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Abstract: Objective: This study was conducted to evaluate the relationship between hearing and cochlear histopathology after arginine vasopressin administration in rats. Methods: A total of 30 Wistar rats were injected with either 0.02unit/g of arginine vasopressin or the same amount of isotonic saline solution. The initial auditory brain stem response threshold was recorded and additional measurements were made at 10, 30, 60, and 90min after injection of arginine vasopressin or isotonic saline solution. The threshold for each timepoint was compared with the initial threshold. Histological quantitative assessment of endolymphatic hydrops in the cochlea was performed using light microscopy and assessment of the basal, intermediate, and marginal cells of the stria vascularis was performed with electron microscopy. Results: The auditory brain stem threshold 60min after arginine vasopressin injection increased significantly in comparison with the initial threshold (P <0.05). Although the index for endolymphatic hydrops in rats administered arginine vasopressin was not different from that in controls (P >0.05), vacuoles in the intermediate cells were increased significantly in the treated rats (P <0.01). Conclusion: Hearing impairment was detected without endolymphatic hydrops in rats administered arginine vasopressin. An increase of vacuoles in the intermediate cells may account for the hearing impairment induced by arginine vasopressin injection. [Copyright &y& Elsevier]

Many large-scale studies revealed that exogenous erythropoietin, erythropoiesis-stimulating agents, have no renoprotective effects. We reported the renoprotective effects of endogenous erythropoietin production on renal function in ischemic reperfusion injury (IRI) of the kidney using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat. The purpose of this study was to investigate the effects of daprodustat on the progression of chronic renal failure. We retrospectively investigated the effects of daprodustat on the progression of chronic renal failure and renal anemia in patients with stages 3a-5 chronic kidney diseases (estimated glomerular filtration rate, eGFR < 60 mL/min/1.73 m2). The results show that daprodustat largely slowed the reduction in eGFR. The recovery of renal function was observed in some patients. Daprodustat is useful not only for renal anemia but also for the preservation of renal function. The renoprotective effect of daprodustat was small in patients with serum creatinine larger than 3–4 mg/dL because of low residual renal function. The appearance of renal anemia would be a sign of the time to start using daprodustat. [ABSTRACT FROM AUTHOR]

Many large-scale studies show that exogenous erythropoietin, erythropoiesis-stimulating agents, lack any renoprotective effects. We investigated the effects of endogenous erythropoietin on renal function in kidney ischemic reperfusion injury (IRI) using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat (ROX). Four h of hypoxia (7% O2) and 4 h treatment by ROX prior to IRI did not improve renal function. In contrast, 24–72 h pretreatment by ROX significantly improved the decline of renal function caused by IRI. Hypoxia and 4 h ROX increased interstitial cells-derived Epo production by 75- and 6-fold, respectively, before IRI, and worked similarly to exogenous Epo. ROX treatment for 24–72 h increased Epo production during IRI by 9-fold. Immunohistochemistry revealed that 24 h ROX treatment induced Epo production in proximal and distal tubules and worked similarly to endogenous Epo. Our data show that tubular endogenous Epo production induced by 24–72 h ROX treatment results in renoprotection but peritubular exogenous Epo production by interstitial cells induced by hypoxia and 4 h ROX treatment did not. Stimulation of tubular, but not peritubular, Epo production may link to renoprotection. [ABSTRACT FROM AUTHOR]

Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting. [ABSTRACT FROM AUTHOR]

The detection of erythropoietin (Epo) protein by Western blotting has required pre‐purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis‐stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG‐bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400‐fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy. [ABSTRACT FROM AUTHOR]

