Targeted disruption of the bradykinin B(2) receptor gene in mice alters the ontogeny of the renin-angiotensin system.

Angiotensin II type 1 (AT(1)) receptor knockout (KO) mice exhibit an activated kallikrein-kinin system (KKS) that serves to attenuate the severity of the renal vascular phenotype in these mice (Tsuchida S, Miyazaki Y, Matsusaka T, Hunley TE, Inagami T, Fogo A, and Ichikawa I, Kidney Int 56: 509-516, 1999). Conversely, gestational high salt suppresses the fetal renin-angiotensin system (RAS) and provokes aberrant renal development in bradykinin B(2)-KO mice (El-Dahr SS, Harrison-Bernard LM, Dipp S, Yosipiv IV, and Meleg-Smith S, Physiol Genomics 3: 121-131, 2000). Thus the cross talk between the RAS and KKS may be critical for normal renal maturation. To further define the developmental interactions between the KKS and RAS, we examined the consequences of B(2) receptor gene ablation on the expression of RAS components. Renal renin mRNA levels are 50% lower in newborn B(2)-KO than wild-type (WT) mice. Also, the age-related decline in renin mRNA is greater in B(2)-KO than WT mice (3.5- vs. 2-fold, P < 0.05). Although renal angiotensinogen (Ao) protein levels are higher in newborn B(2)-KO than WT mice, Ao mRNA levels are not, suggesting accumulation of Ao as a result of decreased renin-mediated cleavage. Similar age-related increases (8-fold) in angiotensin I-converting enzyme (ACE) activity are observed in B(2)-KO and WT mice. Renal AT(1) protein levels are not different in B(2)-KO and WT mice. Furthermore, the developmental increases in renal kallikrein mRNA and enzymatic activity are more pronounced in B(2)-KO compared with WT mice (mRNA: 8- vs. 3-fold; activity: 13- vs. 6-fold, P < 0.05). We conclude that 1) bradykinin stimulates renin gene expression, 2) renal kallikrein is regulated via a negative feedback loop involving the B(2) receptor, and 3) Ao, ACE, and AT(1) are not bradykinin-target genes.

Ureteric bud derivatives express angiotensinogen and AT1 receptors.

Inactivation of the renin-angiotensin system interferes with the morphogenesis of the renal medulla. Thus ureteric bud (UB) derivatives may be a target for angiotensin production and action. To begin to test this hypothesis, we examined the cellular expression of angiotensinogen (Ao) and AT(1) receptor proteins during rat metanephrogenesis by immunohistochemistry. In addition, we tested whether UB-derived cells in culture express the Ao and AT(1) proteins. On embryonic day E15, Ao and AT(1) are expressed in the UB branches and stromal mesenchyme. S-shaped bodies, including the vascular cleft, express AT(1) but not Ao. The metanephric mesenchyme and pretubular aggregates are Ao negative and AT(1) negative. Expression of Ao and AT(1) in UB branches and ampullae is also observed on E16. However, UB expression of Ao is transient and is no longer detectable in the developing distal nephron beyond E17. On E17, both Ao and AT(1) are expressed in capillary loop glomeruli and proximal tubules, whereas UB branches express AT(1) only. By E18, the majority of Ao immunoreactivity is clustered in terminally differentiated proximal tubules, whereas AT(1) receptors are expressed in both proximal and distal nephron segments. The specificity of Ao and AT(1) staining was documented by the elimination/attenuation of immunoreactivity after preadsorption of the primary antibodies with their respective antigens. Consistent with the in vivo findings, the AT(1) protein is abundantly expressed in cellular lysates of mouse UB (E11.5) and IMCD3 (adult) cells. Moreover, AT(1) receptors in UB and IMCD3 cells are functional, since angiotensin II treatment elicits the tyrosine phosphorylation of the mitogen-activated protein kinases, ERK1/2. To our knowledge, this is the first demonstration of Ao and AT(1) protein expression in the developing distal nephron. Angiotensin II may have a paracrine role in the ontogeny of the collecting system.

Bradykinin B2 null mice are prone to renal dysplasia: gene-environment interactions in kidney development.

