Spatial expression of the kallikrein-kinin system during nephrogenesis.

During nephrogenesis, new nephrons are induced in the periphery of the kidney, while maturing nephrons occupy a deeper position in the renal cortex. This centrifugal pattern of maturation is characterized by nephron patterning, establishment of proximal-distal segment identity, tubular and glomerular growth and differentiation, and acquisition of specialized functions. All of these processes are coordinated in time and space with renal vasculogenesis, glomerulogenesis and regional hemodynamic changes. The end-result ensures that tubular structure and function are tightly coordinated with glomerular filtration during normal kidney development. To achieve this delicate task of glomerulotubular balance, the developing kidney produces growth factors and vasoactive hormones that act in a paracrine manner to regulate nephrovascular growth, differentiation and physiological functions. One such paracrine system is the kallikrein-kinin system (KKS), which generates bradykinin (BK) from the cleavage of kininogen by kallikrein. BK activates a G-protein coupled receptor, B2R, to regulate renal blood flow and salt and water excretion. The developing kidney expresses an endogenous KKS. Expression of the KKS components and B2R is intimately coordinated with the terminal differentiation of the distal nephron. Kallikrein marks the onset of connecting tubule development, whereas kininogen and B2R map to the developing ureteric bud branches and maturing collecting ducts.Gene targeting studies indicate that the fetal KKS plays an important role in the maintenance of terminal epithelial cell differentiation.

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.

Angiotensin II-induced hypertension in bradykinin B2 receptor knockout mice.

The present study was performed to examine the role of endogenous bradykinin (BK) in the development of angiotensin II (Ang II)-induced hypertension in mice. BK B2receptor knockout (B2R-/-) and wild-type (B2R+/+) mice (22to 26 g) were infused with either saline (SAL) or Ang II (40ng/min) via an osmotic minipump implanted intraperitoneally. On day 12after implantation, there was no difference in systolic blood pressure (SBP, tail-cuff plethysmography) between SAL/B2R+/+ and SAL/B2R-/- mice(128+/-5 versus 133+/-6 mm Hg, n=24/group). In contrast, SBP was higher on day 12 of infusion in Ang II/B2R-/- than in Ang II/B2R+/+ mice (173+/-6versus 156+/-5 mm Hg; P<0.05, n=27 and 28). Mean arterial pressure (MAP)was also higher in anesthetized Ang II/B2R-/- mice than in Ang II/B2R+/+mice (139+/-3 versus 124+/-3 mm Hg; P<0.05, n=16 and 14). Unlike Ang II, long-term norepinephrine (NE) infusion via an osmotic minipump (45ng/min) caused equivalent increases in SBP in B2R+/+ and B2R-/- mice measured on day 12 after implantation (151+/-4 versus 149+/-5 mm Hg, n=9and 8). MAP also did not differ on day 13 after implantation between NE/B2R+/+ and NE/B2R-/- mice (120+/-6 versus 122+/-4 mm Hg, n=9 and 8). There were no differences in glomerular filtration rate and urinary sodium excretion among the groups. However, renal plasma flow (RPF) was lower in Ang II/B2R-/- mice than in Ang II/B2R+/+ mice (2.34+/-0.06 versus 4.33+/-0.19 mL x min-1 x g-1; P<0.05). Acute inhibition of NO synthase (NOS)with nitro-L-arginine-methyl ester (0.5 microg x g-1 x min-1) in SAL/B2+/+ and SAL/B2-/- mice caused equal increases in MAP (142+/-1 versus 145+/-1 mmHg) and decreases in RPF (2.06+/-0.06 versus 2.12+/-0.15 mL x min-1 x g-1).However, short-term NOS inhibition caused a greater increase in MAP of Ang II/B2R+/+ mice than of Ang II/B2R-/- mice, such that MAP after NOS inhibition in Ang II/B2R+/+ approached that of Ang II/B2R-/- mice (156+/-2versus 159+/-2 mm Hg). These changes were associated with a decrease in RPF in Ang II/B2R+/+ mice to values similar to those of Ang II/B2R-/- mice before NOS inhibition (2.12+/-0.09 versus 2.34+/-0.06 mL x min-1 x g-1). These results demonstrate that the kallikrein-kinin system selectively buffers the vasoconstrictor activity of Ang II. Furthermore, the enhanced susceptibility of B2R-/- mice to Ang II-induced hypertension and renal vasoconstriction is likely due to an impaired ability to release NO by endogenous kinins.

