Kidney RNA Research Articles

Molecular cloning and expression of CCAAT/enhancer binding proteins in Japanese flounder Paralichthys olivaceus

The findings of this study represent the first report, to the authors’ knowledge, of CCAAT/enhancer binding protein (C/EBP) cDNA sequence in a fish species. C/EBP ϵ of Japanese flounder was 1861 bp in length (ORF of 822 bp) encoding for 274 amino acids, with a calculated molecular weight of 30 kDa. Japanese flounder C/EBP β was found to be 1561 bp in length (ORF of 1041 bp), encoding for 347 amino acids and a calculated molecular weight of 39 kDa. These genes were expressed in various fish organs, tissues and secretions. C/EBP ϵ was detected by Northern blot from total RNA of head and posterior kidney, heart and spleen. However, RT-PCR also detected C/EBP ϵ in brain, spleen and peritoneal cavity fluid and peripheral blood leucocyte cDNA. C/EBP β was detected by Northern blot analysis in the head and posterior kidney, spleen, intestine, liver, brain, heart, gill and testis and further found by RT-PCR to be detected in mucus, peritoneal cavity fluid, peripheral blood leucocytes and eye cDNA. Phylogenetic analysis placed the Japanese flounder C/EBP β within the same cluster as previously reported C/EBP β sequences. However, Japanese flounder C/EBP ϵ sequence data were not found to cluster with the three reported mammalian C/EBP ϵ sequences currently available. Understanding C/EBP transcriptional gene control in commercially important fish species may lead to a better control of disease.

Modulation of the two-pore domain acid-sensitive K+ channel TASK-2 (KCNK5) by changes in cell volume.

The molecular identity of K(+) channels involved in Ehrlich cell volume regulation is unknown. A background K(+) conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K(+) channels)-type of two pore-domain K(+) channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K(+) currents in the absence intracellular Ca(2+). Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK(a) in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K(+) > Rb(+) Cs(+) > NH(4)(+) > Na(+) congruent with Li(+), similar to that for the native conductance (I(K, vol)). Clofilium, a quaternary ammonium blocker of I(K, vol), blocked the mTASK-2-mediated K(+) current with an IC(50) of 25 microm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with I(K, vol), and its modulation by changes in cell volume suggest that this two-pore domain K(+) channel participates in the regulatory volume decrease phenomenon.

CUSP/p63 expression in rat and human tissues

The human homolog of KET, p63, bears strong homology to the tumor suppressor p53 and plays an essential role in epithelial development. CUSP, the most abundant cutaneous product of p63, has been identified as an autoantigen in chronic ulcerative stomatitis (CUS). The original report of KET expression at least partially contradicts p63 expression subsequently reported by many different groups. We have examined p63 expression by Northern analysis of RNA from multiple human tissues and by indirect immunofluorescence of rat tissue with CUS patient sera. Northern analysis reveals p63 RNA in skin, thymus, placenta, skeletal muscle, kidney, and lung, with non-transactivating p63 RNA in skin, thymus, and placenta. Reverse transcriptase polymerase chain reaction (rtPCR) assays show abundant non-transactivating p63 RNA, and little to no transactivating p63 RNA, in human basal cell carcinoma as well as in normal skin adjacent to the tumors. p63 RNA expression was not detected in brain, heart, colon, spleen, liver, or small intestine. Immunofluorescence reveals p63 expression in skin, oral epithelium, tongue, kidney, and trachea, but not in liver, large intestine, testis, skeletal muscle, or heart. Focal p63 expression within tissues, the complex array of isoforms encoded by the gene, and the specificity of the probes and antibodies utilized, may all contribute to contradictory accounts of CUSP/ p63 expression.

Renin uptake by the endothelium mediates vascular angiotensin formation.

