Blueprint for discovery in academic medicine: plans, process and outcomes.

By the end of the decade, we had fully implemented most of the recommendations of the Molinoff Report. Our programmatic analysis is summarized in Table 11. While the space needs identified in the Molinoff Report were met by BRB I, II, and III (289,000 nsf as compared [table: see text] to 276,000 nsf as planned), it was possible to provide additional, somewhat unanticipated, research space (111,000 nsf) prior to the end of the decade. The faculty has now developed a research plan for the next decade. It is also important to emphasize that the total faculty grew by 41% [table: see text] over the decade and most of that growth occurred with faculty spending a substantial part of their time in clinical practice. Hence, the dramatic improvement in research funding of over 200% was due largely to the enhanced productivity of our faculty. By taking an organized planning approach deeply seated in the faculty, consistent with Trustee directives and with measurable outcomes, we were successful in growing the research programs within the School of Medicine of the University of Pennsylvania. We believe this particular approach, taken with a focus on multidisciplinary research, [table: see text] was the right one for the 1990s. In the final analysis, it is abundantly clear that outstanding faculty, working in an exciting supportive environment, was the most important factor for success. We are not certain what the right approach will be for the future. Clearly, with the important advances in genomics and information technology, the importance of the team, even if a virtual one world-wide, cannot be overstated. While research is only one mission of the School of Medicine, clearly, our visible success in research played an important role in the overall improvement in the School of Medicine as measured by others. For example, the ranking of the School of Medicine by U.S. News & World Report, perhaps the most widely used ranking by the lay press, went from 10th to 3rd behind only Harvard and Johns Hopkins during the period of the 90s (Table 12).

Mechanisms of chronic hypoxia-induced renal cell growth.

Chronic local tissue hypoxia appears to play an important role in the initiation and progression of chronic renal disease. We examined the effect of local hypoxia on cultured renal tubular epithelial and mesangial cell proliferation, dedifferentiation, and extracellular matrix synthesis. The underlying signaling mechanisms whereby hypoxia induces renal cell growth were evaluated. The roles of protein kinase C, p38 mitogen-activated protein kinase, TGF-beta1, osteopontin, and nitric oxide were determined.

Chronic hypoxia induces LLC-PK1 cell proliferation and dedifferentiation by the activation of protein kinase C.

The effect of chronic hypoxia on the proliferation and dedifferentiation of LLC-PK1 cells was examined. Cultures were exposed either to hypoxia (3% O2) or normoxia (18% O2), and [3H]thymidine incorporation, cell number, and sodium-dependent glucose (Na/Glc) uptakes were assessed. Cultures exposed to hypoxia for 16 h significantly increased [3H]thymidine incorporation followed by a significant increase in cell number both at 24 and 48 h in comparison with respective normoxic controls. Cultures exposed to 24 and 72 h of hypoxia exhibited significant inhibition of Na/Glc uptake when compared with their respective normoxic counterparts. Significant inhibition of cell ATP levels were observed under hypoxic conditions. Acute reoxygenation of hypoxic cells normalized cell ATP levels without any effect on the Na/Glc uptake. Hypoxia also activated protein kinase C (PKC) at 1 and 4 h followed by a subsequent return to baseline with reactivation at 24 h, which remained sustained up to 72 h, suggesting both acute and sustained activation of PKC. Furthermore, the hypoxia-induced alterations in [3H]thymidine incorporation as well as Na/Glc uptake were mitigated by inhibitors of PKC. These results indicate that chronic hypoxia induces both proliferation and dedifferentiation of LLC-PK1 cells mediated, in part, by the activation of PKC.

Chronic hypoxia induces proliferation of cultured mesangial cells: role of calcium and protein kinase C.

The effect of hypoxia on the proliferation of cultured rat mesangial cells was examined. To evaluate the underlying signaling mechanisms, the roles of intracellular calcium ([Ca2+]i) and protein kinase C (PKC) were determined. Quiescent cultures were exposed to hypoxia (3% O2) or normoxia (18% O2), and [3H]thymidine incorporation, cell number, [Ca2+]i, and PKC were assessed. Mesangial cells exposed to 28 h of hypoxia exhibited a significant increase in [3H]thymidine incorporation followed by a significant increase in cell number at 72 h in comparison with respective normoxic controls. Hypoxia induced a biphasic activation of PKC, reflected by translocation of the enzyme activity from cytosol to membrane at 1 h, a return to baseline at 4 and 8 h, with subsequent reactivation from 16 to 48 h. In addition, hypoxia-induced proliferation was prevented by a PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). Cells exposed to hypoxia produced progressive increases in resting [Ca2+]i from 15 to 60 min which remain sustained up to 24 h of examination. Verapamil significantly prevented the hypoxia-induced proliferation, and both verapamil treatment and incubations in a calcium-free medium for 1 h blocked the hypoxia-induced stimulation of [Ca2+]i as well as PKC. These results provide the first in vitro evidence that chronic hypoxia induces proliferation of cultured glomerular mesangial cells, which is mediated by the stimulation of [Ca2+]i and the subsequent activation of PKC.