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.

Roles of ANG II and bradykinin in the renal regional blood flow responses to ACE inhibition in sodium-depleted dogs.

The relative contributions of ANG II and bradykinin (BK) to the renal regional blood flow responses during angiotensin-converting enzyme (ACE) inhibition remain unclear. This study was performed to evaluate renal cortical (CBF) and medullary blood flow (MBF) responses to intrarterial administration of enalaprilat (33 microg. kg(-1). min (-1)) after blockade of the ANG II AT(1 )receptors with candesartan (100 microg) in 7 dogs fed a low-salt diet (0.01%) for 5 days. Laser-Doppler flowmetry was used to measure relative changes in CBF and MBF. Candesartan alone increased CBF (+20 +/- 2%) and MBF (+22 +/- 7%). Enalaprilat infusion after candesartan administration resulted in further increases in both CBF (+21 +/- 5%) and MBF (+41 +/- 8%). However, the relative changes in MBF were significantly greater (P < 0.01) than those in CBF. Administration of the BK B(2) receptor blocker icatibant (300 microg) after enalaprilat returned CBF and MBF to values seen with candesartan alone. These data support a substantive role for BK potentiation during ACE inhibitor-induced renal vasodilation in dogs maintained on a low-sodium diet, with a relatively greater effect on MBF compared to CBF.

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.

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.

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.

Hypoxia induces selective SAPK/JNK-2-AP-1 pathway activation in the nucleus tractus solitarii of the conscious rat.

In the nucleus tractus solitarii, NMDA glutamate receptors are critical to the hypoxic ventilatory response. However, the signal transduction pathways underlying the hypoxic ventilatory response remain undefined. To assess the effect of a moderate hypoxic stimulus (10% O2) on tyrosine phosphorylation of proteins in the nucleus tractus solitarii, tissue lysates were harvested by repeated punch sampling at 0, 1, 10, and 60 min of hypoxia and examined for the presence of phosphorylated tyrosine residues by immunoblotting. Time-dependent phosphotyrosine increases occurred in proteins migrating at regions corresponding to molecular masses of 38-42, 50, 55, and 60 kDa, which were attenuated by pretreatment with the NMDA receptor channel blocker, MK-801. As extracellular signal-regulated kinase (Erk) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) phosphorylation may induce Fos and Jun gene transcription and activator protein-1 (AP-1) DNA binding, the activation of Erk1, Erk2, p38, and SAPK/JNK was examined in the nucleus tractus solitarii and neocortex during hypoxia and following administration of MK-801. Hypoxia enhanced Erk1, Erk2, and p38 activity in the cortex, but not in the nucleus tractus solitarii. Increased phosphorylation of SEK1 and SAPK/JNK-2 occurred in the nucleus tractus solitarii during hypoxia, whereas both SAPK/JNK-1 and SAPK/JNK-2 were recruited in cortex. MK-801 attenuated hypoxia-induced SEK1, SAPK/JNK-2, and AP-1 binding in the nucleus tractus solitarii, and the widespread activation of all MAP kinases in the cortex was also attenuated. We conclude that in conscious rats, a moderate hypoxic stimulus elicits NMDA-dependent widespread mitogen-activated protein kinase activation in cortex, but selective SAPK/JNK-2 and AP-1 activation in the nucleus tractus solitarii, thereby suggesting a functional role for the SAPK/JNK-2-AP-1 pathway.

Regulation of angiotensin II type 1 receptor mRNA and protein in angiotensin II-induced hypertension.