We investigated the role of the vascular endothelium in the local production of angiotensin. Angiotensin release from isolated rat hindquarters perfused with an artificial medium was measured by high-performance liquid chromatography and radioimmunoassay. Perfused hindquarters with endothelium released angiotensin I spontaneously, indicating ongoing renin-angiotensinogen reaction. Endothelium denudation (by a detergent, validated by electron microscopy and by the absence of a vasodilator response to acetylcholine) reduced angiotensin I release by >90%, whereas bilateral nephrectomy 24 hours before perfusion abolished the release completely. Infusion of renin into perfused hindquarters induced sustained local angiotensin I release in the presence of an intact endothelium but not after endothelium denudation. The conversion of angiotensin I to angiotensin II was abrogated by endothelium denudation, whereas the disappearance of angiotensin II was unchanged. Endothelium denudation diminished the pressor response to angiotensin II but abolished the response to renin and angiotensin I. Expression of renin messenger RNA, investigated by reverse-transcription polymerase chain reaction using 4 different primer combinations, was not detected in up to 5 microg vascular RNA, whereas a renin signal was readily detected with 5 ng kidney RNA. The effects of endothelium destruction on Ang I formation support the notion that the endothelium mediates vascular angiotensin formation by taking up renin.

Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.

A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.

The expression of Cox17p in rodent tissues and cells.

Previous works have reported the isolation of a novel polypeptide from porcine heart. Structural analysis has shown that it is a mammalian homologue of Cox17p, believed essential for the assembly of functional cytochrome c oxidase and delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although the human, mouse and porcine homologs of this small protein have already been cloned or purified, the function of Cox17p in the mammalian system has not yet been elucidated. To investigate the physiological function of Cox17p in mammals, we performed Northern blot analysis using probes containing the mouse and rat sequences obtained by RT-PCR. The hybridization signals were detected in all mouse tissues, but notably intense signals were observed in heart, brain and kidney RNA samples. Some of the neuroendocrine and endocrine cell lines showed higher expression levels than fibroblasts. The highest expression level of Cox17p mRNA in mouse brain was observed in the pituitary sample. While in rat heart, Cox17p mRNA expression was detected from early development, in rat brain, embryonic and postnatal changes in the expression were observed. Immunocytochemical analysis showed that Cox17p immunoreactivity was strong in the pituitary cell line, AtT-20. These findings suggested that Cox17p is not only part of the respiratory chain but also involved in brain and endocrine functions.

In vivo gene expression profile analysis of metallothionein in renal cell carcinoma

The antiapoptotic and mitogenic responses of metallothionein (MT) have been well documented in vitro. While MT protein overexpression, frequently encountered in a number of human primary tumors, has been shown to be correlated with disease progression, little information is available on the in vivo isoform expression of MT. In this study we have demonstrated the occurrence of MT proteins and further defined their differential expression profile in human primary renal cell carcinoma (RCC). Pooled normal human kidney RNA and paired biopsy specimens (tumor and control) obtained from 11 patients diagnosed with RCC with tumor grade ranging from 1–3 and a pathological staging of T2–T3 (N0M0) were used for the study. Samples were analyzed for the presence of MT protein using immunohistochemical (IHC) analysis and for MT isoform-specific mRNA expression by reverse transcriptase polymerase chain reaction. Metallothionein protein assumed both cytoplasmic and nuclear staining in cancer cells and was detected in eight of 11 samples (72%) with polyclonal antibodies. The immunoreactivity of MT protein, but not its cellular localization, in RCC specimens suggests a relationship between and advanced disease. While alterations in the basal level of expression of MT-1E, MT-1F and MT-1X genes remained unchanged, significant up-regulation of MT-2A and down-regulation of MT-1A and MT-1G transcripts was observed in RCC tissue specimens when compared with controls. Intriguingly, the paired RCC biopsy specimens had lower MT-1H transcripts than pooled normal human controls. We here provide the first report of the differential expression of MT isoforms in human RCC and that this data further support the role of MT-2A in tumorigenesis.