Congenital abnormalities of the kidney and urinary tract are a common cause of end-stage renal disease in children. Host and environment factors are implicated in the pathogenesis of aberrant renal development. However, direct evidence linking gene-environment interactions with congenital renal disease is lacking. We report an animal model of renal dysgenesis that is dependent on a defined genetic defect and specific embryonic stressor. Specifically, mice that are deficient in the bradykinin type 2 receptor gene (B(2)) and salt loaded during embryogenesis acquire an aberrant kidney phenotype and die shortly after birth. In contrast, B(2) mutant mice maintained on normal sodium intake or salt-loaded wild-type mice do not develop kidney abnormalities. The kidney abnormality is evident histologically on embryonic day 16, shortly after the onset of metanephric B(2) gene expression, and consists of distorted renal architecture, foci of tubular dysgenesis, and cyst formation. The dysplastic tubules are of distal nephron origin [Dolichos biflorus agglutinin (DBA)- and aquaporin-2 (AQP2) positive, and angiotensinogen negative]. Neonatal antihypertensive therapy fails to ameliorate the renal abnormalities, arguing against the possibility that the nephropathy is a consequence of early hypertension. Moreover, the nephropathy is intrinsic to the embryo, because B(2) homozygous offspring from heterozygous parents exhibit the same renal phenotype as offspring from homozygous null parents. Further characterization of the renal phenotype revealed an important genetic background effect since the penetrance of the congenital nephropathy is increased substantially upon backcrossing of 129/BL6 B(2) mutants to a uniform C57BL/6J. We conclude that the type 2 bradykinin receptor is required for the maintenance of metanephric structure and epithelial integrity in the presence of fetal stress. This study provides a "proof-of-principle" that defined gene-environment interactions are a cause of congenital renal disease.

The bradykinin type 2 receptor is a target for p53-mediated transcriptional activation.

The bradykinin type 2 receptor (BK2) is a developmentally regulated G protein-coupled receptor that mediates diverse actions such as vascular reactivity, salt and water excretion, inflammatory responses, and cell growth. However, little is known regarding regulation of the BK2 gene. We report here that the rat BK2 receptor is transcriptionally regulated by the tumor suppressor protein p53. The 5'-flanking region of the rat BK2 gene contains two p53-like binding sites: a sequence at -70 base pairs (P1 site) that is conserved in the murine and human BK2 genes; and a sequence at -707 (P2) that is not. The P1 and P2 motifs bind specifically to p53, as assessed by gel mobility shift assays. Transient transfection into HeLa cells of a CAT reporter construct driven by 1.2-kilobases of the BK2 gene 5'-flanking region demonstrated that the BK2 promoter is dose dependently activated by co-expression of wild-type p53. Co-expression of a dominant negative mutant p53 suppresses the activation of BK2 by wild-type p53. Promoter truncation localized the p53-responsive element to the region between -38 and -94 base pairs encompassing the p53-binding P1 sequence. Furthermore, p53-mediated activation of the BK2 promoter is augmented by the transcriptional co-activators, CBP/p300. Interestingly, removal of the P2 site by truncation or site-directed deletion amplifies p53-mediated activation of the BK2 promoter. These results demonstrate that the rat BK2 promoter is a target for p53-mediated activation and suggest a new physiological role for p53 in the regulation of G protein-coupled receptor gene expression.

Fetal ontogeny and role of metanephric bradykinin B2 receptors.

Previous studies in rats have shown that blockade of bradykinin B2 receptors (B2R) in combination with a high-salt intake during gestation result in poor postnatal survival and long-term hypertension in the offspring. In this study, we examined the fetal ontogeny of B2R and determined the consequences of gestational B2R blockade and high salt on kidney development. B2R gene expression is induced on embryonic day (E16) of fetal metanephrogenesis and remains sustained until term. The earliest expression of the B2R protein is observed on apical membranes of ureteric bud branches and in capillary loop stage glomeruli. By the end of gestation, B2R becomes restricted to more-differentiated tubules in the deep cortex and medulla. Pairs of rats on normal (0.12 mmol/g) or high (0.84 mmol/g) salt diets were mated at 14 weeks of age. The B2R antagonist, Icatibant (previously known as Hoe-140) (300 nmol/kg per day) or saline (vehicle) was infused intraperitoneally during gestation via osmotic minipumps. Fetuses were examined on E20 (n=27-36 per group). No significant differences in litter size or body weight were observed among the groups. Combined high-salt and Icatibant treatment caused aberrant fetal renal development characterized by tubular dysgenesis, widened stromal mesenchyme, and glomerular cysts. The dysgenetic tubules stained positively for the distal nephron lectin, Dolichos biflorus, and exhibited enhanced Bax expression and apoptosis. Renal microvascular development, the number of mature glomeruli, and percentage of proliferating glomerular cells were not affected. Gestational Icatibant or high salt alone had no deleterious effects on fetal nephrogenesis. We conclude that gestational blockade of the kallikrein-kinin system impairs fetal nephrogenesis if combined with an intrauterine stressor such as high-salt intake. B2R may play a protective role during segmental nephron differentiation.