Background: The localization and role of the calcium-sensing receptor (CaSR) along the nephron including the collecting ducts is still open to debate. Methods: Using the quantitative, highly sensitive in situ hybridization technique and a double-staining immunohistochemistry technique, we investigated the axial distribution and expression of CaSR along the nephron in mice (C57B/6J) treated for 6 days with acid or alkali diets. Results: Under control condition, CaSR was specifically localized in the cortical and medullary thick ascending limb of Henle's loop (CTAL and MTAL), macula densa (MD), distal convoluted tubule (DCT), and CCD (TALs, MD > DCT, CCD). Along the CCD, CaSR was co-localized with an anion exchanger type 4 (AE4), a marker of the basolateral membrane of type-B intercalated cell (IC-B) in mice. On the contrary, CaSR was not detected either in principal cells (PC) or in type-A intercalated cell (IC-A). CaSR expression levels in IC-B significantly ( P < 0.005) decreased when mice were fed NHCl (acid) diets and increased when animals were given NaHCO (alkali) diets. As expected, cell heights of IC-A and IC-B significantly ( P < 0.005) increased in the above experimental conditions. Surprisingly, single infusion (ip) of neomycin, an agonist of CaSR, significantly ( P < 0.005) increased urinary Ca excretion without further increasing the hourly urine volume and significantly ( P < 0.05) decreased urine pH. Conclusion: CaSR, cloned from rat kidney, was localized in the basolateral membrane of IC-B and was more expressed during alkali-loading. Its alkali-sensitive expression may promote urinary alkali secretion for body acid-base balance. [ABSTRACT FROM AUTHOR]

Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro . Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro , NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo . These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b. [ABSTRACT FROM AUTHOR]

Highlights: [•] Erythropoietin is produced by cortical nephrons in normal hematopoietic condition. [•] Among cortical nephrons, intercalated cells showed the largest Epo production. [•] Hypoxia induced a large increase in Epo production in peritubular cells. [•] HIF PHD2 may be involved in Epo production by intercalated cells. [•] Regulation of Epo production is different between nephrons and peritubular cells. [Copyright &y& Elsevier]

Highlights: [•] Kir4.1/Kir5.1 heteromers form basolateral K+ channels in renal distal tubules. [•] MAGI-1 facilitates K+-channel formation via interaction with Kir4.1. [•] Kir4.1 A167V mutant cannot interact with MAGI-1 in tubular cells. [•] Mistrafficking of mutant Kir4.1 inhibits its interaction with MAGI-1. [•] Mislocalization of K+ channel causes renal salt wasting in EAST/SeSAME syndrome. [Copyright &y& Elsevier]

Background: Vasopressin V1a receptor (V1aR) null mice have insufficient acid–base balance, but the target cell for V1aR signaling which results in the urinary acidification has not been identified. Methods: By using a quantitative in situ hybridization technique and a double-staining technique with an anti-AQP3 antibody in mice, we investigated the axial distribution and acidosis-induced expression of V1aR mRNA along the nephron. We also investigated the acidosis-induced morphological change in the tubule cells from wild-type and V1aR-null (V1aR −/−) mice. Results: In the normal condition, V1aR mRNA was moderately expressed in the medullary thick ascending limb (MTAL) and highly expressed in the intercalated cell (IC) throughout the collecting duct (CD). However, no expression was observed in the proximal tubule, thin limbs of Henle’s loop, and the principal cell of the CD. Importantly, V1aR mRNA was upregulated significantly both in the TAL and the IC of the CD in the inner stripe of the outer medulla (MTAL is and IC of OMCD is, respectively) when mice were treated with NH 4Cl (0.28 mol/L) for 6 days. Acidosis-induced hypertrophy, which was completely attenuated in V1aR −/− mice, was observed only in the IC of OMCD is ( P < 0.005). In addition, urinary excretion of ammonia (NH 3/NH 4+) was significantly decreased on day 3 ( P < 0.05) and day 6 ( P < 0.005) in the V1aR −/− mice treated with NH 4Cl. Conclusion: In conclusion, the IC of OMCD is may be the target cell stimulated by the vasopressin V1aR axis and contribute to urinary acidification, at least during metabolic acidosis. [ABSTRACT FROM AUTHOR]

