Angiotensin II-AT1–receptor signaling is necessary for cyclooxygenase-2–dependent postnatal nephron generation
2016; Elsevier BV; Volume: 91; Issue: 4 Linguagem: Inglês
10.1016/j.kint.2016.11.003
ISSN1523-1755
AutoresStefanie Frölich, Patrick Slattery, Dominique Thomas, Itamar Goren, Nerea Ferreirós, Boye L. Jensen, Rolf M. Nüsing,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoDeletion of cyclooxygenase-2 (COX-2) causes impairment of postnatal kidney development. Here we tested whether the renin angiotensin system contributes to COX-2–dependent nephrogenesis in mice after birth and whether a rescue of impaired renal development and function in COX-2-/- mice was achievable. Plasma renin concentration in mouse pups showed a birth peak and a second peak around day P8 during the first 10 days post birth. Administration of the angiotensin II receptor AT1 antagonist telmisartan from day P1 to P3 did not result in cortical damage. However, telmisartan treatment from day P3 to P8, the critical time frame of renal COX-2 expression, led to hypoplastic glomeruli, a thinned subcapsular cortex and maturational arrest of superficial glomeruli quite similar to that observed in COX-2-/- mice. In contrast, AT2 receptor antagonist PD123319 was without any effect on renal development. Inhibition of the renin angiotensin system by aliskiren and enalapril caused similar glomerular defects as telmisartan. Administration of the AT1 receptor agonist L162313 to COX-2-/- pups improved kidney growth, ameliorated renal defects, but had no beneficial effect on reduced cortical mass. L162313 rescued impaired renal function by reducing serum urea and creatinine and mitigated pathologic albumin excretion. Moreover, glomerulosclerosis in the kidneys of COX-2-/- mice was reduced. Thus, angiotensin II-AT1–receptor signaling is necessary for COX-2–dependent normal postnatal nephrogenesis and maturation. Deletion of cyclooxygenase-2 (COX-2) causes impairment of postnatal kidney development. Here we tested whether the renin angiotensin system contributes to COX-2–dependent nephrogenesis in mice after birth and whether a rescue of impaired renal development and function in COX-2-/- mice was achievable. Plasma renin concentration in mouse pups showed a birth peak and a second peak around day P8 during the first 10 days post birth. Administration of the angiotensin II receptor AT1 antagonist telmisartan from day P1 to P3 did not result in cortical damage. However, telmisartan treatment from day P3 to P8, the critical time frame of renal COX-2 expression, led to hypoplastic glomeruli, a thinned subcapsular cortex and maturational arrest of superficial glomeruli quite similar to that observed in COX-2-/- mice. In contrast, AT2 receptor antagonist PD123319 was without any effect on renal development. Inhibition of the renin angiotensin system by aliskiren and enalapril caused similar glomerular defects as telmisartan. Administration of the AT1 receptor agonist L162313 to COX-2-/- pups improved kidney growth, ameliorated renal defects, but had no beneficial effect on reduced cortical mass. L162313 rescued impaired renal function by reducing serum urea and creatinine and mitigated pathologic albumin excretion. Moreover, glomerulosclerosis in the kidneys of COX-2-/- mice was reduced. Thus, angiotensin II-AT1–receptor signaling is necessary for COX-2–dependent normal postnatal nephrogenesis and maturation. In mice, and most likely also in man, the late phase of nephrogenesis is strictly dependent on the activity of cyclooxygenase (COX)-2, 1 of 2 COX isoforms that orchestrate the metabolism of arachidonic acid to various bioactive prostanoids.1Smith W.L. DeWitt D.L. Garavito R.M. Cyclooxygenases: structural, cellular, and molecular biology.Annu Rev Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2449) Google Scholar The outstanding role of COX-2 for nephrogenesis is supported by several lines of evidence: First, in COX-2–deficient mice, postnatal kidney development is impaired, manifesting in hypoplastic glomeruli, thinned cortical tissue mass, and the presence of immature glomeruli in the subcapsular nephrogenic zone.2Dinchuk J.E. Car B.D. Focht R.J. et al.Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II.Nature. 