Induction of stress response proteins and experimental renal ischemia/reperfusion.

BACKGROUND: The induction of stress response (heat shock) proteins (HSPs) is a highly conserved response that protects many cell types from diverse physiological and environmental stressors. We tested the hypothesis that the induction of HSPs is protective in experimental renal ischemia/reperfusion injury. METHODS: The effect of prior heat stress was examined in a rat model of renal ischemia. Postischemic renal function, histopathology, myeloperoxidase activity, and mortality were determined in hyperthermia and sham hyperthermia groups. RESULTS: HSP84, HSP70, and HSP22 mRNA were increased after eight minutes but not four minutes of hyperthermia. The induction of HSP84 and HSP70 was blocked by pretreatment with quercetin. Improvement in renal function, mortality, and histologic abnormalities was seen with eight minutes of hyperthermia six hours before ischemia. Protection was dependent on the timing of ischemia relative to heat stress and was not observed when HSPs were not induced. Postischemic increases in renal myeloperoxidase activity were markedly attenuated in the hyperthermia compared with the sham hyperthermia group. CONCLUSION: Endogenous protective mechanisms may be important in renal ischemia/reperfusion injury.

Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain.

To isolate a cDNA encoding Na+/H+ exchanger isoform 5 (NHE5), we screened a human spleen library using exon sequences of the NHE5 gene. Clones spanning 2.9 kilobase pairs were isolated; however, they contained several introns and were missing coding sequences at both the 5' and 3' ends. The missing 5' sequences were obtained by 5'-rapid amplification of cDNA ends and by analysis of an NHE5 genomic clone, and the missing 3' sequences were obtained by 3'-rapid amplification of cDNA ends. Polymerase chain reaction amplification of brain cDNA yielded products in which each of the introns had been correctly excised, whereas the introns were retained in products from spleen and testis, suggesting that the NHE5 transcripts expressed in these organs do not encode a functional transporter. The intron/exon organization of the NHE5 gene was analyzed and found to be very similar to that of the NHE3 gene. The NHE5 cDNA, which encodes an 896-amino acid protein that is most closely related to NHE3, was expressed in Na+/H+ exchanger-deficient fibroblasts and shown to mediate Na+/H+ exchange activity. Northern blot analysis demonstrated that the mRNA encoding NHE5 is expressed in multiple regions of the brain, including hippocampus, consistent with the possibility that it regulates intracellular pH in hippocampal and other neurons.

NHE-1 is the sodium-hydrogen exchanger isoform present in erythroid cells.

Erythrocyte sodium hydrogen exchanger (NHE) represents one of a limited number of sodium entry pathway in erythrocytes. At least five NHE isoforms have been identified, differing in tissue specificity, regulatory characteristics, and pharmacological sensitivities. Although physiological characteristics of erythrocyte NHE suggest that the widely expressed NHE-1 isoform may be present, evidence is not conclusive and does not exclude the existence of other isoforms. In this study, Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses were used to test for five NHE isoforms in erythroid cells. Blood from patients with sickle cell disease was depleted of white blood cells (WBC) by passage through leukocyte filters and cellulose column. RT-PCR performed on WBC depleted reticulocyte RNA using a NHE-1 primer set yielded product a of expected size, the sequence of which was identical to the published human NHE-1 sequence. Northern blot analysis of the reticulocyte RNA using a 1.6 kb probe revealed a message of approximately 5.0 kb in size. RT-PCR analysis of rat kidney RNA using primers specific for NHE isoforms -2, -3, -4 and rat brain RNA using primer specific for NHE-5 isoform yielded products of expected size, whereas WBC depleted RNA under identical conditions yielded no products. These results identify the erythroid isoform of the sodium hydrogen exchanger as NHE-1.

Potassium depletion and acid-base transporters in rat kidney: differential effect of hypophysectomy.

Potassium depletion is involved in the pathophysiology of metabolic alkalosis. In the present study, the expression of renal acid-base transporters that are involved in HCO3- reabsorption was studied in potassium depletion. Rats fed potassium-deficient (KD) diet developed significant hypokalemia at 14 days (serum K+ 1.9 +/- 0.2 in KD vs. 4.2 +/- 0.2 meq/l in control, P < 0.01) but not at 6 days (3.8 +/- 0.3 in KD vs. 4.1 +/- 0.3 meq/l in control, P > 0.05). Kidney mRNA for colonic H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase, cHKA) increased by approximately 3- and 11-fold at 6 and 14 days of KD diet, respectively, indicating that increased expression preceded the onset of hypokalemia. The expression of Na+/H+ exchanger 3 (NHE-3) mRNA and its cognate protein remained unchanged at 6 and 14 days of KD diet. The mRNA levels for NHE-1, NHE-2, and NHE-4 also remained unchanged at 6 and 14 days of KD diet. Hypophysectomized (HPX) rats fed KD diet for 14 days developed similar hypokalemia. However, the expression of cHKA mRNA in the kidney was decreased by approximately 80% in potassium-depleted (HPX+KD) rats (P < 0.01 vs. KD only). Hypophysectomy did not affect the mRNA levels for either gastric H(+)-K(+)-ATPase (gHKA) or NHE isoforms in KD animals. Thus potassium depletion increases expression of cHKA in the kidney but not that of gHKA or NHE isoforms. The signal for this increase appears to precede hypokalemia. Furthermore, the data suggest that pituitary hormone(s) plays an important and novel role in the regulation of cHKA.