Atrial natriuretic peptide (ANP) is known to influence NaCl transport in the medullary thick ascending limbs (MAL), where the largest NaCl reabsorption occurs among distal nephron segments in response to arginine vasopressin (AVP). In the present study, we investigated the effect of ANP on bicarbonate (HCO3−) transport in the MAL using an isolated tubule perfusion technique. The HCO3− concentration was measured using free-flow ultramicro-fluorometer. We first observed basal HCO3− reabsorption in both long- and short-looped MALs (lMALs, and sMALs, respectively). AVP inhibited HCO3− reabsorption in both lMALs and sMALs, whereas ANP did not change HCO3− transport. However, in the presence of AVP, ANP restored the HCO3− reabsorption inhibited by AVP both in lMAL and sMAL. The effects of ANP on HCO3− transport was mimicked by cyclic GMP. The mRNA expression level of the vasopressin V2 receptor in lMALs was significantly higher than in sMALs, whereas expression of the V1a receptor was unchanged. In summary, AVP inhibits HCO3− transport, and ANP counteracts the action of AVP on HCO3− transport both in lMALs and sMALs. [ABSTRACT FROM AUTHOR]

Background: Vasopressin V1a receptor null (V1aR) mice recently showed incomplete urinary concentration due to higher urine volume during control and water diuresis (euhydration), but showed normal response during dehydration (Aoyagi et al., Am J Physiol 295: F100-7, ). Methods: Water balance, plasma vasopressin, plasma and urine osmolality, and aquaporin 2 (AQP2) expression in the kidney of wild-type (WT) and V1aR mice were therefore further examined using improved methods of urine collection (urinary bladder urine). Results: V1aR mice demonstrated a lower urine osmolality (3,360 ± 138 vs. 3,610 ± 47 mOsm/kgHO) and a higher plasma osmolality (354.3 ± 1.3 vs. 342.5 ± 1.5 mOsm/kgHO) after dehydration for 24 h compared to WT mice ( P < 0.05). In contrast, the plasma vasopressin concentration was significantly ( P < 0.001) higher in the V1aR mice (48.8 ± 4.8 vs. 22.1 ± 2.4 pg/ml). On the other hand, although the AQP2 protein expression in the kidney was increased after dehydration, the basal (control) and dehydration-induced AQP2 protein levels were significantly lower in V1aR mice compared to WT mice (by Western blotting). Staining by an anti-AQP2 antibody in the luminal membrane of the collecting ducts was increased in both V1aR and WT mice after dehydration, but was relatively weaker in the V1aR mice (by immunohistochemistry). Moreover, urinary excretion of AQP2 protein, an index of the luminal AQP2 expression, was significantly ( P < 0.05) lower in the V1aR mice. Conclusion: V1aR signaling may be fundamentally important for the expression of AQP2 in the collecting ducts during control conditions and dehydration. [ABSTRACT FROM AUTHOR]

Secretion of HCO3- at the apical side of the epithelial cells of the choroid plexus is an essential step in the formation of cerebrospinal fluid. Anion conductance with a high degree of HCO3- permeability has been observed and suggested to be the major pathway for HCO3- transport across the apical membrane. Recently, it was found that NBC (Na+ /HCO3- co-transporter) 4, an electrogenic member of the NBC family, was expressed in the choroid plexus. We found that a novel variant of the NBC4 [NBC4g/Slc4a5 (solute carrier family 4, sodium bicarbonate cotransporter, member 5)] is almost exclusively expressed in the apical membrane of rat choroid plexus epithelium at exceptionally high levels. RNA interference-mediated knockdown allowed the functional demonstration that NBC4g is the major player in the HCO3- transport across the apical membrane of the choroid plexus epithelium. When combined with a recent observation that in choroid plexus epithelial cells electrogenic NBC operates with a stoichiometry of 3:1, the results of the present study suggest that NBC4g mediates the efflux of HCO3- and contributes to cerebrospinal fluid production. [ABSTRACT FROM AUTHOR]