Inhibition of Na-K-ATPase activity and gene expression by a myeloma light chain in proximal tubule cells.

BACKGROUND: Light chain nephrotoxicity is frequently associated with Fanconi syndrome characterized by amino-aciduria, glycosuria, phosphaturia, and bicarbonaturia. The mechanisms of these transport abnormalities are unknown. To determine the role of Na-K-ATPase, we examined the effects of a lambda-light chain on both the activity and gene expression of Na-K-ATPase in primary cultures of rat proximal tubule cells. METHODS: The lambda-light chain used here was isolated from urine of a patient with multiple myeloma and previously shown to inhibit sodium-dependent phosphate and glucose transport in proximal tubule cells. Na-K-ATPase was determined spectrophotometrically and the gene expression by Northern analysis in cells exposed to light chain. RESULTS: In cells exposed to 200 mumol/L light chain Na-K-ATPase activity was reduced significantly, up to 73%, at 2, 24, and 48 hours compared with control cells (N = 12, P < 0.001). Northern analysis showed that in cells exposed to light chain for 24 and 48 hours the message for the alpha-1 isoform of Na-K-ATPase was suppressed significantly compared with control cells. The messages for GAPDH, beta-actin, and 28 S RNA in light chain exposed cells were also depressed in comparison with control cells. This light chain also significantly inhibited thymidine incorporation by proximal tubule cells in a dose-dependent manner. CONCLUSIONS: These data suggest a general toxicity to cells by this light chain and indicate that inhibitory effects on both the activity and gene expression of Na-K-ATPase may be an important mechanism of light chain cytotoxicity on proximal tubule cells.

Early onset salt-sensitive hypertension in bradykinin B(2) receptor null mice.

Kinins have been implicated in the hemodynamic adaptation to postnatal life. The present study examined the impact of bradykinin B(2) receptor (B(2)R) gene disruption on the postnatal changes in blood pressure (BP) and the susceptibility to early onset salt-sensitive hypertension in mice. B(2)R null (-/-) and wild-type (+/+) mice were fed normal (NS, 1% NaCl) or high (HS, 5% NaCl) salt diets during pregnancy. After birth, the pups remained with their mothers until they were weaned and were subsequently continued on the respective maternal salt intake until 4 months of age. The age-related changes at 3 and 4 months in tail-cuff BP and anesthetized mean arterial pressure at 4 months were not different in NS/B(2)R(-/-) and NS/B(2)R(+/+) mice. However, there was a mild increase in BP in NS/B(2)R(-/-) at 2 months versus NS/B(2)R(+/+). In contrast, HS/B(2)R(-/-) mice manifested early onset and persistent elevations of tail-cuff BP (P<0.05) at 2, 3, and 4 months versus other groups. MAP was also higher in HS/B(2)R(-/-) than HS/B(2)R(+/+), NS/B(2)R(-/-), and NS/B(2)R(+/+) (91+/-3 versus 75+/-5, 74+/-2, and 70+/-2 mm Hg, respectively; P<0.05). Kidney renin and angiotensin type 1 receptor mRNA levels were not different. Additional studies showed that a delay in the initiation of HS until after birth was accompanied by later development of hypertension, although postnatal discontinuation of HS resulted in a gradual return of BP to normal values by 4 months of age. The results demonstrate that (1) kinins protect the developing animal from salt-sensitive hypertension, (2) lack of B(2)R from early development does not alter the maturation of BP under conditions of normal sodium intake, and (3) exposure to a HS diet during fetal life is not sufficient in itself to induce long-term hypertension in either wild-type or B(2)R null mice.