Prostaglandin F2 alpha- and 12-O-tetradecanoylphorbol-13-acetate-induced alterations in the pathways of renal ammoniagenesis.

The mechanisms whereby prostaglandin F2 alpha (PGF2 alpha) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit ammoniagenesis and the reason why they behave differently at pH 7.4, were examined with (15N)glutamine to assess the metabolic pathways and 2'-7'-bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescein, acetoxymethylester (BCECF-AM) to evaluate Na+/H+ antiporter activity. LLC-PK1 cultures were incubated for 1 h in a Krebs-Hensleit bicarbonate buffer of pH 7.4 and pH 6.8 supplemented either with 5-15N- or 2-15N-labeled glutamine, followed by the assessment of (15N)ammonia and (15N)amino acid formation. Exposure of cells to either PGF2 alpha or TPA completely inhibited the low pH-induced increases in (15N)ammonia formation from incubations with 5-15N, reflecting reduced flux through the mitochondrial phosphate-dependent glutaminase, and from (2-15N)glutamine, reflecting reduced flux through the mitochondrial glutamate dehydrogenase pathway. They also qualitatively reversed the acute acidosis-induced changes in (15N)alanine formation and (15N)glutamate accumulation in the media. By contrast only TPA, but not PGF2 alpha, modified glutamine metabolism at pH 7.4. Na+/H+ antiporter activity was assessed under both acidified and basal (pH 7.4) conditions by measuring changes in intracellular pH in cells loaded with BCECF. TPA and PGF2 alpha both stimulated Na+/H+ antiporter activity comparably under acidified conditions. When cells were studied at pH 7.4, TPA but not PGF2 alpha stimulated the Na+/H+ antiporter and increased steady-state intracellular pH.(ABSTRACT TRUNCATED AT 250 WORDS)

PGF2 alpha activation of Na/H antiporter and ammoniagenesis in parent/variant LLC-PK1 cells.

A novel variant of the LLC-PK1 cell line was used to examine directly the mechanism whereby PGF2 alpha and TPA inhibit renal ammoniagenesis. The variant cells, which exhibit a growth pattern and morphology similar to the parent cell line, were isolated by a self selection process utilizing long-term cultures of parent cells maintained under conditions of continuous gentle rocking of the media fluid. Incubation of both parent and variant LLC-PK1 cells for one hour in a glutamine supplemented Krebs-Hensleit media of low pH (pH 6.8) increased ammonia and alanine production in comparison to the basal rates at pH 7.4. The phorbol ester TPA and also PGF2 alpha inhibited the low pH-induced increases in ammonia and alanine formation in parent cells; however, neither TPA nor PGF2 alpha inhibited ammonia or alanine metabolism in variant cells. TPA and PGF2 alpha activated PKC similarly in the parent and variant cells as demonstrated by a significant increase in membrane bound enzyme activity. BCECF labeling of cells indicated that the parent and variant cells possess an amiloride sensitive Na+/H+ antiporter of comparable activity. Exposure of parent cells to PGF2 alpha or TPA resulted in the activation of Na+/H+ antiporter activity. By contrast, neither compound stimulated antiporter activity in variant cells. These studies strongly suggest that PKC mediated activation of the Na+/H+ antiporter accounts for the inhibition of ammonia production produced by both PGF2 alpha and TPA. In addition, this novel variant of LLC-PK1 cells should provide a valuable tool to investigate various normal and pathophysiological functions involving mediation by PKC and/or Na+/H+ antiporter activity.

Chronic hypoxia impairs the differentiation of 3T3-L1 fibroblast in culture: role of sustained protein kinase C activation.

The effect of hypoxia on 3T3-L1 cell differentiation was examined in confluent cultures incubated with differentiation medium (DM) followed by incubation in growth medium (GM). Control cultures remained in GM throughout the incubation period. Eight days after the incubation, cells were assessed either for changes in morphology by staining with Oil Red O/hematoxylin or harvested to measure protein kinase C activity. Morphological examination of stained cells showed almost complete differentiation of normoxic cells to adipocytes when exposed to DM. By contrast hypoxia caused a dramatic inhibition of differentiation under similar media conditions with only 34 +/- 4% of cells accumulating fat deposits. Cultures sustained in GM under normoxic or hypoxic conditions were devoid of any fat deposits, reflecting an undifferentiated phenotype. Normoxic cells exposed to DM exhibited a significantly lower membrane to cytosolic ratio of protein kinase C in comparison with cells maintained in GM, which is consistent with differentiated and undifferentiated phenotypes, respectively. In comparison with normoxic cells incubated in DM, cells exposed to hypoxia under similar media conditions exhibited a significantly higher membrane to cytosolic ratio of protein kinase C, indicating sustained activation of the enzyme. In addition, cells in differentiation medium exposed to hypoxia in the presence of the protein kinase C inhibitors staurosporine or H7 exhibited a significant increase in the number of fat accumulating cells when compared with hypoxic controls. These studies indicate that chronic hypoxia impairs the differentiation of 3T3-L1 cells to adipocytes in association with the sustained activation of protein kinase C, which appears to play a role in mediating this process.

