Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.

Functionally significant polymorphisms in endothelial nitric oxide synthase (eNOS) and reduced vascular eNOS activity have been associated with increased human diabetic nephropathy (DN), but the pathogenic role of eNOS deficiency in the development of DN has not yet been confirmed. This study characterizes the severity of DN in eNOS ؊/؊ mice that were backcrossed to C57BLKS/J db/db mice. Although the severity of hyperglycemia was similar to C57BLKS/J db/db mice, by 26 wk, eNOS ؊/؊ C57BLKS/J db/db mice exhibited dramatic albuminuria, arteriolar hyalinosis, increased glomerular basement membrane thickness, mesangial expansion, mesangiolysis, and focal segmental and early nodular glomerulosclerosis. Even more remarkable, eNOS ؊/؊ C57BLKS db/db exhibited decreases in GFR to levels <50% of that in eNOS ؉/؉ C57BLKS db/db, as confirmed by increased serum creatinine. In summary, eNOS ؊/؊ db/db mice provide the most robust model of type II DN that has been described to date and support a role for deficient eNOS-derived NO production in the pathogenesis of DN.

We have previously shown that in rat renal cortex, cyclooxygenase-2 (COX-2) expression is localized to cTALH cells in the region of the macula densa, and that dietary salt restriction increases COX-2 expression. Administration of the angiotensin converting inhibitor, captopril, further increased COX-2 mRNA and renal cortical COX-2 immunoreactivity, with the most pronounced expression in the macula densa. Administration of an AT1 receptor antagonist, losartan, also significantly increased cortical COX-2 mRNA expression and COX-2 immunoreactivity. Mutant mice homozygous for both Agtr1a and Agtr1b null mutations (Agtr1a -/-,Agtr1b -/-) demonstrated large increases in immunoreactive COX-2 expression inthe cTALH/macula densa. To determine whether increased COX-2expression in response to ACE inhibition mediated increases in renin production, rats were treated with captopril for one week with or without the specific COX-2 inhibitor, SC58236. Plasma renin activity increased significantly in the captropril group, and this increase was significantly inhibited by simultaneous treatment with SC58236. Thus, these studies indicated that angiotensin II inhibitors augment upregulation of renal cortical COX-2 in states of volume depletion, suggesting that negative feedback by the renin-angiotensin system modulates renal cortical COX-2 expression and that COX-2 is a mediator of increased renin production in response to inhibition of angiotension II production.

These results suggest that in an experimental model of diabetes and hypertension, inhibition of COX-2 expression decreases potential mediators of glomerular and tubulointerstitial injury and also decreases biochemical, functional and structural markers of renal injury.

Abstract-Selective cyclooxygenase (COX)-2 inhibitors that are in widespread clinical use were developed to avoid side effects of conventional NSAIDs, including gastrointestinal and renal toxicity. However, COX-2 is constitutively expressed in the kidney and is highly regulated in response to alterations in intravascular volume. COX-2 metabolites have been implicated in maintenance of renal blood flow, mediation of renin release, and regulation of sodium excretion. COX-2 inhibition may transiently decrease urine sodium excretion in some subjects and induce mild to moderate elevation of blood pressure. Furthermore, in conditions of relative intravascular volume depletion and/or renal hypoperfusion, interference with COX-2 activity can have deleterious effects on maintenance of renal blood flow and glomerular filtration rate. In addition to physiological regulation of COX-2 expression in the kidney, increased renal cortical COX-2 expression is seen in experimental models associated with altered renal hemodynamics and progressive renal injury (decreased renal mass, poorly controlled diabetes), and long-term treatment with selective COX-2 inhibitors ameliorates functional and structural renal damage in these conditions. Key Words: COX-2 Ⅲ COX-1 Ⅲ hypertension Ⅲ renin Ⅲ sodium Ⅲ glomerular filtration rate Ⅲ kidney I n the kidney, prostaglandins are important mediators of vascular tone, salt and water balance, and renin release. The rate-limiting enzyme, cyclooxygenase (prostaglandin synthase G 2 /H 2 ) initiates the metabolism of arachidonic acid to prostaglandin (PG) G 2 and subsequently to PGH 2 , which is then further metabolized by tissue-specific isomerases to PGs and thromboxane. There are at least two distinct cyclooxygenases, COX-1 and COX-2, that share Ϸ60% homology 1 but are the products of different genes and have distinct patterns of expression and regulation. COX-1 has been termed "constitutive," because of its wide tissue distribution, while COX-2 has been designated as "inducible" because of its more restricted basal expression and its upregulation by inflammatory and/or proliferative stimuli and its central role in mediation of inflammatory conditions and malignancies. Recently, Chandrasekharan et al characterized a third potential cyclooxygenase isoform, COX-3, a 65-kDa membranebound protein with acetaminophen or paracetamol-sensitive cyclooxygenase activity that represents a splice variant of COX-1. 2 However, other investigators have not confirmed the existence of full-length, catalytically active COX-3 from human genomic clones. Therefore, it remains uncertain whether COX-3 in fact represents the elusive target of paracetamol.Inhibition of COX activity by NSAIDs has been widely used for the treatment of pain and inflammation. Because prostanoids are involved in renal function, nonselective NSAIDs exhibit adverse effects, including salt retention and decreases in glomerular filtration rate (GFR), which may elevate blood pressure (BP) or make pre-existing hypertension worse. 3 Based on the hypothesis...