Bradykinin stimulates the ERK-->Elk-1-->Fos/AP-1 pathway in mesangial cells.

Among its diverse biological actions, the vasoactive peptide bradykinin (BK) induces the transcription factor AP-1 and proliferation of mesangial cells (S. S. El-Dahr, S. Dipp, I. V. Yosipiv, and W. H. Baricos. Kidney Int. 50: 1850-1855, 1996). In the present study, we examined the role of protein tyrosine phosphorylation and the mitogen-activated protein kinases, ERK1/2,in mediating BK-induced AP-1 and DNA replication in cultured rat mesangial cells. BK (10(-9) to 10(-7) M) stimulated a rapid increase in tyrosine phosphorylation of multiple proteins with an estimated molecular mass of 120-130, 90-95, and 44-42 kDa. Immunoblots using antibodies specific for ERK or tyrosine-phosphorylated ERK revealed a shifting of p42 ERK2 to a higher molecular weight that correlated temporally with an increase in tyrosine-phosphorylated ERK2. Genistein, a specific tyrosine kinase inhibitor, prevented the phosphorylation of ERK2 by BK. In-gel kinase assays indicated that BK-induced tyrosine phosphorylation of ERK2 is accompanied by fourfold activation of its phosphotransferase activity toward the substrate PHAS-I (P < 0.05). Furthermore, BK stimulated a 2.5-fold increase (P < 0.05) in phosphorylation of Elk-1, a transcription factor required for growth factor-induced c-fos transcription. In accord with the stimulation of Elk-1 phosphorylation, BK induced c-fos gene expression and the production of Fos/AP-1 complexes. In addition, thymidine incorporation into DNA increased twofold (P < 0. 05) following BK stimulation. Each of these effects was blocked by tyrosine kinase inhibition with genistein or herbimycin A. Similarly, antisense oligodeoxynucleotide targeting of ERK1/2 mRNA inhibited BK-stimulated DNA synthesis. In contrast, protein kinase C inhibition or depletion had no effect on BK-induced c-fos mRNA, AP-1-DNA binding activity, or DNA synthesis. Collectively, these data demonstrate that BK activates the ERK-->Elk-1-->AP-1 pathway and that BK mitogenic signaling is critically dependent on protein tyrosine phosphorylation.

Activation of kininogen expression during distal nephron differentiation.

Previous studies have shown that the epithelial precursors of the connecting tubule and collecting duct express tissue kallikrein and bradykinin B2 receptors, respectively, suggesting the presence of a local kinin-producing/responsive system in the maturing distal nephron. However, evidence for the existence of kininogen in the developing nephron is still lacking. This study examined the spatiotemporal relationships between segmental nephron differentiation and the ontogeny of kininogen and kinins in the rat. Kininogen immunoreactivity is detectable in the metanephros as early as embryonic day 15. In the nephrogenic zone, the terminal ureteric bud branches are the main kinin-expressing segments. Kininogen is also observed in the stromal mesenchyme. In contrast, proximal ureteric bud branches, metanephrogenic mesenchyme, and pretubular aggregates express little or no kininogen. After completion of nephrogenesis, kininogen distribution assumes its classic "adult" pattern in the collecting ducts. Peak kininogen mRNA and protein expression occur perinatally, corresponding to the period of active nephrogenesis in the rat, and declines gradually thereafter. Estimations made by RT-PCR, Western blotting, and radioimmunoassays indicate that renal kininogen mRNA and protein levels are at least 20-fold higher in newborn than adult rats. Likewise, immunoreactive tissue kinin levels are 2.3-fold higher in newborn than adult kidneys (P < 0.05). In summary, the present study demonstrates the activation of kininogen gene expression and kinin production in the developing kidney. The terminal ureteric bud branches and their epithelial derivatives are the principal kinin-producing segments in the maturing nephron. The results suggest an autocrine/paracrine role for the kallikrein-kinin system in distal nephron maturation.

Role of bradykinin B2 receptors in neonatal kidney growth.

