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R C Harris

Publications and source records attributed to R C Harris.

At least 19 recordsLinked to original sources
PubMed1999

Transfection of an active cytochrome P450 arachidonic acid epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor.

A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regio- and enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regio- and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic acid (EET) production (241. 82 ng/10(8) cells, >97% of total EETs), whereas no detectable EETs were seen in cells transfected with vector alone. In F87V BM-3 cells, AA stimulated [3H]thymidine incorporation and increased cell proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002, and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3 cells, which was completely abolished by pretreatment with either 17-ODYA or the phospholipase A2 (PLA2) inhibitor, quinacrine. Compared with vector-transfected cells, F87 BM-3 transfected cells demonstrated marked increases in both the extent and sensitivity of DNA synthesis in response to EGF. These changes occurred in the absence of significant differences in EGF receptor expression. As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a significantly greater extent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in these control cells, neither 17-ODYA nor quinacrine inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V BM-3 cells, both inhibitors reduced ERK tyrosine phosphorylation to levels indistinguishable from that seen in cells transfected with vector alone. These studies provide the first unequivocal evidence for a role for the AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated signaling and mitogenesis and provide compelling evidence for the PLA2-AA-EET pathway as an important intracellular-signaling pathway in cells expressing high levels of cytochrome P450 epoxygenase.

8,11,14-Eicosatrienoic Acid↗
PubMed1999

Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase.

BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.

Catalysis↗
PubMed1999

Coexpression of CD9 augments the ability of membrane-bound heparin-binding epidermal growth factor-like growth factor (proHB-EGF) to preserve renal epithelial cell viability.

BACKGROUND: Transfection of renal epithelial cells (NRK 52E) with membrane-associated heparin-binding epidermal growth factor-like growth factor (proHB-EGF) increased renal epithelial cell survival by promoting cell-cell and cell-extracellular matrix interactions. ProHB-EGF has been shown to form a complex in the plasma membrane with the tetraspanin CD9, an interaction that significantly increases the effectiveness of proHB-EGF as a juxtacrine mitogenic agent. METHODS: We examined whether the coexpression of proHB-EGF and CD9 would increase renal epithelial cell survival. CD9 was stably transfected into NRK 52E cells, either alone (NRKCD9) or together with proHB-EGF (NRKboth). RESULTS: Juxtacrine mitogenic activity of NRKCD9 was no different than in cells transfected with vector alone (NRKvector), but was increased by NRKboth; juxtacrine mitogenic activity by NRKboth was twofold greater than when proHB-EGF was transfected alone (NRKproHB-EGF). When grown in 10% fetal calf serum, growth rates were similar among all transfectants. However, in 1% fetal calf serum, NRKproHB-EGF grew 50% faster than NRKvector or NRKCD9, and NRKboth grew 20% to 50% faster than NRKproHB-EGF at one, two, and three days of culture. NRKproHB-EGF attachment to plastic substratum at one, two, and three hours was 250% greater than that of NRKvector, and NRKboth was 20% to 30% greater than that of NRKproHB-EGF. Coating plates with either poly 2-hydroxyethyl methacrylate or the GRGDTP peptide prevented normal cell-extracellular matrix attachment, and NRKvector or NRKCD9 failed to attach or form cell-cell attachments. NRKproHB-EGF exhibited 300% and NRKboth exhibited 600% greater cell viability under these conditions. Expression of type I and type III collagen mRNA was enhanced similarly in NRKproHB-EGF and NRKboth, but the expression of beta1 integrin was up-regulated only in NRKboth. CONCLUSIONS: Coexpression of proHB-EGF and CD9 may render the renal epithelial cells more resistant to disruption of cell-cell and cell-matrix interactions and could accelerate the re-establishment of these attachments.

Animals↗
PubMed1999

Physiological responses of horses to a treadmill simulated speed and endurance test in high heat and humidity before and after humid heat acclimation.