1995; 378: 406-409Crossref PubMed Scopus (895) Google Scholar, 3Morham S.G. Langenbach R. Loftin C.D. et al.Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse.Cell. 1995; 83: 473-482Abstract Full Text PDF PubMed Scopus (1025) Google Scholar These defects are not obvious at date of birth, but become evident until weaning and result in kidney insufficiency, in addition to glomerular and diffuse interstitial fibrosis.4Norwood V.F. Morham S.G. Smithies O. Postnatal development and progression of renal dysplasia in cyclooxygenase-2 null mice.Kidney Int. 2000; 58: 2291-2300Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Medullar tissue is not affected.4Norwood V.F. Morham S.G. Smithies O. Postnatal development and progression of renal dysplasia in cyclooxygenase-2 null mice.Kidney Int. 2000; 58: 2291-2300Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Second, wild-type mice treated with COX-2 inhibitors in the postnatal phase show similar histomorphologic renal defects.5Kömhoff M. Wang J.L. Cheng H.F. et al.Cyclooxygenase-2-selective inhibitors impair glomerulogenesis and renal cortical development.Kidney Int. 2000; 57: 414-422Abstract Full Text Full Text PDF PubMed Google Scholar, 6Olliges A. Wimmer S. Nusing R.M. Defects in mouse nephrogenesis induced by selective and non-selective cyclooxygenase-2 inhibitors.Br J Pharmacol. 2011; 163: 927-936Crossref PubMed Scopus (18) Google Scholar Third, no apparent renal abnormalities are seen in mice with deficient COX-1.6Olliges A. Wimmer S. Nusing R.M. Defects in mouse nephrogenesis induced by selective and non-selective cyclooxygenase-2 inhibitors.Br J Pharmacol. 2011; 163: 927-936Crossref PubMed Scopus (18) Google Scholar, 7Langenbach R. Morham S.G. Tiano H.F. et al.Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration.Cell. 1995; 83: 483-492Abstract Full Text PDF PubMed Scopus (1041) Google Scholar In humans, in utero exposition to nonsteroidal anti-inflammatory drugs in the second half of gestation (e.g., due to abuse of analgesic drugs or use as long-term tocolytic drugs) can cause renohistologic defects such as small glomeruli, incomplete tubular differentiation, reduced cortical growth, and finally renal failure in the neonate.8Kaplan B.S. Restaino I. Raval D.S. et al.Renal failure in the neonate associated with in utero exposure to non-steroidal anti-inflammatory agents.Pediatr Nephrol. 1994; 8: 700-704Crossref PubMed Scopus (120) Google Scholar, 9van der Heijden B.J. Carlus C. Narcy F. et al.Persistent anuria, neonatal death, and renal microcystic lesions after prenatal exposure to indomethacin.Am J Obstet Gynecol. 1994; 171: 617-623Abstract Full Text PDF PubMed Scopus (86) Google Scholar, 10Antonucci R. Zaffanello M. Puxeddu E. et al.Use of non-steroidal anti-inflammatory drugs in pregnancy: impact on the fetus and newborn.Curr Drug Metab. 2012; 13: 474-490Crossref PubMed Scopus (117) Google Scholar In mice, prenatal inhibition of COX-2 activity does not affect renal development,5Kömhoff M. Wang J.L. Cheng H.F. et al.Cyclooxygenase-2-selective inhibitors impair glomerulogenesis and renal cortical development.Kidney Int. 2000; 57: 414-422Abstract Full Text Full Text PDF PubMed Google Scholar and the postnatal time frame most sensitive toward COX-2 inhibition-induced renal injury has been identified between postnatal day 4 (P4) and P8. This time frame overlaps with transient renal expression of COX-2 and microsomal prostaglandin E synthase (mPGES)-1, leading to enhanced prostaglandin E2 (PGE2) synthetic capacity, whereas COX-1, mPGES-2, and cytosolic PGES (cPGES) remain unaffected.5Kömhoff M. Wang J.L. Cheng H.F. et al.Cyclooxygenase-2-selective inhibitors impair glomerulogenesis and renal cortical development.Kidney Int. 2000; 57: 414-422Abstract Full Text Full Text PDF PubMed Google Scholar, 11Frolich S. Olliges A. Kern N. et al.Temporal expression of the PGE2 synthetic system in the kidney is associated with the time frame of renal developmental vulnerability to cyclooxygenase-2 inhibition.Am J Physiol Renal Physiol. 