Bradykinin/B2 receptor activation regulates renin in M-1 cells via protein kinase C and nitric oxide.

In the collecting duct (CD), the interactions of renin angiotensin system (RAS) and kallikrein-kinin system (KKS) modulate Na+ reabsorption, volume homeostasis, and blood pressure. In this study, we used a mouse kidney cortical CD cell line (M-1 cells) to test the hypothesis that in the CD, the activation of bradykinin B2 receptor (B2R) increases renin synthesis and release. Physiological concentrations of bradykinin (BK) treatment of M-1 cells increased renin mRNA and prorenin and renin protein contents in a dose-dependent manner and increased threefold renin content in the cell culture media. These effects were mediated by protein kinase C (PKC) independently of protein kinase A (PKA) because B2R antagonism with Icatibant and PKC inhibition with calphostin C, prevented these responses, but PKA inhibition with H89 did not modify the effects elicited by the B2R activation. BK-dependent stimulation of renin gene expression in CD cells also involved nitric oxide (NO) pathway because increased cGMP levels and inhibition of NO synthase with L-NAME prevented it. Complementary renin immunohistochemical studies performed in kidneys from mice with conventional B2R knockout and conditional B2R knockout in the CD, showed marked decreased renin immunoreactivity in CD, regardless of the renin presence in juxtaglomerular cells in the knockout mice. These results indicate that the activation of B2R increases renin synthesis and release by the CD cells through PKC stimulation and NO release, which support further the interactions between the RAS and KKS.

Aldosterone regulates microRNAs in the cortical collecting duct to alter sodium transport.

A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.

Protocols and applications of cellular metabolomics in safety studies using precision-cut tissue slices and carbon 13 NMR.

Numerous xenobiotics are toxic to human and animal cells by interacting with their metabolism, but the precise metabolic step affected and the biochemical mechanism behind such a toxicity often remain unknown. In an attempt to reduce the ignorance in this field, we have developed a new approach called cellular metabolomics. This approach, developed in vitro, provides a panoramic view not only of the pathways involved in the metabolism of physiologic substrates of any normal or pathologic human or animal cell but also of the beneficial and adverse effects of xenobiotics on these metabolic pathways. Unlike many cell lines, precision-cut tissue slices, for which there is a renewed interest, remain metabolically differentiated for at least 24-48 h and allow to study the effect of xenobiotics during short-term and long-term incubations. Cellular metabolomics (or cellular metabonomics), which combines enzymatic and carbon 13 NMR measurements with mathematical modeling of metabolic pathways, is illustrated in this brief chapter for studying the effect of insulin on glucose metabolism in rat liver precision-cut slices, and of valproate on glutamine metabolism in human renal cortical precision-cut slices. The use of very small amounts of test compounds allows to predict their toxic effect and eventually their beneficial effects very early in the research and development processes. Cellular metabolomics is complementary to other omics approaches, but, unlike them, provides functional and dynamic pieces of information by measuring enzymatic fluxes.

Differential regulation of the bumetanide-sensitive cotransporter (NKCC2) by ovarian hormones.

The Na-K-2Cl cotransporter (NKCC2) regulates sodium transport along the thick ascending limb of Henle's loop and is important in control of sodium balance, renal concentrating ability and renin release. To determine if there are sex differences in NKCC2 abundance and/or distribution, and to evaluate the contribution of ovarian hormones to any such differences, we performed semiquantitative immunoblotting and immunoperoxidase immunohistochemistry for NKCC2 in the kidney of Sprague Dawley male, female and ovariectomized (OVX) rats with and without 17-beta estradiol or progesterone supplementation. Intact females demonstrated greater NKCC2 protein in homogenates of whole kidney (334+/-29%), cortex (219+/-20%) and outer medulla (133+/-9%) compared to males. Ovarian hormone supplementation to OVX rats regulated NKCC2 in the outer medulla only, with NKCC2 protein abundance decreasing slightly in response to progesterone but increasing in response to 17-beta estradiol. Immunohistochemistry demonstrated prominent NKCC2 labeling in the apical membrane of thick ascending limb cells. Kidney section NKCC2 labeling confirmed regionalized regulation of NKCC2 by ovarian hormones. Localized regulation of NKCC2 by ovarian hormones may have importance in controlling sodium and water balance over the lifetime of women as the milieu of sex hormones varies.

Extracellular glutathione peroxidase (Gpx3) binds specifically to basement membranes of mouse renal cortex tubule cells.