Chronic elevations of circulating angiotensin II (Ang II) cause sustained hypertension and enhanced accumulation of intrarenal Ang II by an AT1 receptor-dependent process. The present study tested the hypothesis that chronic elevations in circulating Ang II regulate AT1 mRNA and protein expression in a tissue-specific manner. Sprague-Dawley rats were infused with Ang II (80 ng/min) or vehicle subcutaneously for 13 days via osmotic minipump. On day 12, systolic blood pressure averaged 186+/-12 mm Hg in Ang II-infused rats compared with rats given vehicle (121+/-2 mm Hg). Plasma renin activity was markedly suppressed in the Ang II-infused rats compared with vehicle-infused rats (0.1+/-0.01 versus 4.9+/-0.9 ng of Ang I. mL-1. h-1; P<0.05). Semiquantitative reverse transcription polymerase chain reaction using rat AT1A- and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)-specific primers was followed by Southern blot hybridization using specific radiolabeled cDNA or oligonucleotide probes. The results showed that the ratios of AT1A/GAPDH mRNA in the kidney (0.19+/-0.05 versus 0. 26+/-0.03) and liver (2.8+/-0.9 versus 3.0+/-0.5) were comparable in Ang II- and vehicle-infused rats. In contrast, AT1A/GAPDH mRNA levels were increased in the adrenal glands of Ang II-infused rats (0.49+/-0.04 versus 0.36+/-0.02; P<0.05). Western blot analysis showed that AT1 protein levels in the kidney and liver were also similar in the two groups. Therefore, these results indicate that renal and liver AT1 receptor gene expression is maintained in Ang II-induced hypertension. The failure to downregulate AT1 receptor mRNA and protein levels thus allows the sustained effects of chronic elevations in Ang II to elicit progressive increases in arterial pressure.

Kinin influences on renal regional blood flow responses to angiotensin-converting enzyme inhibition in dogs.

The relative roles of ANG II and bradykinin (BK) in the regulation of renal medullary circulation have remained unclear. We compared the contributions of ANG II and BK to the renal medullary blood flow (MBF) responses to angiotensin-converting enzyme (ACE) inhibition (enalaprilat, 33 micrograms . kg-1. min-1) in dogs maintained on a normal-salt diet (0.63%, 3 days, n = 14; group 1) with those fed a low-salt diet (0.01%, 5 days, n = 14; group 2), which upregulates both the kallikrein-kinin and the renin-angiotensin systems. MBF responses to ACE inhibition were evaluated either before (n = 7) or after (n = 7) treatment with the BK B2 receptor blocker icatibant (100-300 micergrams) in both groups. Laser-Doppler needle flow probes were used to determine relative changes in MBF and cortical blood flow (CBF). ACE inhibition increased MBF (group 1, 33 +/- 9%, P

The kininogen gene family in obstructive uropathy.

Obstructive uropathy impairs nephron growth and function and is a major cause of end-stage renal disease in both adults and children. The major focus of this review article is to examine the evidence implicating a role for the kallikrein-kinin system in the pathophysiology of obstructive uropathy. Recent in vivo studies using specific kinin receptor antagonists and transgenic animals overexpressing or lacking various components of the kallikrein-kinin system have documented that kinins are involved in the regulation of renal function and blood pressure. Multiple roles have been proposed for kinins in obstructive uropathy. Renal kallikrein gene expression is suppressed in the kidney with chronic (>7 days) complete ureteral obstruction. In contrast, ureteral obstruction stimulates renin expression, creating a state of intrarenal angiotensin excess and kinin deficiency, which plays an important role in mediating the increased renal vascular resistance and decreased renal blood flow in the obstructed kidney. In addition to their hemodynamic effects, kallikrein and kinins influence tubular functions. For example, kallikrein influences urinary acidification in the distal nephron, suggesting that dysregulation of kallikrein expression may contribute to the acidification defect in the obstructed kidney. Also, kinins exert direct diuretic and natriuretic effects in the collecting duct and may be important in mediating the post-obstructive diuresis after the relief of urinary obstruction. The kinin substrate, kininogen, is a potent inhibitor of lysosomal cysteine proteases. Unlike kallikrein, kininogen synthesis is upregulated in the kidneys and liver of animals with urinary obstruction. By neutralizing cysteine proteases, kininogen may protect the tubular epithelium of obstructed nephrons from excessive apoptosis. The beneficial actions of kinins and kininogens on renal hemodynamics, tubular function, and cell survival suggest that strategies aimed at increasing intrarenal kinins, eg, ACE-kininase II inhibitors and kallikrein gene therapy, may represent a useful adjunct in the medical treatment of obstructive uropathy.

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.

Developmental biology of angiotensin-converting enzyme.

Molecular, biochemical, and evolutionary studies indicate that somatic angiotensin-converting enzyme (ACE) is developmentally regulated in a tissue-specific manner. However, many important questions remain unanswered. For example, the regulatory mechanisms that control the cell- and stage-specific expression of ACE remain largely unknown. The nature, location, and role of the enzyme involved in the release of plasma membrane-anchored ACE have not been elucidated. Although the expression and localization of ACE are developmentally regulated, the physiological implications of these changes in segmental nephron differentiation remain to be elucidated. Investigations of the cellular and molecular mechanisms mediating the developmental co-regulation of the renin-angiotensin and Kallikrein-kinin system by ACE are a formidable challenge for future research.