The inducible second isoform of cyclooxygenase (COX-2) that mediates inflammation also is expressed at low levels in normal adult rat kidneys and is upregulated in response to noninflammatory stimuli (R. C. Harris, J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. DuBois, and M. D. Breyer. J. Clin. Invest. 94: 2504–2510, 1994). Roles in morphogenesis are indicated by reported teratogenicity of COX inhibitors and renal dysgenesis in COX-2 knockout mice (J. E. Dinchuk, B. D. Car, R. J. Focht, J. J. Johnston, B. D. Jaffee, M. B. Covington, N. R. Contel, V. M. Eng, R. J. Collins, P. M. Czerniak, A. G. Stewart, and J. M. Trzaskos. Nature 378: 406–409, 1995; S. G. Morham, R. Lagenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C. Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee, and O. Smithies. Cell 83: 473–482, 1995). Blots from developing rat kidneys demonstrated that COX-2 mRNA and immunoreactive protein were present in neonates, peaked in the 2nd and 3rd postnatal weeks and declined to adult levels by the 3rd month. Immunolocalization and in situ hybridization detected intense COX-2 immunoreactivity and mRNA in a subset of thick ascending limb epithelial cells near the macula densa in each developing nephron; after 2 wk the COX-2 gradually waned. These data demonstrate that COX-2 expression is subject to normal developmental regulation and can be sustained over extended periods; they also support the conclusion that metabolites of COX-2 play important roles in the differentiation and early functions of mammalian nephrons.

Abstract-It has been proposed that the macula densa participates in the regulation of increased renin expression in renovascular hypertension (RVH) and that prostaglandins may be among the mediators of macula densa function. We have previously shown that in renal cortex, cyclooxygenase-2 (COX-2) expression is localized to the macula densa and surrounding cortical thick ascending limb and increases in high-renin states, such as salt restriction and angiotensinconverting enzyme inhibition. In the present studies, we examined the effect of the selective COX-2 inhibitor SC58236 on plasma renin activity (PRA) and renal renin expression in RVH in rats. The aorta was coarcted between right and left renal arteries, and animals received either SC58236 or vehicle for 1 week. At day 8, vehicle-treated coarcted rats were hypertensive (mean carotid arterial blood pressure: 138Ϯ3 versus 87Ϯ2 mm Hg in sham-operated controls; nϭ9 to 11; PϽ0.001) and exhibited a disparity of kidney size (ratio left/right kidney: 0.78Ϯ0.04 versus 1.02Ϯ0.02; nϭ9 to 10; PϽ0.001). PRA increased significantly (84.6Ϯ6.5 versus 9.0Ϯ1.4 ng angiotensin I [Ang I] per milliliter per hour; nϭ8 to 9; PϽ0.01). In the coarcted rats, neither renin mRNA expression nor renin activity of the right kidney was altered (renin/GAPDH mRNA: 1.12Ϯ0.05-fold levels in control rats; nϭ6; PϭNS; renin activity: 23.4Ϯ1.8 versus 27.1Ϯ3.4 ng Ang I per hour per milligram protein; nϭ8 to 9; PϭNS). However, the renin mRNA of the left kidney increased to 3.0Ϯ0.6-fold of control (nϭ6), and the renin activity increased to 189.0Ϯ28.6 ng Ang I per hour per milligram protein (nϭ8; PϽ0.01). Expression of COX-2 mRNA and immunoreactive protein increased in the affected left kidney but was not different from control in the unaffected right kidney. SC58236 treatment to coarcted rats did not affect kidney size (ratio left/right kidney: 0.79Ϯ0.06; nϭ9). However, PRA was significantly decreased compared with the vehicle-treated coarcted rats (19.8Ϯ2.8 ng Ang I per milliliter per hour; nϭ9; PϽ0.01). The left kidney renin mRNA and renin content were also decreased (1.7Ϯ0.3-fold control; nϭ6; PϽ0.05; and 45.7Ϯ7.6 ng Ang I per hour per milligram protein; nϭ9; PϽ0.01, respectively), while renin mRNA and renin content of the right kidney were not altered. SC58236 lowered mean arterial blood pressure (122Ϯ5 mm Hg; nϭ14; PϽ0.05 compared with vehicle). A significant correlation was observed between PRA and mean blood pressure (rϭ0.75; PϽ0.01). In summary, these studies indicate that the selective COX-2 inhibitor SC58236 decreases renin production and release in RVH and suggest an important role for COX-2 regulation of the renin-angiotensin system. (Hypertension. 1999;34:96-101.)Key Words: renin Ⅲ cyclooxygenase Ⅲ hypertension, renal Ⅲ macula densa Ⅲ kidney B oth experimental models and clinical experience have indicated that prostaglandins are involved in the regulation of renal renin expression and release. [1][2][3][4] In addition to physiological regulation of renin in response to alterations in intr...