2012; 303: F209-F219Crossref PubMed Scopus (21) Google Scholar The importance of the COX-2/mPGES-1/PGE2 axis for normal renal development is supported by the observation that mice deficient in the PGE2 receptors (EP)2 and EP4 also show defects in nephrogenesis.11Frolich S. Olliges A. Kern N. et al.Temporal expression of the PGE2 synthetic system in the kidney is associated with the time frame of renal developmental vulnerability to cyclooxygenase-2 inhibition.Am J Physiol Renal Physiol. 2012; 303: F209-F219Crossref PubMed Scopus (21) Google Scholar Mechanisms downstream of EP receptor activation responsible for normal renal development are unknown. In adult rodents, COX-2 is necessary for a full renin and angiotensin II (Ang II) response to a reduction in renal blood flow12Wang J.L. Cheng H.F. Harris R.C. Cyclooxygenase-2 inhibition decreases renin content and lowers blood pressure in a model of renovascular hypertension.Hypertension. 1999; 34: 96-101Crossref PubMed Scopus (175) Google Scholar or low luminal NaCl concentration in the loop of Henle.13Traynor T.R. Smart A. Briggs J.P. Schnermann J. Inhibition of macula densa-stimulated renin secretion by pharmacological blockade of cyclooxygenase-2.Am J Physiol. 1999; 277: F706-F710PubMed Google Scholar Taking into account that postnatal activation of the renin-angiotensin system (RAS) has been suggested to be dependent on COX-2 activation,14Stubbe J. Jensen B.L. Bachmann S. et al.Cyclooxygenase-2 contributes to elevated renin in the early postnatal period in rats.Am J Physiol Regul Integr Comp Physiol. 2003; 284: R1179-R1189Crossref PubMed Scopus (35) Google Scholar and renin secretion is stimulated by PGE2/EP2, PGE2/EP4,15Schweda F. Klar J. Narumiya S. et al.Stimulation of renin release by prostaglandin E2 is mediated by EP2 and EP4 receptors in mouse kidneys.Am J Physiol Renal Physiol. 2004; 287: F427-F433Crossref PubMed Scopus (88) Google Scholar and prostacyclin/prostacyclin receptor activation,16Fujino T. Nakagawa N. Yuhki K. et al.Decreased susceptibility to renovascular hypertension in mice lacking the prostaglandin I2 receptor IP.J Clin Invest. 2004; 114: 805-812Crossref PubMed Scopus (98) Google Scholar we hypothesize that components of the RAS such as Ang II may be involved in the COX-2–mediated stage of nephrogenesis. The RAS has been shown to be activated during late fetal and early postnatal life and to be crucial for renal structural development.17Guron G. Friberg P. An intact renin-angiotensin system is a prerequisite for normal renal development.J Hypertens. 2000; 18: 123-137Crossref PubMed Scopus (182) Google Scholar Renin is strongly expressed in prenatal and neonatal rodent kidneys.14Stubbe J. Jensen B.L. Bachmann S. et al.Cyclooxygenase-2 contributes to elevated renin in the early postnatal period in rats.Am J Physiol Regul Integr Comp Physiol. 2003; 284: R1179-R1189Crossref PubMed Scopus (35) Google Scholar Neonatal angiotensin-converting enzyme (ACE) inhibition by enalapril caused prominent papillary atrophy, tubular atrophy and dilatation, and focal glomerulosclerosis in rats.18Guron G. Adams M.A. Sundelin B. Friberg P. Neonatal angiotensin-converting enzyme inhibition in the rat induces persistent abnormalities in renal function and histology.Hypertension. 1997; 29: 91-97Crossref PubMed Scopus (58) Google Scholar Similar renal defects were observed in rats treated neonatally with AT1 antagonist losartan.19Friberg P. Sundelin B. Bohman S.O. et al.Renin-angiotensin system in neonatal rats: induction of a renal abnormality in response to ACE inhibition or angiotensin II antagonism.Kidney Int. 1994; 45: 485-492Abstract Full Text PDF PubMed Scopus (204) Google Scholar Inconsistently, midcortical glomeruli were found to be smaller with fewer capillary loops, whereas juxtamedullary glomeruli appeared normal.20Tufro-McReddie A. Romano L.M. Harris J.M. et al.Angiotensin II regulates nephrogenesis and renal vascular development.Am J Physiol. 1995; 269: F110-F115PubMed Google Scholar However, mice genetically lacking defined components of the RAS such as angiotensinogen (Agt),21Niimura F. Labosky P.A. Kakuchi J. et al.Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.J Clin Invest. 1995; 96: 2947-2954Crossref PubMed Scopus (319) Google Scholar ACE,22Esther Jr., C.R. Howard T.E. Marino E.M. et al.Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility.Lab Invest. 1996; 74: 953-965PubMed Google Scholar or compound Agtr1a-/-/Agtr1b-/- mice23Oliverio M.I. Kim H.S. Ito M. et al.Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.Proc Natl Acad Sci U S A. 1998; 95: 15496-15501Crossref PubMed Scopus (279) Google Scholar display strong renal dysgenesis defined by diminished kidney growth, vascular wall thickening, and atrophy of the inner renal medulla. The present study was aimed to clarify our hypothesis that the RAS is part of the signaling cascade of COX-2–governed kidney development. We focused on the role of RAS during renal cortical development and for the first time provide evidence that administration of a selective AT1 receptor agonist is able to rescue nephrogenic defects and to restore renal function in COX-2-/- mice. Plasma renin concentration (PRC) in the postnatal time phase showed high levels around day P2 going down to low adult levels at P21. A second transient peak was observed around the days P6 to P8 (Figure 1a), which reached significance on P8. Administration of selective COX-2 inhibitor SC-236 significantly suppressed renin release, as shown for P8, P10, and P21, however, was without effect on day P2. In P8, but not in P2 COX-2-/- mice we also observed reduced renin concentrations compared with vehicle-treated control mice (Figure 1a). Quantitative polymerase chain reaction (qPCR) analysis demonstrated a similar time course with a rise in renal renin mRNA expression on days P6 and P8 that was not present in COX-2-/- mice, as shown for P8 (Figure 1b). Administration of telmisartan (0.5 mg/kg/d) to wild-type mice from P1 to P10 caused a decrease in the kidney–body weight ratio (Table 1). Hematocrit, plasma K+, and Ca2+ concentrations were indifferent between wild-type, COX-2-/-, and telmisartan-treated mice. Plasma Na+ and Cl- concentration was elevated on administration of telmisartan.Table 1Kidney–body weight ratio, plasma electrolytes, and hematocrit in wild-type COX-2-/- and telmisartan-treated miceParameterWild typeCOX-2-/-Telmisartan0.5 mg/kgBody weight (g)9.34 ± 0.429.54 ± 0.289.24 ± 0.71Kidney weight (g)0.132 ± 0.0050.090 ± 0.004aP < 0.05 compared with wild-type mice.0.103 ± 0.012aP < 0.05 compared with wild-type mice.Ratio kidney/body weight (× 1000)14.19 ± 0.369.43 ± 0.14aP < 0.05 compared with wild-type mice.11.00 ± 0.58aP < 0.05 compared with wild-type mice.Na+ concentration (mM)134.2 ± 1.1132.8 ± 0.6149.0 ± 0.6aP < 0.05 compared with wild-type mice.,bP < 0.05 compared with COX-2-/- mice.Cl- concentration (mM)103.8 ± 0.798.5 ± 1.5131.0 ± 4.5aP < 0.05 compared with wild-type mice.,bP < 0.05 compared with COX-2-/- mice.K+ concentration (mM)6.74 ± 0.586.75 ± 0.698.90 ± 0.49Ca2+ concentration (mM)1.26 ± 0.061.33 ± 0.101.56 ± 0.08Hematocrit (%)18.20 ± 0.9723.75 ± 0.8522.33 ± 3.53COX, cyclooxygenase; M, mol/l; P1, postnatal day 1.Telmisartan (0.5 mg/kg/d) was administrated for the time course day P1 to P8 and P10, respectively. Vehicle-treated wild-type C57BL6 mice and untreated COX-2-/- mice served as control animals. Plasma samples were analyzed on day P8, ratios of kidney–body weight were determined on day P21. Data show means ± SEMs; n = 5 mice for plasma analysis and n = 8 to 10 mice for kidney–body weight ratio.a P < 0.05 compared with wild-type mice.b P < 0.05 compared with COX-2-/- mice. Open table in a new tab COX, cyclooxygenase; M, mol/l; P1, postnatal day 1. Telmisartan (0.5 mg/kg/d) was administrated for the time course day P1 to P8 and P10, respectively. Vehicle-treated wild-type C57BL6 mice and untreated COX-2-/- mice served as control animals. Plasma samples were analyzed on day P8, ratios of kidney–body weight were determined on day P21. Data show means ± SEMs; n = 5 mice for plasma analysis and n = 8 to 10 mice for kidney–body weight ratio. To evaluate renal cortical defects, we focused on size of glomeruli, cortical thickness, which gives an estimation of the subcapsular cortical growth, and the amount of superficial glomeruli to give a measure for maturational arrest of newly formed nephrons in the outer cortex. Morphology of kidney tissue on P21 demonstrated that postnatal inhibition of the AT1 receptor by telmisartan from P1 to P10 caused defects in cortical kidney development quite similar to that observed in COX-2-/- mice (exemplarily shown in Figure 2a). Histomorphologic