Male and female employment in the textile industry in relation to miscarriage and preterm delivery.

To address potential reproductive hazards in textile manufacturing, we conducted a community-based case-control study in central North Carolina. Miscarriage cases were identified from medical records (280 interviewed cases): preterm delivery cases and term, normal birth weight controls (454 and 605, respectively) were identified from area hospitals. Exposures were based on job title, an interview concerning textile-related exposures, expert imputation of exposure based on job titles and interviews, and self-reported exposures by women. Relative to women and men working in nonhazardous occupations, workers in the textile industry were not at increased risk of miscarriage or preterm delivery, with the possible exception of preterm delivery among women and men employed in sectors other than knitting and yarn mills and men employed in yarn mills. Inferred exposures to specific agents were also not associated with adverse pregnancy outcome. Subject to uncertainty in exposure assessment and nonresponse, these data indicate an absence of adverse effects of the textile workplace environment on these pregnancy outcomes.

Forests of hope: Papua New Guinea. Saying "No" to Asian loggers.

PIP: In the pristine rainforest that covers the steep Bainings mountains of the Gazelle Province in East New Britain, inhabitants live in isolation, with very few roads, first aid posts or schools, and rare opportunities to earn cash for essentials like medicines, rice and kerosene. However, the local Baining people have been able to resist the bribes of the Asian loggers. These loggers have been persistent, returning every now and then with more promises. In view of this, Max Henderson, a naturalized Papua New Guinean, established the Pacific Heritage Foundation to provide an alternative for villagers who wanted an entry into the cash economy. They run at a risk though of being tricked by logging companies to give up their forests for a fraction of the market value. Its main focus is on running environmental awareness campaigns, timber skill courses, as well as providing a market for villagers' timber. Papua New Guinea government may have implemented anti-logging rules and policies, but it is not behind what it says, rather, they act as ambassadors for the exploiters for their benefit.^ieng

Molecular cloning and physical and genetic mapping of a novel human Na+/H+ exchanger (NHE5/SLC9A5) to chromosome 16q22.1.

A human genomic clone for a novel fifth member of the Na+/H+ exchanger (NHE) family, NHE5 (gene symbol SLC9A5), has been isolated and partially sequenced. The deduced amino acid sequence of two exons, containing 154 codons, exhibits 59-73% identity to the other members of the NHE family, with closest similarity to NHE3. Northern blot analysis demonstrated that the NHE5 gene is expressed in brain, testis, spleen, and skeletal muscle. Fluorescence in situ hybridization analysis of a cosmid containing NHE5 to human metaphase chromosomes localized the NHE5 gene to the cytogenetic interval 16q21-q22. A panel of somatic cell hybrids containing various portions of chromosome 16 was used to refine further the placement of NHE5 within band 16q22.1. A polymorphic dinucleotide (GT/CA)n repeat contained in the NHE5 cosmid was identified and developed into a microsatellite PCR marker. This was typed in a subset of the CEPH (Centre d'Etude du Polymorphisme Humain) families to place it on a genetic map of the human genome. Pairwise linkage analysis of this marker showed that it was linked to marker D16S421 with a maximal lod score of 35.21 at a recombination fraction (theta) of 0.000, in complete concordance with its chromosomal localization by physical mapping. Multipoint linkage analysis placed NHE5 between the flanking markers D16S421 and D16S512. The cloning of this new member of the sodium hydrogen exchanger family, its chromosomal localization, and the discovery of a polymorphic marker for it now make it feasible to study the possible involvement of this gene in disorders of Na+/H+ transport.

Physician, heal thyself. Developing a hospital-based physician well-being committee.

This article describes the development of a physician well-being committee at the Sir Mortimer B. Davis-Jewish General Hospital. It discusses the issue of physician stress, outlines the committee's mandate, and describes the various activities and services that were implemented.

Regulation of prostaglandin endoperoxide synthase gene expression in rat mesangial cells by interleukin-1 beta.