Cyclooxygenase-2-selective inhibitors impair glomerulogenglandins in renal development. These renal abnormaliesis and renal cortical development.ties range from oliguria to renal dysgenesis and have Background. Antenatal exposure to nonsteroidal anti-inflambeen seen particularly when NSAIDs were administered matory drugs (NSAIDs) has been associated with renal dysgenbefore the 32nd week of gestation [1, 2]. Similar findings esis in humans.were observed in offspring of rhesus monkeys treated Methods. These studies characterized cyclooxygenase-2 (COX-2) versus COX-1-selective inhibition on nephrogenesis with indomethacin during gestation [3]. Avner and coin the rodent using histomorphometry, immunohistology, and workers showed that prostaglandin E 1 (PGE 1 ) is necesin situ hybridization. sary for maximal growth and differentiation of meta-Results. Administration of a COX-2-selective inhibitor nephroi in culture [4]. Taken together, these data suggest (SC58236), started during pregnancy until weaning, signifian important role for cyclooxygenase (COX)-mediated cantly impaired development of the renal cortex and reduced glomerular diameter in both mice and rats. An identical phenoprostaglandin formation in renal development.type was demonstrated in COX-2 Ϫ/Ϫ mice. In contrast to its Two isoforms of COX are known to exist. COX-1 is effects on the developing kidney, a COX-2 inhibitor had no expressed constitutively and considered a housekeeping effect on glomerular volume in adult mice. This effect was gene, having important roles in the maintenance of epispecific for COX-2 because maternal administration of a COXthelial integrity. COX-2 was identified as an immediate 1-selective inhibitor (SC58560) did not affect renal development despite significantly inhibiting gastric mucosal prostaglan-early-response gene, up-regulated in fibroblasts treated din E 2 (PGE 2 ) synthesis in pups. The expression of COX-2 with tumor-promoting phorbol esters [5]. COX-2 is not immunoreactivity peaked in the first postnatal week and was constitutively expressed in many tissues and is only oblocalized to S-shaped bodies and the macula densa in the cortex. served after they are exposed to cytokines and growth Treatment with a COX-2 inhibitor during this period (from factors. However, COX-2 is expressed constitutively in postnatal day 0 to day 21) severely reduced glomerular diamefetal and adult kidney in all species examined [6][7][8][9]. Gene ter, whereas treatment limited to pregnancy did not affect glomerular size.knockout studies show that COX-1 disruption does not Conclusion. These data demonstrate an important role for interfere with normal renal development [10]. In con-COX-2 activity in nephrogenesis in the rodent, and define a trast, COX-2-deficient mice exhibit renal dysgenesis asspecific time period of susceptibility to these effects. sociated with hypoplastic glomeruli [11, 12]. These structural renal abnormalities in COX-2 null mice have not been quantitatively defined. Furthermore, the effect of Reports of renal dysgenesis in ...