Abstract: ATP-sensitive K+ (KATP) channel subunits SUR2A and SUR2B in the rat brain were investigated by RT-PCR assay, western blot analysis, in situ hybridization histochemistry, and immunohistochemical staining. The results show that the mRNA and protein of SUR2A and SUR2B are expressed in whole rat brain extracts and selected regions. SUR2 mRNA is widely expressed in many neurons and glial cells as revealed by in situ hybridization histochemistry. Immunohistochemical staining shows SUR2A to be widely expressed in neurons of the brain, especially in the large pyramidal neurons and their main dendrites in the neocortex and in the Purkinje cells of the cerebellar cortex. In contrast to SUR2A, SUR2B is potently expressed in small cells in the corpus callosum and cerebellar white matter, but is also weakly expressed in some neurons. Double immunostaining shows SUR2B to be localized in astrocytes and oligodendrocytes, while SUR2A is only localized in oligodendrocytes. These results suggest that SUR2A might be mainly a regulatory subunit of the KATP channel in most neurons and part of oligodendrocytes, while SUR2B might be mainly a regulatory subunit of the KATP channel in astrocytes, oligodendrocytes, and some neurons. [Copyright &y& Elsevier]

The article reports on a study on transgenic mice to analyze the role of Dock7 protein in Schwann cell differentiation and myelination. The study found that this Guanine-Nucleotide Exchange Factor protein negatively regulates Schwann cell differentiation and the onset of myelination both in vitro and in vivo. The study found that the downregulation of the enzyme c-Jun N-terminal kinases due to Dock7 knockdown in turn led to this negative effect on myelination.

Background: The calcium (Ca)-activated potassium (K) channel is an alternative K-secretory pathway in the apical membranes of the distal nephrons of adrenalectomized (ADX) animals. As a potential approach for estimating intracellular Ca increase, we investigated normal and ADX mice to determine whether dietary K intake would stimulate the expression of the calbindin D28k protein, a cytosolic Ca-binding protein, along the distal nephron consisting of the early and late portions of the distal convoluted tubule (DCT1 and DCT2, respectively), the CNT, and CCD. Methods: ADX mice received a control diet plus either 0.3% NaCl solution (C) or a 0.3% NaCl plus 3% KCl solution (HK) for 7 days before the experiment. Results: The mean plasma K concentration and pH were significantly ( P < 0.001) higher (7.9 ± 0.3 mEq/l) and lower (7.28 ± 0.02) in the K-loaded ADX mice than in the control ADX mice. The mean urinary K excretion (mEq/day) and urine flow (ml/day) increased significantly ( P < 0.0001) from 0.47 ± 0.07 (C) to 4.80 ± 0.57 (HK) and from 1.1 ± 0.2 (C) to 8.8 ± 1.0 (HK). Urinary Ca excretion significantly ( P < 0.005 and P < 0.05, respectively) increased in K-loaded normal and ADX mice compared with control normal and ADX mice. Immunofluorescence studies revealed that the relative staining of calbindin was 167.0 ± 15.4%, 291.3 ± 13.8%, and 206.3 ± 11.3% for DCT1, DCT2/CNT, and CCD of normal control mice, respectively. These values increased significantly ( P < 0.0001) only in DCT2/CNT (574.8 ± 42%) of the K-loaded ADX mice. Conclusion: Upregulation of calbindin in the late distal tubule suggests that Ca-dependent K transport may function as an alternative mechanism for urinary K excretion in ADX mice. [ABSTRACT FROM AUTHOR]

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys. [ABSTRACT FROM AUTHOR]