analysis revealed concentration-dependent renal defects by telmisartan with strongest effects at a dose of 0.5 mg/kg/d resulting in reduction of the glomerular diameter by about 30% (Figure 2b), thinning of the subcapsular cortex by about 50% (Figure 2c), and an increase in the relative amount of superficial glomeruli by about 230% (Figure 2d). The glomerular defects were indifferent to defects observed in COX-2-/- mice, whereas cortical growth and maturation of superficial glomeruli were less impaired than COX-2-/- mice were. Telmisartan treatment results in a steep dose-response curve. At a dose of 0.4 mg/kg/d, only glomerulus size was significantly affected, and at 0.25 mg/kg/d, none of the morphologic parameters were affected. In dosages of 1 mg/kg/d and higher, postnatal administration of telmisartan was lethal (data not shown). Juxtamedullary glomeruli known to be developed prenatally, were also affected by postnatal telmisartan treatment resembling observations in COX-2-/- (Figure 2e). Inhibition of the RAS at different steps by postnatal administration of aliskiren as well as enalapril also caused a significant reduction in glomerular size in wild-type mice (Table 2).Table 2Effect of inhibition of various components of RAS on size of glomeruliTreatmentDiameter of glomeruli (μm)Vehicle46.73 ± 0.51Aliskiren (50 mg/kg/d)40.85 ± 0.29aP < 0.05 compared with vehicle.Enalapril (0.1 mg/kg/d)39.45 ± 0.25aP < 0.05 compared with vehicle.Telmisartan (0.5 mg/kg/d)33.47 ± 1.00aP < 0.05 compared with vehicle.P1, postnatal day 1; RAS, renin-angiotensin system.Wild-type mice C57BL6 were treated with the indicated substances from days P1 to P10. Kidneys were removed and processed for histomorphometric analysis. Data show means ± SEMs; n = 6 mice.a P < 0.05 compared with vehicle. Open table in a new tab P1, postnatal day 1; RAS, renin-angiotensin system. Wild-type mice C57BL6 were treated with the indicated substances from days P1 to P10. Kidneys were removed and processed for histomorphometric analysis. Data show means ± SEMs; n = 6 mice. Whereas telmisartan administration from postnatal day P3 to P8 and P3 to P10 reduced glomerulus size as to be indistinguishable from COX-2-/- mice, and also caused significant thinning of the subcapsular cortical tissue, exposure time from P1 to P3 neither affected glomerular size nor cortical thickness (Figure 3a–c). In line with these observations, exposure to telmisartan from P1 to P10 caused similar defects as starting telmisartan administration on P3, albeit the effect on arrest of superficial glomeruli was slightly stronger but was not significantly different to the P3 to P8 treatment scheme (Figure 3a–c). Therefore, in following experiments telmisartan was administrated from P3 to P8. To study whether telmisartan may have affected COX-2 or mPGES-1 activity and thereby caused impaired kidney development we analyzed their mRNA expression on postnatal day P6 and PGE2 excretion on postnatal day P8. As expected under treatment with vehicle, a rise in COX-2 and in mPGES-1 mRNA expression and also in urinary PGE2 excretion was observed with age (Figure 4a–c). Administration of telmisartan did not suppress these changes, on the contrary it amplified COX-2 and mPGES-1 expression as well as PGE2 excretion (Figure 4a–c). Moreover, renal tissue concentration of PGE2 was significantly higher following treatment with telmisartan (Figure 4d). Immunologic detection of COX-2 protein revealed increased staining signals associated with epithelial cells of cortical thick ascending limb of Henle loop including cells of macula densa following telmisartan treatment (Figure 4e). Only faint signals that were unaltered in the telmisartan group were observed in kidney medulla (data not shown). In the next set of experiments we questioned whether the dependency on Ang II during renal development is proprietary to AT1 receptors or also involves angiotensin receptor type AT2. In contrast to telmisartan, application of 10 mg/kg/d PD123319, a selective antagonist for the AT2 receptor, was without any effect on glomerulus size (Figure 5a), thickness of cortex corticis (Figure 5b) or amount of superficial glomeruli (Figure 5c). Determination of Ang II plasma concentration revealed a