Chronic effect of parathyroid hormone on NHE3 expression in rat renal proximal tubules

The most abundant Na+/H+ exchanger in the apical membrane of proximal tubules is the type 3 isoform (NHE3), and its activity is acutely inhibited by parathyroid hormone (PTH). In the present study, we investigate whether changes in protein abundance as well as in mRNA levels play a significant role in the long-term modulation of NHE3 by PTH. Three groups of animals were compared: (1) HP: animals submitted to hyperparathyroidism by subcutaneous implantation of PTH pellets, providing threefold basal levels of this hormone (2.1 U. h-1); (2) control: sham-operated rats in which placebo pellets were implanted; (3) PTX: animals submitted to hypoparathyroidism by thyroparathyroidectomy followed by subcutaneous implantation of thyroxin pellets, which provided basal levels of thyroid hormone. After eight days, we measured bicarbonate reabsorption in renal proximal tubules by in vivo microperfusion. NHE3 activity was also measured in brush border membrane (BBM) vesicles by proton dependent uptake of 22Na. NHE3 expression was evaluated by Northern blot, Western blot and immunohistochemistry. Bicarbonate reabsorption in renal proximal tubules was significantly decreased in HP rats. Na+/H+ exchange activity in isolated BBM vesicles was 6400 +/- 840, 9225 +/- 505, and 12205 +/- 690 cpm. mg-1. 15 s-1 in HP, sham, and PTX groups, respectively. BBM NHE3 protein abundance decreased 39.3 +/- 8.2% in HP rats and increased 54.6 +/- 7.8% in PTX rats. Immunohistochemistry showed that expression of NHE3 protein in apical BBM was decreased in HP rats and was increased in PTX rats. Northern blot analysis of total kidney RNA showed that the abundance of NHE3 mRNA was 20.3 +/- 1.3% decreased in HP rats and 27. 7 +/- 2.1% increased in PTX. Our results indicate that the chronic inhibitory effect of PTH on the renal proximal tubule NHE3 is associated with changes in the expression of NHE3 mRNA levels and protein abundance.

Lack of in vivo function of osteopontin in experimental anti-GBM nephritis.

Osteopontin (Opn) is a potent chemoattractant for mononuclear cells that is upregulated in various inflammatory states of the kidney. Opn is believed to contribute to mononuclear cell infiltration and renal injury. The importance of Opn was examined in vivo in rapidly progressive glomerulonephritis in Opn knockout mice. Glomerulonephritis was induced by intravenous injection of rabbit anti-mouse glomerular basement membrane antiserum in mice that had been presensitized to rabbit IgG. Immunologic responsiveness to rabbit IgG (assessed by cutaneous delayed-type hypersensitivity and antibody titers) showed no significant difference between wild-type and Opn -/- mice. Proteinuria was also similar in both groups. Glomerular crescent formation was not different in Opn +/+ and -/- groups (26 +/- 6% versus 29 +/- 7%). Tubulointerstitial infiltration was assessed qualitatively and showed no significant difference between the two genotypes. Formation of thrombi in the glomerular capillaries on a scale from 0 to 3 also showed no significant difference (1.3 +/- 0.3 for Opn +/+ and 1.4 +/- 0.3 for Opn -/- mice). Northern blot analysis of total kidney RNA showed a 5.1-fold increase of Opn expression in Opn +/+ mice compared with untreated controls and the absence of expression in Opn -/- mice, as expected. Regulated upon activation, normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 mRNA levels were also markedly upregulated with no significant difference between the two strains, excluding compensatory overexpression of these two chemokines in Opn -/- mice. It is concluded that the known upregulation of Opn in murine anti-glomerular basement membrane nephritis does not significantly contribute to the glomerular and tubulointerstitial mononuclear cell infiltration in this model.

Coexpression of neuropilin-1, Flk1, and VEGF164in developing and mature mouse kidney glomeruli

Neuropilin-1, a neuronal cell surface semaphorin III receptor protein important for axonal guidance in developing peripheral nervous system efferents, has also been identified as a vascular endothelial growth factor (VEGF) receptor on endothelial cells. To evaluate its expression in kidney, we carried out RT-PCR on newborn and adult total renal RNAs. A 403-bp product, which was predicted to be that from neuropilin-1 mRNA, was found in both samples. Nucleotide sequencing confirmed that these products encoded neuropilin-1. Northern analysis of newborn and adult kidney RNA showed specific hybridization to appropriately sized bands of approximately 6 kb. In situ hybridization with a mouse-specific antisense neuropilin-1 (35)S-cRNA probe showed distinct glomerular localization on sections from both newborns and adults. Similar patterns of hybridization were seen in sections treated with antisense cRNA probes against another VEGF receptor, Flk1, and with VEGF probes. However, the VEGF hybridization signal was markedly less in adult glomeruli than those for neuropilin-1 and Flk1. Because neuropilin-1 specifically binds VEGF(165) in humans, we carried out RT-PCR on mouse kidney RNA with primers that amplified the three alternatively spliced isoforms of VEGF mRNA. Our analysis showed that for both newborn and adult kidneys, the relative abundance of VEGF mRNA was VEGF(164) >> VEGF(120) > VEGF(188). We conclude that the expression of neuropilin-1, in conjunction with Flk1 and VEGF(164), jointly contributes to the development and maintenance of glomerular capillaries.