To investigate whether horses were able to acclimate to conditions of high temperature and humidity, 5 horses of different breeds were trained for 80 min on 15 consecutive days on a treadmill at 30 degrees C and 80%RH. Training consisted of a combination of long duration low-intensity exercise, medium duration medium intensity exercise and short duration high intensity exercise. Between training sessions the horses were maintained at 11+/-3 degrees C and 74+/-2%RH. Before (PRE-ACC) and after acclimation (POST-ACC) the horses undertook a simulated Competition Exercise Test (CET), designed to represent the Speed and Endurance Test of a 3-day event, at 30 degrees C/80%RH. Maximal oxygen uptake (VO2PEAK) was not changed following acclimation (PRE-ACC 141+/-8 ml/min/kg bwt vs. POST-ACC 145+/-9 ml/min/kg bwt [STPD], P>0.05). Following acclimation, 4 of the 5 horses were able to complete a significantly greater amount of Phase D in the CET (PRE-ACC 6.3+/-0.3 min vs. POST-ACC 7.3+/-0.3 min, P<0.05; target time = 8 min). Resting body temperatures (pulmonary artery [TPA], rectal [TREC] and tail-skin [TTSK] temperatures) were all significantly lower following acclimation. During exercise, metabolic heat production (M) and heat dissipation (HD), for the same exercise duration, were both significantly lower following acclimation (P<0.05), although heat storage (HS) was significantly higher (P<0.05). The higher heat storage following acclimation was associated with a lower TTSK for a given TPA and a decreased total fluid loss (% bodyweight, P<0.05). Plasma volume was not changed following acclimation. The relationship of sweating rate (SR) to TPA or TTSK on either the neck or the gluteal region was not significantly altered by acclimation, although the onset of sweating occurred at a lower TPA or TTSK following acclimation (P<0.05). The horses in the present study showed a number of physiological adaptations to a period of 15 days of exposure to high heat and humidity consistent with a humid heat acclimation response. These changes were mostly similar to those reported to occur in man and other species and were consistent with thermal acclimation and an increased thermotolerance, leading to an improved exercise tolerance. It is concluded that a 15 day period of acclimation is beneficial for horses from cooler and or drier climates, that have to compete in hot humid conditions and that this may redress, to some extent, the decrement in exercise tolerance seen in nonacclimated horses and reduce the risk of heat related disorders, such as heat exhaustion.

Acid-Base Equilibrium↗
PubMed1999

Cyclooxygenase-2 inhibition decreases renin content and lowers blood pressure in a model of renovascular hypertension.

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 angiotensin-converting 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: 138+/-3 versus 87+/-2 mm Hg in sham-operated controls; n=9 to 11; P<0.001) and exhibited a disparity of kidney size (ratio left/right kidney: 0.78+/-0.04 versus 1.02+/-0.02; n=9 to 10; P<0.001). PRA increased significantly (84.6+/-6.5 versus 9.0+/-1.4 ng angiotensin I [Ang I] per milliliter per hour; n=8 to 9; P<0.01). In the coarcted rats, neither renin mRNA expression nor renin activity of the right kidney was altered (renin/GAPDH mRNA: 1.12+/-0.05-fold levels in control rats; n=6; P=NS; renin activity: 23.4+/-1.8 versus 27.1+/-3.4 ng Ang I per hour per milligram protein; n=8 to 9; P=NS). However, the renin mRNA of the left kidney increased to 3.0+/-0.6-fold of control (n=6), and the renin activity increased to 189.0+/-28.6 ng Ang I per hour per milligram protein (n=8; P<0.01). 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: 0.79+/-0.06; n=9). However, PRA was significantly decreased compared with the vehicle-treated coarcted rats (19.8+/-2. 8 ng Ang I per milliliter per hour; n=9; P<0.01). The left kidney renin mRNA and renin content were also decreased (1.7+/-0.3-fold control; n=6; P<0.05; and 45.7+/-7.6 ng Ang I per hour per milligram protein; n=9; P<0.01, respectively), while renin mRNA and renin content of the right kidney were not altered. SC58236 lowered mean arterial blood pressure (122+/-5 mm Hg; n=14; P<0.05 compared with vehicle). A significant correlation was observed between PRA and mean blood pressure (r=0.75; P<0.01). 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.

Animals↗
PubMed1999

Angiotensin II attenuates renal cortical cyclooxygenase-2 expression.

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.

Angiotensin II↗
PubMed1999

Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization.

Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth factor receptor internalization. The type 1 angiotensin II receptor (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT1R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 microM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 microM] or tyrphostin B-48 (TB-48) [50 microM], were assayed for acid-resistant specific 125I-Ang II binding, a measure of Ang II internalization. Both Gen (n = 7) and TB-48 (n = 3) inhibited AP 125I-Ang II internalization (80+/-7% inhibition; p<0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP 125I-Ang II endocytosis (n = 3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n = 5) or herbimycin A [150 ng/ml] (n = 4) also inhibited MSC 125I-Ang II internalization (82+/-11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in MSC and selectively decreased AP Ang II internalization in LLCPKAT1R cells suggesting that AP AT1R in LLCPKAT1R cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.

Angiotensin II↗
PubMed1998

Epoxyeicosatrienoic acids and their sulfonimide derivatives stimulate tyrosine phosphorylation and induce mitogenesis in renal epithelial cells.

In our present studies utilizing a well characterized proximal tubule cell line, LLCPKcl4, we determined that all four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) stimulated [3H]thymidine incorporation, with 14,15-EET being the most potent. In contrast, no mitogenic effects were seen with arachidonic acid, other cP450 arachidonate metabolites (12R-hydroxyeicosatetraenoic acid (12R-HETE), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), or 20-HETE), or lipoxygenase metabolites (5S-HETE, leukotriene B4, or lipoxin A4). We found that their metabolically more stable sulfonimide (SI) analogs (11,12-EET-SI and 14,15-EET-SI) were also potent mitogens. In addition 14,15-EET-SI also increased cell proliferation as well as expression of both c-fos and egr-1 mRNA. The protein kinase C and A inhibitors, H-7 and H-8, or the cyclooxygenase inhibitor, indomethacin, had no effect upon 14, 15-EET-induced [3H]thymidine incorporation, but the selective tyrosine kinase inhibitor, genistein, significantly inhibited it. Immunoprecipitation and immunoblotting demonstrated increased tyrosine phosphorylation of PI3-kinase and epidermal growth factor receptor (EGFR) within 1 min of EET administration. EETs also stimulated association of PI3-kinase with EGFR. PI3-kinase inhibitors, wortmannin and LY 294002, markedly inhibited 14, 15-EET-SI-stimulated [3H]thymidine incorporation. In addition, 14, 15-EET-SI administration stimulated tyrosine phosphorylation of src homologous and collagen-like protein (SHC) and association of SHC with both growth factor receptor-binding protein (GRB2) and EGFR. Mitogen-activated protein kinase was also activated within 5 min. Pretreatment of the cells with the mitogen-activated protein kinase kinase inhibitor, PD98059, inhibited the 14,15-EET-SI-stimulated [3H]thymidine incorporation. Moreover, immunoblotting indicated that 14,15-EET stimulated tyrosine phosphorylation of the specific pp60(c-src) substrate p120 and c-Src association with EGFR. 14, 15-EET increased src kinase activity within 1 min. Our data indicate that EETs are potent mitogens for renal epithelial cells, and the mitogenic effects of the EETs are mediated, at least in part, by the activation of Src kinase and initiation of a tyrosine kinase phosphorylation cascade.

8,11,14-Eicosatrienoic Acid↗
PubMed1998

Angiotensin induces the urinary peristaltic machinery during the perinatal period.

The embryonic development of mammalian kidneys is completed during the perinatal period with a dramatic increase in urine production, as the burden of eliminating nitrogenous metabolic waste shifts from the placenta to the kidney. This urine is normally removed by peristaltic contraction of the renal pelvis, a smooth muscle structure unique to placental mammals. Mutant mice completely lacking angiotensin type 1 receptor genes do not develop a renal pelvis, resulting in the buildup of urine and progressive kidney damage. In mutants the ureteral smooth muscle layer is hypoplastic and lacks peristaltic movements. We show that angiotensin can induce the ureteral smooth muscles in organ cultures of wild-type, but not mutant, ureteral tissues and that, in wild-type mice, expression of both renal angiotensin and the receptor are transiently upregulated at the renal outlet at birth. These results reveal a new role for angiotensin in the unique cellular adaptations of the mammalian kidney to the physiological stresses of postnatal life.