The kallikrein-kinin system is developmentally expressed in newborn kidneys. In addition, bradykinin (BK) is mitogenic in cultured glomerular mesangial cells. However, the role of endogenous BK in postnatal renal development has not been defined. In this study, the role of the BK-B2 receptor in neonatal kidney growth in the rat was examined. RNA blot analysis and semiquantitative reverse transcription-polymerase chain reaction showed that BK-B2 mRNA levels were approximately 30- to 40-fold higher in newborn than adult kidneys. Treatment of newborn rats with the selective BK-B2 antagonist, Hoe 140 (600 micrograms/kg per day, sc), from days 1 through 14 of life significantly reduced body weight, kidney-to-body weight ratios, and kidney DNA content, compared with saline-treated controls. Hoe 140 treatment had no effect on kidney protein or RNA content or the expression of transforming growth factor-beta mRNA. The growth retardation induced by BK-B2 blockade was observed only in the kidney and, to a lesser extent, in the heart. BK-B2 blockade had no effect on renal growth in adult rats, suggesting that these effects are developmentally regulated. In contrast to Hoe 140 treatment, neonatal protein undernutrition resulted in a generalized reduction in kidney DNA, RNA, and protein contents; increased renal transforming growth factor-beta gene expression; and decreased renal kallikrein expression and enzymatic activity. The results suggest that activation of BK-B2 receptor expression in the neonatal kidney plays an important role in the regulation of DNA synthesis during the latter stages of nephrogenesis.

Bradykinin stimulates c-fos expression, AP-1-DNA binding activity and proliferation of rat glomerular mesangial cells.

An important role for bradykinin (BK) in nephrogenesis has been suggested based on impairment of renal growth in developing rats treated with a kinin antagonist. However, direct effects of BK on renal cell mitogenesis have not been reported. In the present study, we examined the mitogenic effects of BK on cultured rat mesangial cells. Transcripts encoding BK-B2 receptors were detected in quiescent and proliferating mesangial cells by reverse transcription-coupled polymerase chain reaction. In quiescent mesangial cell cultures (0.5% FCS for 48 hr), BK (10(-9) to 10 (-7)M) caused a significant increase in DNA synthesis (3H-thymidine incorporation into DNA) and cell number. BK-induced DNA synthesis was preceded by activation of c-fos gene expression and both of these effects were inhibited by Hoe-140, a specific BK-B2 antagonist. Electrophoretic gel mobility shift assays revealed enhanced binding of AP-1 complexes to a consensus AP-1 DNA sequence in BK-stimulated cells. Gel supershift assays confirmed that the AP-1 complexes contained the fos protein. These data document a direct mitogenic effect of BK, acting on B2 receptors, on mesangial cells.

Differential effects of ureteral obstruction on rat kininogen gene family.

The precursors of kinins, K-kininogens and T-kininogens (KG), are encoded by separate genes that display 90% nucleotide sequence homology. Despite their homology, K-KG and T-KG genes are differentially regulated. The K-KG gene is expressed constitutively and encodes high- and low-molecular-weight KG, the precursors of the vasoactive nonapeptide bradykinin. In contrast, the T-KG gene is inducible, and its protein is a potent thiol-protease inhibitor. Given their potential role in the regulation of blood pressure, renal hemodynamics, and the response to inflammation and tissue injury, K-KG and T-KG gene expression in rats subjected to chronic (1 or 5 wk) unilateral ureteral obstruction (UUO), a maneuver that suppresses renal kallikrein synthesis to 25% of controls, has been examined. Northern and slot blots of total liver and kidney RNA were probed with oligonucleotides complementary to either T-KG or K-KG mRNA under high-stringency conditions. Steady-state levels of hepatic T-KG mRNA were increased in the UUO compared with sham-operated rats--2.7-fold at 1 wk and 4.1-fold at 5 wk (P < 0.05). Western blot analysis revealed that the 68-kd T-KG protein was up-regulated 2.5- to 3-fold in the liver of UUO rats (P < 0.05). In marked contrast, the abundance of high (2.3-kb)- and low (1.6-kb)-molecular-weight splicing transcripts of hepatic pre-K-KG mRNA was not altered at either time after UUO.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of somatic angiotensin-converting enzyme.