Characterization of parathyroid hormone/parathyroid hormone-related protein receptor and signaling in hypercalcemic Walker 256 tumor cells.

Parathyroid hormone (PTH)-related protein (PTHrP) is the main factor responsible for humoral hypercalcemia of malignancy. Both PTH and PTHrP bind to the common type I PTH/PTHrP receptor (PTHR), thereby activating phospholipase C and adenylate cyclase through various G proteins, in bone and renal cells. However, various normal and transformed cell types, including hypercalcemic Walker 256 (W256) tumor cells, do not produce cAMP after PTHrP stimulation. We characterized the PTHrP receptor and the signaling mechanism upon its activation in the latter cells. Scatchard analysis of PTHrP-binding data in W256 tumor cells revealed the presence of high affinity binding sites with an apparent K(d) of 17 nM, and a density of 90 000 sites/cell. In addition, W256 tumor cells immunostained with an anti-PTHR antibody, recognizing its extracellular domain. Furthermore, reverse transcription followed by PCR, using primers amplifying two different regions in the PTHR cDNA corresponding to the N- and C-terminal domains, yielded products from W256 tumor cell RNA which were identical to the corresponding products obtained from rat kidney RNA. Consistent with our previous findings on cAMP production, 1 microM PTHrP(1-34), in contrast to 10 microg/ml cholera toxin or 1 microM isoproterenol, failed to affect protein kinase A activity in W256 tumor cells. However, in these cells we found a functional PTHR coupling to G(alpha)(q/11), whose presence was demonstrated in these tumor cell membranes by Western blot analysis. Our findings indicate that W256 tumor cells express the PTHR, which seems to be coupled to G(alpha)(q/11). Taken together with previous data, these results support the hypothesis that a switch from the cAMP pathway to the phospholipase C-intracellular calcium pathway, associated with PTHR activation, occurs in malignant cells.

Lack of effect of beta-naphthoflavone on induction of Nramp genes in adult rainbow trout Oncorhynchus mykiss

Natural resistance-associated macrophage protein (Nramp) genes in rainbow trout, Oncorhynchus mykiss, were identified and characterized. The greatest mRNA level encoding these genes was in the developing ovary of rainbow trout. We evaluated the response of these genes to a certain aromatic hydrocarbon receptor (AHR) agonist. Adult rainbow trout were treated with β-naphthoflavone (BNF) (50 and 100 mg/kg) for 48 h. Using reverse-transcriptase polymerase chain reaction with ovary and head kidney RNA and specific α and β Nramp primers, a 400 bp Nramp-α- and a 400 bp Nramp-β-specific cDNA were obtained. There were no changes in the α and β Nramp mRNA levels in the ovary following BNF administration. CYP1A1 mRNA was increased in the ovary and kidney, suggesting the presence of AHR in rainbow trout ovary, while the AHR agonist produced no effect on Nramp mRNAs.

DNA sequence and functional characterization of the human and rat epidermal growth factor promoter: regulation by cell growth

Epidermal growth factor (EGF) regulates cell growth and differentiation through intracellular transduction networks activated by its tyrosine kinase receptor, EGFR. In this report we describe the structure and DNA sequence of transcriptional control regions from both human and Wistar-rat single copy EGF genes and their functional analysis in epithelial cell cultures. By sequence comparison we show these proximal gene regions have remained conserved in evolution to −640 (relative to the rodent mRNA initiation site), where similarity is interrupted by a rodent interspersed-repeat element (SINE). Transcript mapping reveals complexity in EGF initiation site selection: whereas a single rat liver initiation site (+1) appears 30 bp 3′ to the TTTAA element, an additional upstream site is detected in kidney RNA at −14. In contrast, in human RNA a single initiation is observed, which is displaced 12 bp 3′ to the rodent RNA terminus. Both promoters were defined in transient expression assays. Our results show the human promoter to be at least 20-fold more active than the equivalent rodent sequence, although both are activated during cell proliferation and negatively regulated in contact inhibited and quiescent cultures. The results indicate EGF gene expression and cell division are temporally linked, suggesting its promoter comprises a growth responsive regulatory domain.