Angiotensin II↗
PubMed1998

Prostaglandins in macula densa function.

Cyclooxygenase (COX)-2 mRNA and immunoreactive protein localize to the macula densa and adjacent cortical thick ascending limb in renal cortex, and chronic NaCl restriction increases expression of this enzyme. These findings suggest an integral role for eicosanoids generated by macula densa-associated COX-2 in mediating renin release. As selective inhibitors of COX-2 become available, it will be important to assess their effects on the renin-angiotensin system and glomerular hemodynamics.

Animals↗
PubMed1998

Adenosine and hypoxanthine transport in horse erythrocytes: evidence for a polymorphism in the transport of hypoxanthine via a sodium-dependent cotransporter.

The inward transport of two purines, adenosine and hypoxanthine, at 37 degrees C by horse erythrocytes was compared. No mediated transport of adenosine was detected in horse erythrocytes, nor was saturable, high-affinity binding of the potent facilitated-diffusion inhibitor nitrobenzylthioinosine demonstrable in horse erythrocyte membranes. In contrast, erythrocytes from most horses possessed a saturable sodium-dependent hypoxanthine transporter (apparent K(m), 100 +/- 28 microM; Vmax, 0.20 +/- 0.08 mmol (l cells)-1 h-1; means +/- S.E.M., n = 5). Guanine inhibited hypoxanthine influx (apparent Ki, 24 +/- 6 microM), but adenine and xanthine had no effect. Unlike human erythrocytes, no sodium-independent hypoxanthine transporter was detected in horse erythrocytes. There are, however, a small number of animals (approximately 15%) whose erythrocytes fail to transport hypoxanthine. This variation appears to be under genetic control, but the precise nature of the control is unknown.

Adenosine↗
PubMed1998

Constitutive expression of cyclooxygenase-2 in rat vas deferens.

Prostaglandins, lipoid substances discovered in human semen as modulators of uterine muscle contractility, are known to play significant roles in virtually all mammalian organ systems, but their male reproductive functions are unclear. Cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis, occurs in two isoforms distinguished on the basis of constitutive (COX-1) or inducible (COX-2) expression patterns in mammalian tissues. However, in the adult rat male reproductive system, immunohistochemistry and Western and Northern analysis showed that COX-2 is the predominant isoform and is heavily localized to the epithelium of the distal vas deferens, where constitutive expression is manyfold greater than in any other organs of the body. COX-2 is not detected in the proximal one-half of the vas nor in the testis, epididymis, seminal vesicles, or prostate. Elimination of luminal sperm by vasectomy does not affect COX-2 levels, whereas castration severely depletes COX-2 and androgen replacement after castration restores COX-2, indicating that COX-2 expression in the vas is androgen dependent. Because the distal vas also comprises an extensive submucosal venous plexus connected to the penile corpora cavernosa, prostaglandins from the vas may play a role in erection.

Animals↗
PubMed1998

Young SHR express increased type 1 angiotensin II receptors in renal proximal tubule.