Glutathione peroxidase-3 (Gpx3), also known as plasma or extracellular glutathione peroxidase, is a selenoprotein secreted primarily by kidney proximal convoluted tubule cells. In this study Gpx3(-/-) mice have been produced and immunocytochemical techniques have been developed to investigate Gpx3 metabolism. Gpx3(-/-) mice maintained the same whole-body content and urinary excretion of selenium as did Gpx3(+/+) mice. They tolerated selenium deficiency without observable ill effects. The simultaneous knockout of Gpx3 and selenoprotein P revealed that these two selenoproteins account for >97% of plasma selenium. Immunocytochemistry experiments demonstrated that Gpx3 binds selectively, both in vivo and in vitro, to basement membranes of renal cortical proximal and distal convoluted tubules. Based on calculations using selenium content, the kidney pool of Gpx3 is over twice as large as the plasma pool. These data indicate that Gpx3 does not serve in the regulation of selenium metabolism. The specific binding of a large pool of Gpx3 to basement membranes in the kidney cortex strongly suggests a need for glutathione peroxidase activity in the cortical peritubular space.

Vasopressin V2 receptor expression along rat, mouse, and human renal epithelia with focus on TAL.

In renal epithelia, vasopressin influences salt and water transport, chiefly via vasopressin V(2) receptors (V(2)Rs) linked to adenylyl cyclase. A combination of vasopressin-induced effects along several distinct portions of the nephron and collecting duct system may help balance the net effects of antidiuresis in cortex and medulla. Previous studies of the intrarenal distribution of V(2)Rs have been inconclusive with respect to segment- and cell-type-related V(2)R expression. Our study therefore aimed to present a high-resolution analysis of V(2)R mRNA expression in rat, mouse, and human kidney epithelia, supplemented with immunohistochemical data. Cell types of the renal tubule were identified histochemically using specific markers. Pronounced V(2)R signal in thick ascending limb (TAL) was corroborated functionally; phosphorylation of Na(+)-K(+)-2Cl(-) cotransporter type 2 (NKCC2) was established in cultured TAL cells from rabbit and in rats with diabetes insipidus that were treated with the V(2)R agonist desmopressin. We found solid expression of V(2)R mRNA in medullary TAL (MTAL), macula densa, connecting tubule, and cortical and medullary collecting duct and weaker expression in cortical TAL and distal convoluted tubule in all three species. Additional V(2)R immunostaining of kidneys and rabbit TAL cells confirmed our findings. In agreement with strong V(2)R expression in MTAL, kidneys from rats with diabetes insipidus and cultured TAL cells revealed sharp, selective increases in NKCC2 phosphorylation upon desmopressin treatment. Macula densa cells constitutively showed strong NKCC2 phosphorylation. Results suggest comparably significant effects of vasopressin-induced V(2)R signaling in MTAL and in connecting tubule/collecting duct principal cells across the three species. Strong V(2)R expression in macula densa may be related to tubulovascular signal transfer.

The effect of fennel essential oil on uterine contraction as a model for dysmenorrhea, pharmacology and toxicology study.

Increasing the ectopic uterine motility is the major reason for primary dysmenorrhea. This motility is the basis for several symptoms including for pain is the main complaints of patients with primary dysmenorrhea. There are several mechanisms, which initiate dysmenorrhea. Therefore, different compounds can be employed to control its symptoms. In long-term therapy, combination of oestrogens and progestins may be useful. In short-term therapy, dysmenorrhea sometimes non-steroidal anti-inflammatory drugs (NSAIDs) are used. Most of NSAIDs in long-term therapy show severe adverse effects. In an attempt to find agents with less adverse effect the fennel essential oil (FEO) was chosen for this investigation. In this article, effects of FEO on the uterine contraction and estimation of LD(50) in rat were described. For assessment of pharmacological effects on the isolated rat uterus, oxytocin (0.1, 1 and 10 mu/ml) and prostaglandin E(2) (PGE(2)) (5x10(-5) M) were employed to induce muscle contraction. Administration of different doses of FEO reduced the intensity of oxytocin and PGE(2) induced contractions significantly (25 and 50 microg/ml for oxytocin and 10 and 20 microg/ml PGE(2), respectively). FEO also reduced the frequency of contractions induced by PGE(2) but not with oxytocin. LD(50) of FEO was obtained in the female rats by using moving average method. The estimated LD(50) was 1326 mg/kg. No obvious damage was observed in the vital organs of the dead animals.

Intrarenal distribution of the colonic H,K-ATPase mRNA in rabbit.

