Generation of Superoxide and In Vitro Inactivation of Viruses by Aldopentoses

Ren-1 renin is synthesized in the kidney of every mouse. Ren-2 renin has been observed in the submandibular gland (SMG) of male mice carrying two renin genes. However, it is not known if Ren-2 renin is in the kidney and blood of the two-renin gene mice. In this study, a direct ELISA for Ren-2 renin (SMG renin) was established by a sandwich method. This ELISA could measure the Ren-2 active renin in the range from 1 to 100ng and distinguish Ren-2 active renin from not only Ren-1 renin but also Ren-2 prorenin. By a combination of this assay system and conventional methods, the pro-form as well as the active form of Ren-2 renin was found in the kidney and plasma of male AKR mice carrying two-renin genes.     

Close physical linkage of the murine Ren-1 and Ren-2 loci

In addition to the Ren-1 gene common to all mice, some inbred strains carry a second copy of the renin structural gene, Ren-2. These two loci are tightly linked genetically on mouse chromosome one. We have used pulsed field gel electrophoresis (PFGE) to study the physical arrangement of the two renin genes in the inbred strain DBA/2. PFGE mapping permitted the construction of a restriction map of the Ren loci spanning roughly 120 Kb. The results indicate that the genes are transcribed in the same relative direction, that Ren-2 lies upstream relative to Ren-1, and that the respective coding sequences are separated by approximately 20 Kb.     

Evolution and Variation of Renin Genes in Mice

Inbred strains of mice carry Ren-1, a gene encoding the thermostable Renin-1 isozyme. Ren-1 is expressed at relatively low levels in mouse submandibular gland and kidney. Some strains also carry Ren-2, a gene encoding the thermolabile Renin-2 isozyme. Ren-2 is expressed at high levels in the mouse submandibular gland and at very low levels, if at all, in the kidney. Ren-1 and Ren-2 are closely linked on mouse chromosome 1, show extensive homology in coding and noncoding regions and provide a model for studying the regulation of gene expression. An investigation of renin genes and enzymatic activity in wild-derived mice identified several restriction site polymorphisms as well as putative variants in renin gene expression and protein structure. The number of renin genes carried by different subpopulations of wild-derived mice is consistent with the occurrence of a gene duplication event prior to the divergence of M. spretus (2.75–5.5 million yr ago). This conclusion is in agreement with a prior estimate based upon comparative sequence analysis of Ren-1 and Ren-2 from inbred laboratory mice.     

Physical Characterization of Genetic Rearrangements at the Mouse Renin Loci

Many inbred strains of mice have a single locus encoding renin, Ren-1, whereas other inbred strains have two tandemly linked loci, Ren-1 and Ren-2. Each of these renin genes in inbred mice exhibits a unique pattern of tissue-specific expression. As a prerequisite to understanding the structural basis for the expression differences, we have physically characterized the sequence organization of this chromosomal region in both types of strains. Pulsed field gel electrophoresis was initially used to compare the long-range structure of this region in C57BL/6 (Ren-1) and DBA/2 (Ren-1 + Ren-2) mice. The structure in both inbred strains is extremely similar, except for an additional 30 kb containing Ren-2 in DBA/2 mice. The boundaries of the extra 30-kb segment were sequenced and compared to homologous sequences flanking the Ren-1 alleles. This analysis identified the precise recombination site, and also the presence of a large insertion, between the renin loci in DBA/2. The renin gene duplication apparently resulted from recombination between sequences sharing little homology, suggesting that nonhomologous chromosomal breakage and rejoining may have been involved mechanistically in the event.     

Renin enhancer is critical for control of renin gene expression and cardiovascular function

The important cardiovascular regulator renin contains a strong in vitro enhancer 2.7 kb upstream of its gene. Here we tested the in vivo role of the mouse Ren-1c enhancer. In renin-expressing As4.1 cells stably transfected with Ren-1c promoter with or without enhancer, expression of linked beta-geo reporter, stable expression, and colony formation were dependent on the presence of the enhancer. We then generated mice carrying a targeted deletion of the enhancer (REKO mice) and found marked depletion of renin in renal juxtaglomerular and submandibular ductal cells, as well as hyperplasia of macula densa cells. Plasma creatinine was increased, but electrolytes were normal. Male REKO mice implanted with telemetry devices had 9 +/- 1 mm Hg lower mean arterial pressure (p < 0.001), which was partly normalized by a high NaCl diet. Locomotor activity was lower, and baroreflex sensitivity was normal. Markedly reduced mean arterial pressure variability in the midfrequency band indicated a contribution of reduced sympathetic vasomotor tone to the hypotension. In conclusion, the renin enhancer is critical for renin gene expression and physiological sequelae, including response to alteration in salt intake. The REKO mouse may be useful as a low renin expression model.     