The neuropeptide hormone arginine-vasopressin (AVP) is well known to exert its antidiuretic effect via the vasopressin V2 receptor (V2R), whereas the role of the vasopressin V1a receptor (V1aR) in the kidney remains to be clarified. Previously, we reported decreased plasma volume and blood pressure in Via receptor-deficient (V1aR) mice (Koshimizu T, Nasa Y, Tanoue A, Oikawa R, Kawahara Y, Kiyono Y, Adachi T, Tanaka T, Kuwaki T, Mon T. Proc Natl Acad Sci USA 103: 7807-78 12, 2006). In this study, we investigated the role of V1aR in urine concentration, renal function, and the renin-angiotensin system (RAS) using V1aR-/- mice. Urine volume of V1aR-/- mice was greater than that of wild-type mice, particularly when water was loaded, while the glomerular filtration rate (GFR), urinary NaCl excretion, AVP-dependent cAMP generation, V2R, and aquaporin 2 (AQP2) expression in the kidney were lower, indicating that the diminished GFR and V2R-AQP2 system led to impaired urinary concentration in V1aR-/- mice. Since the GFR and V2R-AQP2 system are regulated by RAS, we analyzed renin and angiotensin II in V1aR-/- mice and found that the plasma renin and angiotensin II were decreased. The expression of renin in granule cells was decreased in V1aR-/- mice, which led to a decreased level of plasma renin. In addition, the expression of renin stimulators such as neuronal nitric oxide synthase and cyclooxygenase-2 in macula densa (MD) cells, where V1aR was specifically expressed, was decreased in V1aR-/- mice. These data indicate that AVP regulates body fluid homeostasis and GFR via the V1aR in MD cells by activating RAS and subsequently the V2R-AQP2 system. [ABSTRACT FROM AUTHOR]

The hair follicle bulge area is an abundant, easily accessible source of actively growing, pluripotent adult stem cells. Nestin, a protein marker for neural stem cells, also is expressed in follicle stem cells and their immediate, differentiated progeny. The fluorescent protein GFP, whose expression is driven by the nestin regulatory element in transgenic mice, served to mark the follicle cell fate. The pluripotent nestin-driven GFP stem cells are positive for the stem cell marker CD34 but negative for keratinocyte marker keratin 15, suggesting their relatively undifferentiated state. These cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In vivo studies show the nestin-driven GFP hair follicle stem cells can differentiate into blood vessels and neural tissue after transplantation to the subcutis of nude mice. Equivalent hair follicle stem cells derived from transgenic mice with β-actin-driven GFP implanted into the gap region of a severed sciatic nerve greatly enhance the rate of nerve regeneration and the restoration of nerve function. The follicle cells transdifferentiate largely into Schwann cells, which are known to support neuron regrowth. Function of the rejoined sciatic nerve was measured by contraction of the gastrocnemius muscle upon electrical stimulation. After severing the tibial nerve and subsequent transplantation of hair follicle stem cells, walking print length and intermediate toe spread significantly recovered, indicating that the transplanted mice recovered the ability to walk normally. These results suggest that hair follicle stem cells provide an important, accessible, autologous source of adult stem cells for regenerative medicine. [ABSTRACT FROM AUTHOR]

Permanent isolated proximal renal tubular acidosis (pRTA) with ocular abnormalities is a systemic disease with isolated pRTA, short stature and ocular abnormalities. We identified a novel homozygous deletion of nucleotide 2,311 adenine in the kidney type Na+/HCO3- cotransporter (kNBC1) cDNA in a patient with permanent isolated pRTA. This mutation is predicted to result in a frame shift at codon 721 forming a stop codon after 29 amino acids anomalously transcribed from the SLC4A4 gene. Cosegregation of this mutation with the disease was supported by heterozygosity in the parents of the affected patient. The absence of this mutation in 156 alleles of 78 normal individuals indicates that this mutation is related to the disease and is not a common DNA sequence polymorphism. When injected into Xenopus oocytes, the mutant cRNA failed to induce electrogenic transport activity. In addition, immunofluorescence and Western blot analysis failed to detect the expression of the full-length protein in mutant-injected oocytes. Our results expand the spectrum of kNBC1 mutations in permanent isolated pRTA with ocular abnormalities and increase our understanding of the renal tubular mechanism that is essential for acid-base homeostasis. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-12 plays a key role in determining the immune response pattern that results in maturation of Th0 to Th1 and Th2. To investigate the correlation between intracellular redox state and IL-12 production in macrophages, cells from the mouse cell line J774A.1 were treated with reagents modulating the glutathione-redox couple before stimulation with lipopolysaccharide (LPS). It was found that the glutathione reductase inhibitor, 1,3-bis (2-chloroethyl)-1-nitrosourea, markedly augmented LPS induced IL-12p40 production particularly when it was added for 24 h before LPS stimulation, whereas the glutathione-synthesis inhibitor, L-buthionine-(S,R)-sulfoximine, suppressed IL-12p40 production. The profile of IL-12p40 augmentation correlated well with the profile of intracellular glutathione oxidation (GSSG) and the activation profile of nuclear transcription factor κB (NF-κB), suggesting that GSSG is important in NF-κB activation which leads to IL-12p40 production. Our results indicate that the glutathione-redox couple plays an important role in the augmented production of IL-12p40 and thus in influencing immune response patterns. [ABSTRACT FROM AUTHOR]