BACKGROUND: Evidence suggests that the colonic H,K-ATPase isoform is expressed in the kidney and that a mRNA species highly homologous to the rat and guinea pig HKalpha2 is expressed in the cortical collecting duct (CCD) of the rabbit. The goals of this study were to determine if this mRNA is the rabbit homologue of HKalpha2 or a novel isoform and to determine intrarenal distribution of the HKalpha2 mRNA in rabbit. METHODS: 5'-RACE and Dye Deoxy Terminator chemistry were used to determine the full-length sequence of the rabbit HKalpha2 mRNA. The intrarenal distribution of HKalpha2 mRNA was determined in microdissected nephron segments, connecting tubule (CNT), and CCD cells isolated by immunodissection, as well as in the three cell types of the CCD. Principal cells and alpha- and beta-intercalated cells were isolated by fluorescence-activated cell sorting. HKalpha2 mRNA levels were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) or single-nephron RT-PCR (SN-RTPCR). RESULTS: The full-length sequence of the rabbit kidney HKalpha2 mRNA was determined. This transcript is identical to the one expressed in rabbit distal colon. In microdissected nephron segments, strong HKalpha2 amplicons were present in the CNT, CCD, and outer medullary collecting duct (OMCD), whereas no signal was detected in the proximal tubule, distal convoluted tubule, think ascending limb, and inner medullary collecting duct. Roughly comparable levels of HKalpha2 mRNA were present in all three CCD cell types, and the highest levels were observed in a subpopulation most likely corresponding to CNT cells. CONCLUSIONS: These results suggest that the HKalpha2 mRNA is expressed in rabbit collecting duct is identical in size and sequence to the one expressed in rabbit distal colon. HKalpha2 mRNA in the rabbit kidney is selectively expressed in the CNT, CCD, and OMCD, and all three collecting duct subtypes express its mRNA.

Immunolocalization of the secretory isoform of Na-K-Cl cotransporter in rat renal intercalated cells.

Two bumetanide-sensitive ion cotransporters that carry Na+, K+, and Cl- in a coupled fashion have been identified. One type, the "absorptive" isoform, carries these ions across the apical plasma membrane of the thick ascending limb of Henle's loop. Another isoform, the "secretory" cotransporter, has been identified in a number of epithelial tissues by physiological means, but its sites of expression in the kidney have not been fully characterized. Complementary DNA believed to code for the secretory isoform (called "BSC2" or "NKCC1") have recently been cloned. This study used a specific affinity-purified antipeptide antibody to this protein for immunolocalization in the rat kidney. Immunoblot studies using this antibody show abundant immunoreactivity against bands of 140-190 and 120 kd in the parotid gland, colon, and stomach, sites where the secretory form of the cotransporter has been identified by physiological techniques. This distribution supports the hypothesis that this isoform represents the secretory form of the cotransporter. Studies in the kidney revealed that the same bands are associated with membrane fractions chiefly in the outer medulla. Immunolocalizations show that immunoreactivity is selectively and intensely localized to the basolateral plasma membrane of a subfraction of outer medullary collecting duct cells. An independently produced monoclonal antibody (T4) specific for Na-K-Cl cotransporter displays the same localization. Dual localizations of cotransporter antibody with respect to antibody specific for principal cells (aquaporin-2) and intercalated cells (band 3 and H(+)-ATPase) show that cotransporter immunoreactivity is localized to alpha-intercalated cells of the outer medullary collecting duct in the rat. This distinctive localization suggests that the secretory form of the cotransporter may play a role in renal NH4+ and/or acid secretion by this cell type.

Ryanodine receptor expression in the kidney and a non-excitable kidney epithelial cell.

An oligonucleotide probe to a conserved 3' region within the three identified ryanodine receptor-calcium release channel isoforms hybridized to a single clone from a rabbit kidney cDNA library. The kidney clone encoded the carboxyl-terminal 338 amino acids within the putative transmembrane domain of the type 2 ryanodine receptor sequence. Reverse transcriptase-polymerase chain reaction with isoform-specific oligonucleotide primers demonstrated the presence of the type 2 ryanodine receptor transcript in rabbit kidney, as well as in a non-excitable cell line, LLC-RK1, derived from rabbit kidney epithelial cells. Amplification by rapid amplification of 5' cDNA ends indicated the kidney type 2 ryanodine receptor transcript extended >7000 base pairs from the stop codon and is therefore not homologous to the short RyR-1 transcript of approximately 2500 base pairs previously observed in rabbit brain. [3H]Ryanodine binding and immunoblot analysis with a type 2 ryanodine receptor-specific antibody demonstrated that the native type 2 ryanodine receptor protein is expressed in the kidney. These observations suggest that the type 2 ryanodine receptor isoform may play a functional role in regulating intracellular calcium homeostasis in non-excitable cells.

Perlecan and basement membrane-chondroitin sulfate proteoglycan (bamacan) are two basement membrane chondroitin/dermatan sulfate proteoglycans in the Engelbreth-Holm-Swarm tumor matrix.

The presence of proteoglycans bearing galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue-specific differences in their distribution.

Effect of transition metal ions on lipid peroxidation of rabbit renal cortical mitochondria.

Effect of transition metal ions on lipid peroxidation in rabbit renal cortical mitochondria was studied. The lipid peroxidation of mitochondria was promoted by the metal ions tested in the following order of intensity; Fe2+, Hg2+ and Cu2+. Sodium selenite inhibited Fe2+ or Hg2+-induced lipid peroxidation, but it had no influence on the lipid peroxidation stimulated by Cu2+. This result suggests that the mechanism of the stimulatory effect on lipid peroxidation of Cu2+ may be different from that of Fe2+ or Hg2+ in mitochondria.