significantly lower concentration in COX-2-/- mice on day P8 compared with wild-type mice (23.40 ± 6.72 pg/ml vs. 40.22 ± 3.92 pg/ml, n = 9, P = 0.04). The following experiments were designed to clarify whether renal defects in COX-2-/- mice were reversible. For this purpose, AT1 selective agonist L16231324Wan Y. Wallinder C. Johansson B. et al.First reported nonpeptide AT1 receptor agonist (L-162,313) acts as an AT2 receptor agonist in vivo.J Med Chem. 2004; 47: 1536-1546Crossref PubMed Scopus (44) Google Scholar was administrated to COX-2-/- mice from postnatal day P1 to P10. Compared with vehicle-treated COX-2-/- mice, L162313 agonist caused a recovery of glomerulogenesis demonstrated by a significant increase in glomerular size that was indistinguishable from that of wild-type mice (Figure 6a and b). Also the pathologic accumulation of superficial glomeruli decreased significantly, albeit not to control level (Figure 6c). Moreover, size of juxtamedullary glomeruli completely recovered (Figure 6d). However, no significant effect of L162313 was seen regarding thickness of subcapsular cortex (Figure 6e). The amelioration of kidney developmental defects in COX-2-/- mice was also reflected by an increase in kidney–body weight ratio following administration of L162313 (body weight: 8.26 ± 0.415 g; kidney weight: 0.091 ± 0.005 g) (Figure 7a). In COX-2-/- mice renal defects are associated with kidney insufficiency at older age, demonstrated by reduced glomerular filtration rate and thereby elevated levels of serum creatinine and serum urea.4Norwood V.F. Morham S.G. Smithies O. Postnatal development and progression of renal dysplasia in cyclooxygenase-2 null mice.Kidney Int. 2000; 58: 2291-2300Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 25Ahmetaj-Shala B. Kirkby N.S. Knowles R. et al.Evidence that links loss of cyclooxygenase-2 with increased asymmetric dimethylarginine: novel explanation of cardiovascular side effects associated with anti-inflammatory drugs.Circulation. 2015; 131: 633-642Crossref PubMed Scopus (62) Google Scholar Therefore, we questioned whether AT1 agonist L162313 may also rescue kidney function in aged COX-2-/- mice treated from day P1 to P10 with L162313. Compared with vehicle-treated COX-2-/- mice, a significant fall in urea (Figure 7b) as well as in serum creatinine levels (Figure 7c) was measured on P90, indicating an improvement of kidney function. Moreover, pathologically enhanced albumin excretion observed in COX-2-/- mice was normalized by L162313 (Figure 7d). A further pathologic characteristic of renal abnormalities in COX-2-/- mice is glomerulosclerosis.11Frolich S. Olliges A. Kern N. et al.Temporal expression of the PGE2 synthetic system in the kidney is associated with the time frame of renal developmental vulnerability to cyclooxygenase-2 inhibition.Am J Physiol Renal Physiol. 2012; 303: F209-F219Crossref PubMed Scopus (21) Google Scholar Treatment of COX-2-/- pups with L162313 significantly reduced periglomerular and intraglomerular staining with Sirius red (Figure 7e–g). The antifibrotic effect of L162313 was further substantiated by reduced pathologically enhanced expression of α-smooth muscle actin in COX-2-/- kidneys on the protein and mRNA levels (Figure 7h and i). Our findings provide clear evidence that components of RAS, in particular Ang II-AT1 receptor interaction, are a crucial part of the COX-2–dependent signaling pathway leading to normal kidney development. Using this knowledge, we were able to partially rescue the developmental renal defects in neonatal COX-2-/- pups by administration of the AT1 receptor agonist L162313 and to improve long-term renal filtration. In support of a developmental role of Ang II after birth, PRC rose transiently around postnatal days P6 to P8, most likely due to the elevated renin mRNA levels observed. High PRC values in contrast to mRNA expression around P2 may be a result of the typical birth peak also seen in rats. An explanation may be given by the release of renin during delivery and also as a postnatal adaptation to establish and maintain effective circulating volume despite low Na intake. These findings confirm previous studies on the postnatal time course of renin.14Stubbe J. Jensen B.L. Bachmann S. et al.Cyclooxygenase-2 contributes to elevated renin in the early postnatal period in rats.Am J Physiol Regul Integr Comp Physiol. 