Background. Coordinated expression of ROMK (luminal K+ channel in the thick ascending limb and the collecting duct) and Na+ ,K+ -ATPase has been demonstrated to be involved in the postnatal development of renal K+ excretion; however, the developmental expression of the basolateral K+ channel Kir7.1 is unknown. The purpose of this study was to elucidate the possible involvement of Kir7.1 in the maturation of renal K+ excretion. Methods. Developmental changes in the renal K+ excretion under the condition of K+ overload was investigated by collecting urine from neonatal rats infused with K+ (KCl solution). RNase protection analysis was used to elucidate the expression of Kir7.1 and Na+ ,K+ -ATPase mRNA in pre- and postnatal rats, and the expression of Kir7.1 and ROMK mRNA at 7, 14, and 21 days. Western blotting of Kir7.1, and immunohistochemistry of Kir7.1 and ROMK were used to determine their protein expression. Results. The ratio of urinary K+ excretion to K+ load increased between 7 and 14 days after birth. In addition, half excretion time of K+ load gradually decreased through the experimental period of 7 and 21 days. Na+ ,K+ -ATPase mRNA levels showed a peak of up-regulation at birth that remained elevated. ROMK1 mRNA levels significantly increased between 7 and 14 days. On the other hand, Kir7.1 mRNA and protein levels significantly increased between 14 and 21 days. Kir7.1 protein in the thick ascending limb was first recognized at 7 days, whereas its expression in the distal convoluted tubule and the cortical collecting duct was found in 21-day-old neonates. Conclusion. Our results suggest that Kir7.1 is involved in the development of renal K+ excretion between 14 and 21 days after birth under the condition of K+ overload. [ABSTRACT FROM AUTHOR]

Kir6.2, a subunit of the ATP-sensitive K+ channel (KATP), was localized in adult rat brain by immunohistochemistry and in situ hybridization. The Kir6.2 mRNA was widely expressed in most rat brain neuronal populations and nuclei examined, intensely in the mitral cell layer and tufted cells of the olfactory bulb, pontine nucleus, pontine reticular nucleus, motor and spinal trigeminal nuclei and cuneate nuclei of the brain stem, moderately in the neocortex and cerebellar Purkinje cells, and weakly in the granular cell layer of the olfactory bulb and the granular layer of the cerebellum. In addition, glial cells also expressed the Kir6.2 gene weakly in the corpus callosum and cerebellar white matter. This wide localization of the gene was quite similar to that of Kir6.2 protein. Double stainings with anti-GFAP and anti-Kir6.2 antibodies were performed in this study. Glial cells showing immunoreactivity to both anti-Kir6.2 and anti-GFAP were confirmed to be astrocytes, and those showing only immunoreactivity to anti-Kir6.2 but not to anti-GFAP were presumed to be oligodendrocytes and confirmed by immunoelectron microscopy. Thus, it may be concluded that both oligodendrocytes and astrocytes contain Kir6.2. Under the electron microscope, we showed in vivo for the first time that the immunoreactive products were localized in the endoplasmic reticulum and Golgi apparatus as well as the plasma membranes of neurons and glial cells. [Copyright &y& Elsevier]

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin–angiotensin–aldosterone system (RAS). [ABSTRACT FROM AUTHOR]