Bovine NAD+-Dependent Isocitrate Dehydrogenase: Alternative Splicing and Tissue-Dependent Expression of Subunit 1

NAD+-dependent isocitrate dehydrogenase (IDH), a key regulatory enzyme in the Krebs cycle, is a multi-tetrameric enzyme. At least three of the subunits in the core tetramer of mammals are unique gene products. Subunits 1/beta and 2/gamma are considered to be regulatory, while subunits 3,4/alpha, comprising half the tetramer, are catalytic. The full sequence was obtained for the major subunit 1 cDNA in bovine heart, IDH 1-A. A second cDNA, rare in heart, was also identified (IDH 1-B). Differences in the two were confined to the 3'-region, suggesting alternative splicing. Screening of brain, kidney, and liver RNA showed the presence of IDH 1-A and 1-B and a third major species, IDH 1-C. Amplification of bovine genomic DNA by PCR across the regions of difference produced a single product. Comparison of the genomic and mRNA sequences showed that IDH 1-A resulted from splicing of exon W to exon Y, eliminating intron w, exon X, and intron x. IDH 1-B was formed by splice junctions between exon W, exon X, and exon Y. IDH 1-C resulted from splicing of exon W to exon X and subsequent retention of intron x. The 2 proteins predicted from these 3 mRNAs are identical over their first 357 residues. Protein IDH 1-A, resulting from a termination codon within exon Y, contains an additional 26 residues. Proteins IDH 1-B and 1-C derive from a common termination codon within exon X and contain an additional 28 residues. The two C-terminal regions differ notably in the number and nature of charged residues, resulting in proteins with a charge difference of 3.2 at pH 7.0. Subunit 1 sequences previously reported from other species grouped with one or the other of the bovine proteins. No evidence was found for alternative splicing in subunit 3,4/alpha. The results of the present study, together with recent work on the 2/gamma subunit [Brenner,V., Nyakatura, G., Rosenthal, A., and Platzer, M. (1998) Genomics 44, 8], indicate that the regulatory subunits of the enzyme, but not the catalytic, possess alternatively spliced forms varying in C-terminal properties with tissue-specific expression. The finding is suggestive of a mechanism for modulation of allosteric regulation tailored to the needs of different tissues.

Cloning and sequence analysis of rainbow trout LMP 2 cDNA and differential expression of the mRNA

A Rainbow trout Oncorhynchus mykiss (L.) clone encoding the low molecular weight proteasome LMP 2 has been cloned and sequenced from rainbow trout leukocyte cells that had been stimulated with the mitogen phytohemagglutinin (PHA). The cDNA was 1254bp and had an open reading frame of 648bp encoding 217 amino acids. The sequence has a high degree of identity with other published LMP 2 sequences varying from 84·4% identity with medaka (Oryzias latipes) to 61·4% identity with Xenopus laevis. Northern blot analysis showed the LMP 2 mRNA to be expressed in a wide range of tissues, with two transcripts in gill, spleen and anterior kidney RNA. Up regulation of the LMP 2 mRNA transcript was shown in cells that were maintained in primary culture following stimulation with the PHA mitogen for 24h. A 3·7-fold increase in mRNA was observed between control and stimulated cells.

Renal effects of uroguanylin and guanylin in vivo.