Angiotensin-converting enzyme or kininase II (ACE-KII) plays a central role in the control of circulating and tissue levels of angiotensin II and kinins. Both peptides have been implicated in the regulation of renal function and growth during normal development. We tested the hypothesis that the developing rat kidney expresses ACE-KII mRNA transcripts and the active enzyme and evaluated whether the developmental expression of the ACE-KII gene is related to changes in circulating angiotensin II and tissue kallikrein. ACE-KII mRNA and enzymatic activity were low in the newborn kidney; peak expression occurred on days 15 and 20 of postnatal life (16-fold versus day 1). In extrarenal tissues, ACE-KII activity and mRNA levels were also low during the newborn period in the following order of abundance: lung > kidney > aorta > heart. The lung showed a higher age-related increase in active ACE-KII and mRNA abundance (15-fold) than heart and aorta (activity, 3- to 4-fold; mRNA, 6- to 10-fold). The developmental profile of ACE-KII correlated temporally with changes in circulating angiotensin II and tissue kallikrein. Plasma angiotensin II levels were 2.5-fold higher in newborn than adult rats, whereas renal and extrarenal kallikrein-like activity increased twofold to fivefold from birth to adulthood. These results demonstrate that the ACE-KII gene is developmentally regulated in a tissue-specific manner. Tissue kinin generation and degradation, reflected by kallikrein and ACE-KII activities, are coordinately regulated during development, whereas circulating angiotensin II and tissue ACE-KII change in a reciprocal manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Renin, angiotensinogen, and kallikrein gene expression in two-kidney Goldblatt hypertensive rats.

An imbalance in the activity of the vasopressor renin-angiotensin and vasodepressor kallikrein-kinin systems may play an important role in the pathogenesis of hypertension after unilateral renal artery constriction. To test this hypothesis, we examined the expression of the renin, angiotensinogen (Ao), and tissue kallikrein genes 7 and 25 days after placement of a 0.25-mm clip on the left renal artery of rats. One week after clipping, renin mRNA levels were 4.6-fold higher in the clipped and 50% lower in the nonclipped kidneys compared with kidneys from sham-operated rats. At 25 days, renin mRNA levels in the clipped kidneys were not different from sham kidneys, but were suppressed to almost undetectable levels in the nonclipped kidneys. Steady-state Ao mRNA levels in the clipped kidneys were not different from those of nonclipped or sham kidneys at either 7 or 25 days. However, at 25 days, Ao mRNA levels were lower in the liver (70%), left ventricle (55%), and aorta (45%) of clipped than sham-operated rats. The expression of the renal kallikrein gene was unchanged at 7 days and was suppressed by 50% at 25 days. These results are consistent with the notion that activation of the intrarenal renin-angiotensin system occurs during the initial phase of the two-kidney, one-clip hypertension model. The renal kallikrein gene, in marked contrast to renin, becomes downregulated in the chronic phase. The differential regulation of renin-angiotensin and kallikrein genes may be an important pathogenetic factor in renovascular hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular aspects of kallikrein and kininogen in the maturing kidney.

Kinins are vasoactive paracrine peptides which participate in a wide range of functions, including the regulation of local organ blood flow, systemic blood pressure, transepithelial water and electrolyte transport, cellular growth, capillary permeability and inflammatory response, and pain. The recent introduction of specific bradykinin receptor subtype antagonists has greatly advanced our understanding of the role of the kallikrein-kinin system (KKS) in various physiological and disease states. However, a major gap remains in our knowledge of the role of kinins in early development. In this review, evidence is presented that the developing nephron expresses both tissue kallikrein and kininogen, and that the genes encoding the components of the KKS are subject to considerable developmental regulation. The activity of the intrarenal kinin-generating system is lowest in the developing kidney and increases with age. Completion of nephrogenesis is characterized by a marked surge in intrarenal kallikrein synthesis and gene transcription. Maturation is associated with redistribution of intrarenal kallikrein and its messenger RNA from the inner to outer cortical nephrons following the centrifugal pattern of nephron development. Challenges for the future include delineation of the direct role of kinins in the maturation of renal functions and elucidation of the molecular mechanisms underlying the developmental expression of the KKS.