A potential role for the renin-angiotensin system (RAS) in the development and/or maintenance of hypertension in the genetic model of rat hypertension, spontaneously hypertensive rats (SHR), has been suggested by studies indicating that treatment of immature animals with angiotensin-converting enzyme (ACE) inhibitors prevents subsequent development of hypertension. Because young SHR also demonstrate RAS-dependent increased sodium retention, we examined proximal tubule type 1 angiotensin II receptor (AT1R) mRNA expression in young (4 wk) or adult (14 wk) SHR compared with age-matched Wistar-Kyoto (WKY) rats. Proximal tubules were isolated by Percoll gradient centrifugation, and AT1R mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). At 14 wk, when SHR had established hypertension [mean arterial blood pressure (MAP) of SHR vs. WKY: 145 +/- 6 vs. 85 +/- 5 mmHg, n = 14-15], there were no differences in proximal tubule AT1R mRNA levels [SHR vs. WKY: 79 +/- 14 vs. 72 +/- 14 counts/min (cpm) per cpm mutant AT1R per cpm beta-actin x 10(-6), n = 6; not significant (NS)]. In contrast, in 4 wk SHR, at a time of minimal elevations in blood pressure (MAP: 70 +/- 8 vs. 63 +/- 3), SHR proximal tubule AT1R mRNA levels were 263 +/- 30% that of WKY (143 +/- 18 vs. 60 +/- 11 cpm per cpm of mutant AT1R per cpm beta-actin x 10(-6), n = 8; P < 0.005). We have recently shown that chronic ACE inhibition decreases proximal tubule AT1R expression and have also shown that chronic L-3,4-dihydroxyphenylalamine (L-DOPA) administration inhibits AT1R expression in adult Sprague-Dawley proximal tubule and cultured proximal tubule, and this inhibition is mediated via Gs-coupled DA1 receptors. When 3-wk-old animals were given L-DOPA or captopril for 1 wk, MAP was not altered (70 +/- 8 vs. 60 +/- 4 or 61 +/- 5 mmHg), but proximal tubule AT1R mRNA was no longer significantly different between SHR and WKY (68 +/- 9 vs. 38 +/- 7 or 20 +/- 3 vs. 47 +/- 15 cpm per cpm of mutant AT1R per cpm beta-actin x 10(-6)), due to a significant decrease in proximal tubule AT1R expression in SHR (P < 0.005, compared with untreated SHR). Immunoreactive proximal tubule AT1R expression also was increased in 4 wk SHR and was reversed with captopril or L-DOPA treatment. Therefore, these results indicate that young, but not adult, SHR have increased expression of proximal tubule AT1R and that chronic L-DOPA or captopril treatment decreased the elevated AT1R expression to control levels. These results provide further support for an important role of the RAS in the development of hypertension in SHR.

Angiotensin II↗
PubMed1998

Selective increase of cyclooxygenase-2 expression in a model of renal ablation.

Previous studies have suggested a possible role for prostaglandins (PGs) in mediating alterations in nephron structure and function ensuing after renal ablation. Two isoforms of cyclooxygenase (COX) have been described: constitutive (COX-1) and inducible (COX-2). We examined expression of these isoforms following subtotal renal ablation (5/6 ablation, RA) in rats. In renal cortex, COX-2 mRNA and immunoreactive protein (IP) increased progressively compared with sham-operated littermates. In contrast, there were no significant changes in COX-1 mRNA expression. In normal kidney, cortical COX-1 IP was immunolocalized predominantly to mesangial cells and collecting tubules, whereas COX-2 IP was found in a subset of cortical thick ascending limb of Henle's loop (CTAL) cells in the region of the macula densa (MD). Following RA, significantly increased COX-2 IP was detected in the MD and surrounding CTAL cells. In addition, fainter immunoreactive COX-2 was detected in scattered visceral epithelial cells and mesangial cells of the glomerulus. Immunoblotting of isolated glomeruli demonstrated a selective increase of glomerular immunoreactive COX-2 expression following RA. No change of COX-1 expression was seen. To determine COX activity, isolated glomeruli were incubated with arachidonic acid and PGE2 measured by enzyme immunoassay (EIA). Compared with sham, glomeruli from 2 wk RA produced significantly more PGs. SC-58560, a selective COX-1 inhibitor, did not inhibit PG production in the remnant glomeruli at concentrations up to 10(-4) M, whereas SC-58236, a relatively selective COX-2 inhibitor, significantly inhibited PG production by RA glomeruli. In preliminary studies, to define mechanisms of altered expression of glomerular COX-2, rat mesangial cells were incubated with serum from sham or 2 wk RA. There were significant increases in COX-2 expression in response to 2 wk RA serum. In summary, these results indicate selective increases in renal cortical COX-2 expression following renal ablation.

Analysis of Variance↗
PubMed1997

The membrane-bound form of heparin-binding epidermal growth factor-like growth factor promotes survival of cultured renal epithelial cells.