Ontogeny of the intrarenal kallikrein-kinin system: proposed role in renal development.

The kallikrein-kinin system (KKS) plays an important role in the regulation of renal function. Endogenous kinins modulate renal microvascular resistance, medullary blood flow, and distal nephron sodium and water reabsorption. All the components of the KKS, including tissue kallikrein, kininogen, kininase II, and kinin receptors are expressed within the kidney, establishing a paracrine system capable of controlling local nephron functions. In this review, data will be presented demonstrating that the developing kidney expresses an endogenous, functionally active KKS. Molecular studies have shown that gene expression of the renal KKS in the rat is activated postnatally, and that the intrarenal distribution of KKS components is subject to developmental control. Furthermore, the developmental expression of KKS appears to be regulated primarily at the transcriptional level. Ontogenetic studies have also revealed that the bradykinin B-2 receptor gene is overexpressed in the developing rat kidney. As kinins are potent vasoactive and growth-promoting factors, it is proposed that endogenous kinins mediate developmental renal growth and differentiation, and modulate the maturational changes which occur in renal hemodynamics.

Regulation of angiotensin II AT1 and AT2 receptors in neonatal ureteral obstruction.

Chronic unilateral ureteral obstruction (UUO) in early development activates the intrarenal renin-angiotensin system and leads to profound renal vasoconstriction, renal growth arrest, and interstitial fibrosis. To investigate the response of the AT1 and AT2 subtypes of the angiotensin II (ANG II) receptors to UUO, Sprague-Dawley rats underwent UUO or control sham operation in the first 48 h of life and were studied 1-28 days later. Renal mRNA for renin, AT1 and AT2 receptor, and receptor binding and distribution were determined. In contrast to controls, renin mRNA increased from 14 to 28 days in the obstructed kidney. After ipsilateral UUO, AT1 mRNA was suppressed at 1 day, but had increased compared with controls at 28 days. AT2 receptor mRNA fell rapidly in all kidneys from 1 to 3 days of age, after which it remained undetectable. Compared with the intact opposite kidney, AT2 mRNA was suppressed in the obstructed kidney 1 day after UUO. Compared with controls, AT1 and AT2 receptor binding was decreased by ipsilateral UUO at 1 day, whereas AT1 binding was increased at 28 days. Renal ANG II content was increased in the obstructed compared with the intact opposite kidney 28 days after UUO. In view of the increase in renal renin and angiotensin II production resulting from UUO, increased renal AT1 mRNA and receptor binding are likely to contribute to the vasoconstriction and interstitial fibrosis of the neonatal kidney after prolonged UUO.

Immunohistochemical localization of ANG II AT1 receptor in adult rat kidney using a monoclonal antibody.

Molecular and functional studies have suggested that AT1 receptors are present in most nephron segments, yet direct demonstration of AT1 at these sites is lacking. The present study was performed to determine the intrarenal localization of the AT1 receptor utilizing a monoclonal anti-peptide (amino acid residues 8-17) antibody (6313/G2) in adult male Sprague-Dawley rats. Western blot analysis of kidney protein extracts showed a predominant 41-kDa immunoreactive band corresponding to the molecular weight of the deduced cDNA sequence. To determine optimal fixation conditions, kidney tissues were immersion fixed in Bouin's solution, 10% buffered Formalin, or 4% paraformaldehyde. Specificity of immunostaining was documented by preadsorption of the antibody with the immunogenic peptide sequence. Prominent AT1 immunostaining was visualized in the proximal tubule brush-border and basolateral membranes. In addition, distal tubules, cortical and medullary collecting ducts, and the renal arterial vasculature exhibited specific immunoreactivity. Glomerular staining for AT1 was observed in mesangial cells and podocytes. Macula densa cells stained positively. Similar localization of the AT1 receptor was obtained using the three tissue fixation methods, although the intensity of vascular and glomerular staining was highest in Bouin-fixed tissues. The present study demonstrates that the AT1 receptor is more widely distributed along the nephron than previously described and includes renal vascular smooth muscle and proximal and distal epithelial sites.