Upregulation of renin-angiotensin system and downregulation of kallikrein in obstructive nephropathy.

The purpose of this study was to delineate the effects of prolonged (1 and 5 wk) unilateral ureteral obstruction (UUO) on the intrarenal renin-angiotensin and kallikrein-kinin systems in the rat. Systolic blood pressure (SBP) and plasma angiotensin (ANG) II levels were significantly higher at 1 and 5 wk of obstruction than in sham-operated groups. Also, plasma renin activity and ANG I levels were elevated at 1 wk (P < 0.05), and plasma angiotensin-converting enzyme (ACE)-kininase II activity was elevated at 5 wk (P < 0.05). Blockade of ANG II receptors with losartan (Dup 753) prevented the rise in SBP after UUO and normalized SBP in chronically hypertensive UUO rats. Renin mRNA levels and ANG II content were elevated in the obstructed kidneys at 1 and 5 wk compared with sham-operated kidneys (P < 0.05). ACE-kininase II activity was elevated in both the obstructed and contralateral kidneys at 5 wk compared with sham-operated kidneys (P < 0.05). In marked contrast to renin, total immunoreactive kallikrein contents and tissue kallikrein mRNA levels in the obstructed kidneys were reduced to 25% of sham-operated kidneys both at 1 and 5 wk (P < 0.001). The results indicate that urinary obstruction activates renin and suppresses kallikrein gene expression. Activation of ACE-kininase II by UUO also serves to enhance intrarenal ANG II generation and kinin degradation. The results implicate ANG II overproduction and kinin deficiency in the pathogenesis of UUO-induced hypertension and intrarenal vasoconstriction.

Differential developmental expression of the rat kininogen genes.

The rat liver expresses two low molecular weight kininogens (T-KG and K-KG). Although they share 90% of the nucleotide sequence in their 5' flanking regions, T- and K-KG genes are differentially regulated. The T-KG gene is inducible, and its protein is a potent thiol-protease inhibitor. In contrast, K-KG gene is expressed constitutively and encodes the precursor of the vasoactive nonapeptide bradykinin. To further elucidate the differential regulation of T- and K-KG genes, we examined their developmental expression in the Sprague-Dawley rat. Northern blots of total liver RNA were probed with oligonucleotides complementary to T and K-KG mRNA under high-stringency conditions. A single T-KG mRNA (1.8 kb) and two K-KG mRNA species (1.6 and 2.3 kb) were consistently detected at all ages studied. Steady state T-KG mRNA levels increased 3.5-fold at birth and remained high during the 1st week of postnatal life only to decline thereafter. T-KG immunoreactivity in the liver and plasma determined by Western blot analysis paralleled T-KG mRNA expression. In marked contrast, K-KG mRNA expression was not altered during the transition from fetal to neonatal life, nor was it affected by postnatal maturation. The results demonstrate that the fetal rat liver synthesizes kininogens and that T- and K-KG genes are differentially regulated during development. Up-regulation of T-KG synthesis after birth may serve a protective function in the newborn via its antiprotease activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiology of rob beta-lactamase among ampicillin-resistant Haemophilus influenzae isolates in the United States.

We surveyed 161 clinical isolates of ampicillin-resistant, beta-lactamase-producing isolates of Haemophilus influenzae obtained between 1975 and 1985 to determine whether they produced TEM-1 or Rob beta-lactamase. Plasmid DNA was obtained from a Rob-producing isolate, F990, and a plasmid (pBR322) known to encode TEM-1. Both plasmids were labeled with 32P and hybridized to whole cell DNA obtained from the clinical isolates. All 161 isolates hybridized with one of the plasmid probes and could be classified as TEM-1- or Rob-producing isolates. Analysis of the distinctive pH profiles of the two beta-lactamases was used to confirm the findings of the DNA hybridization assay. Overall, 13 (8%) isolates obtained from patients in California, North Carolina, Tennessee, Missouri, Louisiana, and Mississippi produced the Rob beta-lactamase. The remaining isolates elaborated the TEM-1 enzyme. We conclude that ampicillin resistance in H. influenzae may be mediated by the production of Rob beta-lactamase and that the occurrence of this enzyme is not limited to the two isolates described to date.