2003; 284: R1179-R1189Crossref PubMed Scopus (35) Google Scholar, 26Machura K. Steppan D. Neubauer B. et al.Developmental renin expression in mice with a defective renin-angiotensin system.Am J Physiol Renal Physiol. 2009; 297: F1371-F1380Crossref PubMed Scopus (20) Google Scholar The increase in renin expression and plasma renin occurred simultaneously with a markedly enhanced COX-2 and mPGES-1 expression, congruent to previous results.11Frolich S. Olliges A. Kern N. et al.Temporal expression of the PGE2 synthetic system in the kidney is associated with the time frame of renal developmental vulnerability to cyclooxygenase-2 inhibition.Am J Physiol Renal Physiol. 2012; 303: F209-F219Crossref PubMed Scopus (21) Google Scholar This observation is in agreement with the known role of COX-2–derived prostanoids, most likely PGE2, to stimulate renin secretion via PGE2 receptor types EP2 and EP4 in adult mice.15Schweda F. Klar J. Narumiya S. et al.Stimulation of renin release by prostaglandin E2 is mediated by EP2 and EP4 receptors in mouse kidneys.Am J Physiol Renal Physiol. 2004; 287: F427-F433Crossref PubMed Scopus (88) Google Scholar, 27Friis U.G. Stubbe J. Uhrenholt T.R. et al.Prostaglandin E2 EP2 and EP4 receptor activation mediates cAMP-dependent hyperpolarization and exocytosis of renin in juxtaglomerular cells.Am J Physiol Renal Physiol. 2005; 289: F989-F997Crossref PubMed Scopus (53) Google Scholar In conformity with this, Cox-2 gene knockout or suppression of COX-2 enzymatic activity by selective inhibitor SC-236 flattens the transient renin peak. Unaltered PRC peak by COX-2 inhibition or COX-2 depletion on day P2 indicates that COX-2 most likely is not involved in the very early abundance of renin at delivery. Large numbers of COX-2 expressing cells observed in cortical thick ascending limb of Henle loop epithelial cells and the perimacula densa region of developing nephrons28Zhang M.Z. Wang J.L. Cheng H.F. et al.Cyclooxygenase-2 in rat nephron development.Am J Physiol. 1997; 273: F994-F1002PubMed Google Scholar might be the probable explanation for higher levels of prostanoids, such as PGE2, necessary to drive development and maturation of the nephrons. Disruption of the RAS at any step caused impairment of nephrogenesis associated with reduced glomerular size, thinning of the renal cortical tissue, and accumulation of mostly premature superficial glomeruli in the subcapsular zone. Compared with inhibition of AT1, the effect of renin and ACE inhibition on glomerular size was less profound (also at higher dosages [data not shown]). This indicates that under these experimental conditions, suppression of Ang II–AT1 signaling in mice pups is not fully achieved. Also for man, it has been shown that the most complete blockade of the RAS can be achieved by pharmacologic interruption at the level of the angiotensin receptor.29Price D.A. De'Oliveira J.M. Fisher N.D. Hollenberg N.K. Renal hemodynamic response to an angiotensin II antagonist, eprosartan, in healthy men.Hypertension. 1997; 30: 240-246Crossref PubMed Scopus (56) Google Scholar Of note, RAS inhibition is beneficial in obesity-related glomerulopathy, featuring proteinuria, glomerulomegaly, and progressive glomerulosclerosis.30D'Agati V.D. Chagnac A. de Vries A.P. et al.Obesity-related glomerulopathy: clinical and pathologic characteristics and pathogenesis.Nat Rev Nephrol. 2016; 12: 453-471Crossref PubMed Scopus (343) Google Scholar It remains uncertain whether renin, ACE, or inhibition at the step of AT1 is clinically more useful. Regarding the strong effects of telmisartan in our study, we assume that in obesity-related glomerulopathy, AT1 antagonism might be most promising. Size of juxtamedullary glomeruli known to be developed early in prenatal nephrogenesis, was also affected by telmisartan. Therefore, we assume that not only the development of newly formed glomeruli in the subcapsular nephrogenic zone is impaired by inhibition of the AT1 receptor, but also maturation of already formed glomeruli. Furthermore, postnatal antagonism of AT1 before day
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