In primary cultures of rat mesangial cells from passage 3 to 6, interleukin-1 beta (IL-1) induced a time-dependent increase in prostaglandin E2 (PGE2) formation and release into the extracellular medium. This increase was associated with a dramatic upregulation of the steady-state levels of mRNA for the prostaglandin endoperoxide synthetase (PES)-2 gene transcript as demonstrated by Northern analysis. In contrast, there did not appear to be a significant increase in the mRNA levels for a 2.8-kb transcript for the PES-1 gene. At 18 h of exposure to IL-1, the steady-state level of message for PES-2 remained elevated at 50% of the 2-h time point. Culturing the cells in the presence of cycloheximide and IL-1 demonstrated a superinduction of the PES-2 message without any change in PES-1 message. The tumor-promoting phorbol ester, phorbol myristate acetate (PMA), was also associated with an upregulation of the message for the PES-2 gene and did not influence the levels of the message for the PES-1 gene as demonstrated by Northern analysis. Dexamethasone (Dex) inhibited to control levels the induction by PMA, but the induction of the message by IL-1 was only inhibited 30%. Despite 70% of the message being present by 2 h of induction, Dex was capable of totally inhibiting the inductive effect of IL-1 with respect to PGE2 biosynthesis. Immunocytochemical studies demonstrated a dramatic induction of PES-2 protein by IL-1, which was inhibited by Dex. The data suggest that Dex inhibits the translation of the PES-2 protein.

Amplification of the arachidonic acid cascade: implications for pharmacologic intervention.

The signal transduction pathway that results in prostaglandin production is thought to occur in a stepwise manner that involves agonist-stimulated action of a phospholipase that releases the second messenger arachidonic acid from membrane phospholipids. Prostaglandin endoperoxide synthase (PGH synthase) then converts arachidonic acid to the prostaglandin precursors. Further delineation of this cascade has recently occurred with the identification of two distinct prostaglandin endoperoxide synthases, PGH synthase-1 and PGH synthase-2. There is evidence that PGH synthase-1 may have broad cellular expression and may be constitutively expressed in most cells. In contrast, PGH synthase-2 expression may be more limited and has been shown to be stimulated by a variety of cytokines and growth factors. Dexamethasone inhibits the expression of an early response gene, TIS10, which is homologous to PGH synthase-2. The exact mechanism of PGH synthase-2 gene regulation in mesangial cells is unknown; however, it may be a potential site for pharmacologic intervention. Regulation of PGH synthase-2 could in turn modulate prostaglandin production and temper the production of extracellular matrix and thus scar formation that occurs in a wide variety of inflammatory renal diseases.

Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro.

A hematopoietic cell (CFU-B1) capable of producing blast cell containing colonies in vitro was detected using a semisolid culture system. The CFU-B1 has the capacity for self-renewal and commitment to a number of hematopoietic lineages. Monoclonal antibody to the human progenitor cell antigen-1 (HPCA-1) and a monoclonal antibody against the major histocompatibility class II antigen (HLA-DR) were used with fluorescence activated cell sorting to phenotype the CFU-B1. The CFU-B1 was found to express My10 but not HLA-DR antigen; experiments using complement-dependent cytotoxicity to eliminate DR positive cells confirmed this finding. Pretreatment of marrow cells with two chemotherapeutic agents, 5-fluorouracil and 4-hydroperoxycyclophosphamide facilitated detection of CFU-B1 derived colonies, while diminishing or totally inhibiting colony formation by other hematopoietic progenitor cells. CFU-B1-derived colony formation was dependent upon the addition of exogenous hematopoietic growth factors. Media conditioned either by the human bladder carcinoma cell line 5637 or lectin stimulated leukocytes, as well as recombinant granulocyte-macrophage colony stimulating factor, interleukin 3 or interleukin 1 alpha promoted blast cell colony formation. By contrast, neither recombinant erythropoietin, recombinant interleukin 4, purified macrophage colony stimulating factor or recombinant granulocyte colony-stimulating factor alone promoted blast cell colony formation.

Regulation of proinsulin synthesis in isolated rat islets.

(1) A system is described for studying the short-term effects of agents on proinsulin synthesis in vitro, as measured by the incorporation of [3H]leucine into isolated proinsulin. (2) Of the agents tested, glucose has the most marked, and apparently earliest, effect on proinsulin synthesis. (3) The adenyl cyclase system participates in the regulation of proinsulin synthesis since exogenous cyclic AMP, glucagon, and caffeine are stimulatory. When cyclic AMP is added to the medium in the presence of glucose, it is the most potent agent acting on the adenyl cyclase-phosphodiesterase system. (4) The addition of NADPH to isolated rat islets inhibits proinsulin and Bulk Protein synthesis in vitro.