DNA insertions distinguish the duplicated renin genes of DBA/2 and M. hortulanus mice.

In a survey of inbred and wild mouse DNAs for genetic variation at the duplicate renin loci, Ren-1 and Ren-2, a variant Not I hybridization pattern was observed in the wild mouse M. hortulanus. To determine the basis for this variation, the structure of the M. hortulanus renin loci has been examined in detail and compared to that of the inbred strain DBA/2. Overall, the gross features of structure in this chromosomal region are conserved in both Mus species. In particular, the sequence at the recombination site between the linked Ren-1 and Ren-2 loci was found to be identical in both DBA/2 and M. hortulanus, indicating that the renin gene duplication occurred prior to the divergence of ancestors of these mice. Renin flanking sequences in M. hortulanus, however, were found to lack four DNA insertions totaling approximately 10.5 kb which reside near the DBA/2 loci. The postduplication evolution of the mouse renin genes is thus characterized by a number of insertion and/or deletion events within nearby flanking sequences. Analysis of renin expression showed little or no difference between these mice in steady state renin RNA levels in most tissues examined, suggesting that these insertions do not influence expression at those sites. A notable exception is the adrenal gland, in which DBA/2 and M. hortulanus mice exhibit different patterns of developmentally regulated renin expression.     

DNA insertions distinguish the duplicated renin genes of DBA/2 andM. hortulanus mice

In a survey of inbred and wild mouse DNAs for genetic variation at the duplicate renin loci,Ren-1 andRen-2, a variantNot I hybridization pattern was observed in the wild mouseM. hortulanus. To determine the basis for this variation, the structure of theM. hortulanus renin loci has been examined in detail and compared to that of the inbred strain DBA/2. Overall, the gross features of structure in this chromosomal region are conserved in bothMus species. In particular, the sequence at the recombination site between the linkedRen-1 andRen-2 loci was found to be identical in both DBA/2 andM. hortulanus, indicating that the renin gene duplication occurred prior to the divergence of ancestors of these mice. Renin flanking sequences inM. hortulanus, however, were found to lack four DNA insertions totaling approximately 10.5 kb which reside near the DBA/2 loci. The postduplication evolution of the mouse renin genes in thus characterized by a number of insertion and/or deletion events within nearby flanking sequences. Analysis of renin expression showed little or no difference between these mice in steady state renin RNA levels in most tissues examined, suggesting that these insertions do not influence expression at those sites. A notable exception is the adrenal gland, in which DBA/2 andM. hortulanus mice exhibit different patterns of developmentally regulated renin expression.     

The nucleotide sequence of a mouse renin-encoding gene, Ren-1d, and its upstream region

The renin-encoding genes have been cloned from high (Ren-1d, Ren-2d)- and low (Ren-1c)-renin-producing strains of mice (DBA/2J and C57BL/10). Each of the genes is approx. 9.6 kb in length and consists of nine exons and eight introns. The entire nucleotide sequence of the Ren-1d gene has been determined and the 5'-flanking regions of the three genes, Ren-1c, Ren-1d and Ren-2d, have been compared. The significance of several potential regulatory signals found in the DNA is discussed.     