To understand whether expression of membrane-anchored heparin binding epidermal growth factor (proHB-EGF) is involved in renal epithelial cell survival, rat membrane-bound HB-EGF precursor was stably transfected into a renal epithelial cell line, NRK 52E cells (NRKproHB-EGF). When exposed to 10% fetal calf serum (FCS), there were no differences in growth rates among wild-type (WT), vector-transfected (NRKvector), and NRKproHB-EGF. However, when cells were grown in the presence of 1% FCS, the growth rate of NRKproHB-EGF was 65% faster. When confluent cell monolayers were exposed to H2O2 or etoposide, WT or NRKvector exhibited significant apoptotic bodies and DNA laddering; in contrast, NRKproHB-EGF were resistant to both stimuli, as indicated by increased cell viability and marked decrease of apoptotic bodies and DNA laddering. When plated at high density onto plastic dishes without FCS, WT and NRKvector formed few attachments, did not proliferate, and underwent apoptosis. By day 3, no cells survived. Addition of exogenous recombinant HB-EGF (10(-8) M) to WT or NRKvector increased cell survival by <10% and incubation with conditioned media of NRKproHB-EGF had no effect. In contrast, NRKproHB-EGF attached and formed epithelial colonies, although they did not proliferate. After 3 days, cell viability was 84% of the initial cell number plated, and no evidence of apoptosis was present. When plated in 10% FCS, NRKproHB-EGF attachment to plastic substratum at 1, 2, and 3 h was 250% greater than that of WT or NRKvector. Addition of exogenous recombinant human HB-EGF to WT or NRKvector increased attachment by <50%. When grown on poly(2-hydroxyethyl methacrylate) or in the presence of the integrin receptor-blocking peptide GRGDTP, neither WT nor NRKvector attached to the substratum or formed cell-cell attachments. Compared with WT or NRKvector, NRKproHB-EGF exhibited 300% greater cell viability on either poly(2-hydroxyethyl methacrylate)-coated dishes or in the presence of GRGDTP and formed cell clusters. When plated at low density (1 x 10(3) cells/1.5-cm dish) or at high density in the presence of an anti-HB-EGF blocking antibody, NRKproHB-EGF failed to form epithelial colonies. Addition of formalin fixed NRKproHB-EGF promoted EGF receptor tyrosine phosphorylation in quiescent A431 cells and stimulated DNA synthesis and prevented H2O2-induced apoptosis in renal epithelial cells. These results indicate that membrane-bound HB-EGF promotes renal epithelial cell survival, possibly by promoting cell-matrix and cell-cell interactions. The failure of either conditioned media or exogenous HB-EGF to reproduce these findings suggests that juxtacrine or tightly coupled paracrine interactions underlie this cytoprotection.

Animals↗
PubMed1997

Production of heparin binding epidermal growth factor-like growth factor in the early phase of regeneration after acute renal injury. Isolation and localization of bioactive molecules.

We have recently reported that heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA is induced in the rat kidney after acute ischemic injury. The present studies were designed to investigate whether bioactive HB-EGF protein is also produced in response to renal injury induced by either ischemia/reperfusion or aminoglycosides. Heparin-binding proteins were purified from kidney homogenates by heparin affinity column chromatography using elution with a 0.2-2.0 M gradient of NaCl. A single peak of proteins that eluted at 1.0-1.2 M NaCl was detected in the postischemic kidney within 6 h of injury. This eluate fraction stimulated DNA synthesis in quiescent Balb/c3T3, RIE, and NRK-52E cell lines, all of which are responsive to the epidermal growth factor family of mitogenic proteins. The EGF receptor of A431 cells was also tyrosine phosphorylated by this eluate peak. Furthermore, immunoblotting with a polyclonal antibody against rat HB-EGF indicated that the eluate peak contained immunoreactive proteins of 22 and 29 kD mol wt, consistent with the reported sizes of the secreted form and membrane anchored form of HB-EGF, respectively. Immunohistochemical studies revealed that HB-EGF was produced predominantly in distal tubules in kidneys injured either by ischemia/reperfusion or aminoglycoside administration. We also found that during metanephric development immunoreactive HB-EGF was detected in the ureteric bud as early as E14.5 and persisted in structures arising from the ureteric bud throughout embryogenesis. These results suggest that in response to acute injury, HB-EGF is produced predominantly in distal tubules and that endogenous HB-EGF may be an important growth factor involved in renal epithelial cell repair, proliferation, and regeneration in the early stages of recovery after acute renal injury, as well as in nephrogenesis.

Aminoglycosides↗