Uroguanylin and guanylin are newly discovered endogenous heat-stable peptides that bind to and activate a membrane bound guanylyl cyclase signaling receptor (termed guanylyl cyclase C; GC-C). These peptides are not only found in blood but are secreted into the lumen of the intestine and effect a net secretion of electrolytes (Na+, K+, Cl-, HCO3-) and fluid into the intestine via a cyclic guanosine-3', 5'-monophosphate (cGMP) mechanism. GC-C is also the receptor for Escherichia coli heat-stable enterotoxin (STa) and activation by STa results in a diarrheal illness. Employing mouse renal in vivo models, we have demonstrated that uroguanylin, guanylin, and STa elicit natriuretic, kaliuretic, and diuretic effects. These biological responses are time- and dose-dependent. Maximum natriuretic and kaliuretic effects are observed within 30-40 min following infusion with pharmacological doses of the peptides in a sealed-urethra mouse model. Our mouse renal clearance model confirms these results and shows significant natriuresis following a constant infusion of uroguanylin for 30 min, while the glomerular filtration rate, plasma creatinine, urine osmolality, heart rate, and blood pressure remain constant. These data suggest the peptides act through tubular transport mechanisms. Consistent with a tubular mechanism, messenger RNA-differential display PCR of kidney RNA extracted from vehicle- and uroguanylin-treated mice show the message for the Na+/K+ ATPase gamma-subunit is down-regulated. Interestingly, GC-C knockout mice (Gucy2c -/-) also exhibit significant uroguanylin-induced natriuresis and kaliuresis in vivo, suggesting the presence of an alternate receptor signaling mechanism in the kidney. Thus, uroguanylin and guanylin seem to serve as intestinal and renal natriuretic peptide-hormones influencing salt and water transport in the kidney through GC-C dependent and independent pathways. Furthermore, our recent clinical probe study has revealed a 70-fold increase in levels of urinary uroguanylin in patients with congestive heart failure. In conclusion, our studies support the concept that uroguanylin and guanylin are endogenous effector peptides involved in regulating body salt and water homeostasis.

Rat muscle fructose-1,6-bisphosphatase: cloning of the cDNA, expression of the recombinant enzyme, and expression analysis in different tissues.

The 1282 bp cDNA of an isoenzyme of fructose-1,6-bisphosphatase was cloned from rat muscle. It shows 70% positional identity to the cDNA of rat liver fructose-1,6-bisphosphatase and is clearly the product of a gene different from that coding for the liver enzyme. After cloning of the coding region of the rat muscle fructose-1,6-bisphosphatase cDNA in an expression vector, the recombinant enzyme could be detected in E. coli cell-free extracts by activity determination and Western blotting. Overexpressed fructose-1,6-bisphosphatase was found to be allosterically inhibited by AMP comparably to the enzyme isolated from rat muscle. Analysis of steady-state mRNA levels of various rat tissues with reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting revealed one or the two fructose-1,6-bisphosphatase isoenzyme mRNAs in most tissues tested with significant quantitative differences. Quantitative PCR using a homologous competitor showed that 1 microg of total RNA of rat muscle contains 1.7 x 10(6) molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 x 10(4) copies of this message were found per microg total RNA of heart and kidney, respectively.

Altered sodium pump alpha and gamma subunit gene expression in nephron segments from hypertensive rats.

To determine the qualitative and quantitative expression of alpha and gamma sodium pump subunits in whole kidney and nephron segment RNA from Sprague Dawley rats, spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. A novel reverse transcription polymerase chain reaction technique was devised which provides accurate and precise measurement of the number of molecules of specific transcript abundance, a measurement of gene expression. This allows the quantitative comparison of multiple samples across multiple subjects and, since the estimates are accurate rather than relative, can also be used to make quantitative comparisons across expressed genes, such as isoforms and subunits of the heterotrimeric renal sodium pump. We examined which catalytic isoforms were expressed and then quantified transcript abundance in whole kidney and convoluted and straight segments of the proximal tubule. Alpha 1 and gamma transcripts, but not alpha 2, alpha 3 or alpha 4 isoforms, were consistently observed in nephron segments. Levels of alpha 1 were lower in kidney RNA from 15-16-week-old SHR than in WKY rats of the same age (P = 0.001), but were not different between SHR and WKY in 4-5-week-old animals. No significant difference was observed in gamma subunit abundance in kidney RNA from 4-5-week-old animals; however, at 15-16 weeks, the expression in SHR was one-third that in WKY rats (P = 0.003). In proximal convoluted tubules from 4-5-week-old animals, the level of alpha 1 RNA expression was lower (P = 0.03) in SHR than in WKY rats. In addition, levels of alpha 1 in proximal straight tubule from the 4-5-week-old SHR were also lower than in WKY rats (P = 0.02). This difference was even greater in 15-16-week-old animals: in SHR, alpha 1 expression was less than 20% of the level of expression in WKY rats (P = 0.0003). Expression of the gamma subunit exhibited a similar pattern of downregulation in SHR. In RNA from proximal convoluted tubules and proximal straight tubules from both 4-5- and 15-16-week-old animals, expression of the gamma subunit was demonstrated to be significantly lower in SHR than in WKY rats. The results indicate a coordinate reduction in the abundance of sodium pump alpha and gamma subunits in the proximal tubules of SHR, which occurs early during the development of hypertension.

