCRF群において, BUN, クレアチニンと有意な相関を認めなかった. また, 75g経口ブドウ糖負荷試験において正常パターン, および耐糖能異常を示した群のフルクトサミンは, 各々, 2.81±0.34, 3.02±0.45mmol/lで有意差を認めなかった. DM群において, フルクトサミンはHbA1, HbA1cと有意な正の相関関係を認めた. また, DMHD群2例, DMPD群2例における経時的検討にて, フルクトサミンと過去2週間の血糖の間の相関係数は, 各々, r=0.90, 0.50, 0.85, 0.73で3例において有意であった.以上より, フルクトサミンは腎不全に影響されず, 糖尿病性腎症患者の血糖コントロールに有用な指標となると考えられた.

Cloning, tissue distribution, and intrarenal localization of ClC chloride channels in human kidney. Two kidney-specific chloride channels, ClC-K1 and ClC-K2, have been isolated from rat kidney. In the present study, we sought to isolate human homologue of rat ClC-K2 chloride channel that was present in the thick ascending limb of Henle's loop and collecting ducts. Human kidney cDNA library was screened with the whole rat ClC-K2 cDNA probe. Two highly homologous but not identical cDNAs were isolated and sequenced. Northern analysis showed that both clones were expressed only in kidney among various human tissues, demonstrating that kidney-specific ClC family members were also present in human kidney. Because both clones had almost the same nucleotide identity (˜80%) with rat ClC-K2, we could not determine by sequence alone which human clone corresponded to rat ClC-K2. Accordingly, we performed reverse transcription PCR using dissected human nephron segments and identified the site of expression of each clone in human nephron segments. One clone was only expressed in the thin limb of Henle's loop and the other was expressed in glomeruli, proximal tubules, and collecting ducts. We identified the latter clone as human ClC-K2 based on the localization of rat ClC-K1 and ClC-K2. Identification of human ClC-K2 clone will be of help in understanding the genetic involvement of chloride channel in disorders of chloride transport such as Bartter's syndrome.

"Avian-type" renal medullary tubule organization causes immaturity of urine-concentrating ability in neonates. Background While neonatal kidneys are not powerful in concentrating urine, they already dilute urine as efficiently as adult kidneys. To elucidate the basis for this paradoxical immaturity in urine-concentrating ability, we investigated the function of Henle's loop and collecting ducts (IMCDs) in the inner medulla of neonatal rat kidneys. Methods Analyses of individual renal tubules in the inner medulla of neonatal and adult rat kidneys were performed by measuring mRNA expression of membrane transporters, transepithelial voltages, and isotopic water and ion fluxes. Immunofluorescent identification of the rCCC2 and rCLC-K1 using polyclonal antibodies was also performed in neonatal and adult kidney slices. Results On day 1, the transepithelial voltages (V Ts ) in the thin ascending limbs (tALs) and IMCDs were 14.6 ± 1.1mV ( N = 27) and -42.7 ± 6.1mV ( N = 14), respectively. The V Ts in the thin descending limbs (tDLs) were zero on day 1. The V Ts in the tALs were strongly inhibited by luminal bumetanide or basolateral ouabain, suggesting the presence of a NaCl reabsorption mechanism similar to that in the thick ascending limb (TAL). The diffusional voltage (V D ) of the tAL due to transepithelial NaCl gradient was almost insensitive to a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The V Ts in the IMCDs were strongly inhibited by luminal amiloride. On day 1, both the tDL and tAL were impermeable to water, indicating the water impermeability of the entire loop. Diffusional water permeability (P dw ) and urea permeabilities (P urea ) in the IMCDs indicated virtual impermeability to water and urea on day 1. Stimulation by vasopressin (1nmol/L) revealed that only P dw was sensitive to vasopressin by day 14. A partial isoosmolar replacement of luminal urea by NaCl evoked negligible water flux across the neonatal IMCDs, indicating the absence of urea-dependent volume flux in the neonatal IMCD. These transport characteristics in each neonatal tubule are similar to those in quail kidneys. Identification of mRNAs and immunofluorescent studies for specific transporters, including rAQP-1, rCCC2, rCLC-K1, rENaC β subunit, rAQP-2, and rUT-A1, supported these findings. Conclusion We hypothesize that the renal medullary tubule organization of neonatal rats shares a tremendous similarity with avian renal medulla. The qualitative changes in the organization of medullary tubules may be primarily responsible for the immature urine-concentrating ability in mammalian neonates.

During dehydration, protein kinase A phosphorylates aquaporin 2 (AQP2) at serine 256 and this is essential for apical membrane sorting of AQP2 in the collecting ducts. A-kinase anchoring proteins (AKAPs) bind protein kinase A and protein phosphatases conferring substrate specificity to these enzymes and localize them to the appropriate intracellular compartment. We found that AKAP220 bound to AQP2 in a yeast two-hybrid screen. Further, it was highly localized to the papilla compared to other regions of the kidney. Using double immunofluorescence and immunoelectron microscopy we found that AKAP220 co-localized with AQP2 in the cytosol of the inner medullary collecting ducts. Forskolin-mediated phosphorylation of AQP2, transiently expressed in COS cells, was increased by AKAP220 co-expression. Our results suggest that AKAP220 may be involved in the phosphorylation of AQP2 by recruiting protein kinase A.

Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP) promotes AT1R internalization along with suppression of pathological activation of tissue AT1R signaling. However, the functional significance of ATRAP in renal sodium handling and blood pressure regulation under pathological stimuli is not fully resolved. Here we show the blood pressure of mice with a gene-targeted disruption of ATRAP was comparable to that of wild-type mice at baseline. However, in ATRAP-knockout mice, angiotensin II-induced hypertension was exacerbated and the extent of positive sodium balance was increased by angiotensin II. Renal expression of the sodium-proton antiporter 3, a major sodium transporter in the proximal tubules, urinary pH, renal angiotensinogen production, and angiotensin II content was unaffected. Stimulation of the renal expression and activity of the epithelial sodium channel (ENaC), a major sodium transporter in the distal tubules, was significantly enhanced by chronic angiotensin II infusion. The circulating and urinary aldosterone levels were comparable. The blood pressure response and renal ENaC expression by aldosterone were not affected. Thus, ATRAP deficiency exacerbated angiotensin II-mediated hypertension by pathological activation of renal tubular AT1R by angiotensin II. This directly stimulates ENaC in the distal tubules and enhances sodium retention in an aldosterone-independent manner.

This paper proposes a cost-effective phased-array radar with an extremely small number of antennas and reduced signal processing complexity compared with a conventional 2D phased-array radar. The key techniques to realize the feature are 1D linear Tx and Rx antenna arrays located orthogonally to each other and the combination of analog-beamforming Tx and digital-beamforming Rx. The effectiveness of the proposed phased-array radar was verified by simulation and 3D spatial profiling experiments. The experiments were performed using a 79GHz prototype radar system that we developed. A phase shifter which was robust against process/temperature variations and whose phase error was reduced to smaller than 2.0 degrees was designed, fabricated in 28nm CMOS process, and used for the analog-beamforming Tx of the prototype radar. A contactless vital sign monitoring system which tracks and continuously monitors the target person in the room was demonstrated using the prototype radar system.