Influence of the adenosine A1 receptor on blood pressure regulation and renin release

The present study was performed to investigate the role of adenosine A1 receptors in regulating blood pressure in conscious mice. Adenosine A1-receptor knockout (A1R-/-) mice and their wild-type (A1R+/+) littermates were placed on standardized normal-salt (NS), high-salt (HS), or salt-deficient (SD) diets for a minimum of 10 days before telemetric blood pressure and urinary excretion measurements in metabolic cages. On the NS diet, daytime and nighttime mean arterial blood pressure (MAP) was 7-10 mmHg higher in A1R-/- than in A1R+/+ mice. HS diet did not affect the MAP in A1R-/- mice, but the daytime and nighttime MAP of the A1R+/+ mice increased by approximately 10 mmHg, to the same level as that in the A1R-/-. On the SD diet, day- and nighttime MAP decreased by approximately 6 mmHg in both A1R-/- and A1R+/+ mice, although the MAP remained higher in A1R-/- than in A1R+/+ mice. Although plasma renin levels decreased with increased salt intake in both genotypes, the A1R-/- mice had an approximately twofold higher plasma renin concentration on all diets compared with A1R+/+ mice. Sodium excretion was elevated in the A1R-/- compared with the A1R+/+ mice on the NS diet. There was no difference in sodium excretion between the two genotypes on the HS diet. Even on the SD diet, A1R-/- mice had an increased sodium excretion compared with A1R+/+ mice. An abolished tubuloglomerular feedback response and reduced tubular reabsorption can account for the elevated salt excretion found in A1R-/- animals. The elevated plasma renin concentrations found in the A1R-/- mice could also result in increased blood pressure. Our results confirm that adenosine, acting through the adenosine A1 receptor, plays an important role in regulating blood pressure, renin release, and sodium excretion.     

Granulation rescue and developmental marking of juxtaglomerular cells using "piggy-BAC" recombination of the mouse ren locus

Mice lacking a functional Ren-1(d) gene exhibit a complete lack of renal juxtaglomerular cell granulation and atypical macula densa morphology. Transgenic mice carrying a 145-kilobase BAC clone encompassing the Ren-1(d) and Ren-2 loci were generated, characterized, and backcrossed with Ren-1(d-/-) mice. Homozygous Ren-1(d)-null mice expressing the BAC clone exhibited complete restoration of normal renal structure. Homologous recombination in Escherichia coli was used to generate a modified version of the BAC clone, in which an IRESbeta-geo cassette was inserted specifically into the Ren-1(d) gene. When introduced into the germline, the modified clone provided a marker for juxtaglomerular cell differentiation and beta-geo was expressed appropriately in juxtaglomerular cells throughout development. Parallel backcross experiments onto the Ren-1(d)-null background demonstrated that the juxtaglomerular cells expressed the modified Ren-1(d) locus in the absence of regranulation. These data demonstrate that the nongranulated cells constitute bona fide juxtaglomerular cells despite their altered morphology, that overexpression of renin-2 cannot compensate for the loss of renin-1(d), and that primary structural differences between the two isoforms are responsible for the differences in granulation. The use of BAC modification as part of functional complementation studies illustrates the potential for in vivo molecular dissection of key physiological mechanisms.     

N-linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells

Most mouse inbred strains carry two renin genes, Ren-1 and Ren-2, Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.     

The Bradykinin B2 receptor is required for full expression of renal COX-2 and renin

Angiotensin converting enzyme (ACE) inhibition leads to increased levels of bradykinin, cyclooxygenase-2 (COX-2), and renin. Since bradykinin stimulates prostaglandin release, renin synthesis may be regulated through a kinin-COX-2 pathway. To test this hypothesis, we examined the impact of bradykinin B2 receptor (B2R) gene disruption in mice on kidney COX-2 and renin gene expression. Kidney COX-2 mRNA and protein levels were significantly lower in B2R-/- mice by 40-50%. On the other hand, renal COX-1 levels were similar in B2R-/- and +/+ mice. Renal renin protein was 61% lower in B2R-/- compared to B2R+/+ mice. This was accompanied by a significant reduction in renin mRNA levels in B2R-/- mice. Likewise, intrarenal angiotensin I levels were significantly lower in B2R-/- mice compared to B2R+/+ mice. In contrast, kidney angiotensin II levels were not different and averaged 261+/-16 and 266+/-15fmol/g in B2R+/+ and B2R-/- mice, respectively. Kidney angiotensinogen, AT1 receptor and ACE activity were not different between B2R+/+ and B2R-/- mice. The results of these studies demonstrate suppression of renal renin synthesis in mice lacking the bradykinin B2R and support the notion that B2R regulation of COX-2 participates in the steady-state control of renin gene expression.