Regulation of the genes for 11beta-hydroxysteroid dehydrogenase type 1 and type 2 in the kidney of the Dahl rat.

An isoenzyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), 11beta-HSD-2 confers aldosterone specificity on the mineralocorticoid receptor (MR) and is found collocated in renal cortical collecting duct cells with the MR. To investigate whether the salt sensitivity of the Dahl salt-sensitive (S) rat is due to 11beta-HSD deficiency, we measured 11beta-HSD-1 and 11beta-HSD-2 mRNA levels in the kidneys of Dahl-S and Dahl salt-resistant (R) rats. In addition, we studied the effects of gender, age and dietary sodium on expression of mRNA for the two isoforms. S and R rats were placed on low- or high-sodium (HNa) diets and sacrificed after 33 and 115 days. Rat kidney RNA was isolated and 11beta-HSD-1 and 11beta-HSD-2 mRNA levels were measured on Northern filter hybridization using isoform-specific probes. No strain differences were observed in the mRNA expression of the two isoforms of 11beta-HSD under any of the experimental conditions. No gender or age differences were observed in 11beta-HSD-2 mRNA but HNa diet almost doubled 11beta-HSD-2 mRNA (P<0.0009). 11beta-HSD-1 mRNA levels were consistently higher, more than double, in male rats versus females rats (P<0.0001), and in the 115-day-old rats versus the 33-day-old rats (P<0.0001). Dietary sodium intake did not affect 11beta-HSD-1 mRNA levels. There is no difference in the expression of the two isoforms of 11beta-HSD in the kidneys of the S and R rats, which might explain the salt sensitivity and higher blood pressure of the S rat. Renal 11beta-HSD-1 mRNA levels are higher in male than in female rats, and in the older rats of both strains. In the kidney, the 11beta-HSD-2 gene is regulated by sodium status but is not affected by gender or age.

Developmental expression of protein phosphatase 2A in the kidney.

Although a number of growth and transcription factors are known to regulate renal growth and development, the signal transduction molecules necessary to mediate these developmental signals are relatively unknown. Therefore, the activity and mRNA and protein expression of the signal transduction molecule protein phosphatase 2A (PP2A) were examined during rat kidney development. Northern analysis of total kidney RNA or Western analysis of kidney protein homogenates from embryonic day 15 to 90-d-old adults demonstrated developmental regulation of the catalytic, major 55-kD B regulatory subunit and A structural subunit with the highest levels of expression in late embryonic and newborn kidneys. Similarly, okadaic acid-inhibitable phosphatase enzyme activity was highest in the embryonic and newborn kidney. To map cell-specific expression of PP2A in the developing kidney, in situ hybridization with a catalytic subunit digoxigenin-labeled cRNA was performed on embryonic day 20 and newborn kidneys. PP2A was found predominately in the nephrogenic cortex and particularly in the developing glomeruli and non-brush border tubules in the embryonic day 20 and newborn kidneys. Similarly, immunocytochemistry with a specific PP2A catalytic subunit polyclonal anti-peptide antibody demonstrated catalytic subunit protein particularly concentrated in the podocytes of glomeruli in the newborn kidney. In the adult kidney, PP2A protein was no longer detectable except in the nuclei of distal tubular cells. Therefore, the developmental regulation of PP2A activity and protein during kidney development and its mapping to the nephrogenic cortex, developing glomeruli, and tubules suggests a role for PP2A in the regulation of nephron growth and differentiation.