Metabolic substrates alter attachment and differentiated functions of proximal tubule cell culture.

Proximal tubules cultured in vitro gradually lose their differentiated functions. Because standard culture media lacks several substrates important for renal proximal tubule oxidative metabolism, whether a mixture of substrates including butyrate, alanine, and lactate (BAL) would modify growth and/or differentiated function of proximal tubular cells in culture was examined. Tubules cultured in media supplemented with 2 mM butyrate, alanine, and lactate exhibited enhanced attachment but did not exhibit an altered growth rate. Higher levels of phosphoenolpyruvate carboxykinase and leucine-amino peptidase were sustained, although these activities were still diminished in comparison with that in fresh tubules. Sodium-dependent glucose uptake and dome formation--other reflections of epithelial cell differentiated function--also were enhanced. These studies demonstrate that the substrates used to culture proximal tubules can modify both their attachment and their manifestation of differentiated function in culture.

The intensity of acidosis differentially alters the pathways of ammoniagenesis in LLC-PK1 cells.

Utilizing [5-15N] and [2-15N]-labeled glutamine and gas chromatography mass spectrometry methodology, we examined the pathways of ammoniagenesis under basal and acute acidotic conditions of pH 7.0 and pH 6.8, respectively. LLC-PK1 cultures were incubated for one hour with gentle rocking in a bicarbonate buffer of pH 7.4, pH 7.0, or pH 6.8 supplemented either with [5-15N] or [2-15N] glutamine at 37 degrees C in a 5% CO2/95% air incubator atmosphere. Incubation of cultures with [5-15N] glutamine at pH 7.4 resulted in a substantial amount of 15N ammonia formation which was not significantly altered by incubations at pH 7.0. By contrast, exposure to pH 6.8 significantly increased 15N ammonia formation in comparison with its production at pH 7.0 or 7.4. However, 15N ammonia production from [2-15N] glutamine was significantly stimulated at pH 7.0 and was further increased at pH 6.8. Incubation of the cells with [2-15N] glutamine resulted in a substantially lower amounts of 15N ammonia formation than produced with [5-15N] glutamine. Alanine formation from [2-15N] glutamine increased significantly at pH 7.0; but in contrast to 15N ammonia formation, pH 6.8 had no additional stimulatory effect on 15N alanine formation. Cells incubated with [2-15N] glutamine resulted in a significant decrement in 15N glutamate production at both pH 7.0 and 6.8 when compared with pH 7.4. 15N aspartate formation was unaltered by the changes in media pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxia-mediated impaired differentiation by LLC-PK1 cells: evidence based on the protein kinase C profile.

We recently reported that mild hypoxia in LLC-PK1 cells, grown in standard fashion under a still layer of overlying medium at 5% CO2/18% O2 environment, result in decreased oxidative metabolism and impaired differentiated functions in comparison to adequately oxygenated cultures maintained either under a higher oxygen (36% O2) environment or conditions of continuous rocking of the media fluid. In the present study, subcellular distribution of a regulatory enzyme protein kinase C (PKC) was examined between hypoxic still and normoxic rocked LLC-PK1 cells. Subconfluent cultures of hypoxic LLC-PK1 cells exhibited significantly lower and predominantly membrane-bound PKC activity in comparison to mostly cytosolic localization of this enzyme in normoxic rocked cells. One hour of exposure of adequately oxygenated-rocked LLC-PK1 cells with the phorbol ester TPA, a dedifferentiating agent that did not effect the cell ATP content, resulted in significant inhibition of dome formation and sodium-dependent glucose transport activity, a partial loss of pH-responsive ammoniagenesis, and almost complete translocation of protein kinase C activity from cytosol to the membrane pool; all of which resembles the behavior of hypoxic still cultured cells. In addition, acute re-oxygenation of hypoxic still cultures by rocking the media fluid for one hour resulted in an increase in cell ATP content to the cellular levels of ATP observed in normoxic rocked cells. However, all the parameters of differentiation were unaffected by re-oxygenation. These studies support the notion that hypoxia can act in some primary fashion, independent of its effects on energy metabolism, to impair cellular differentiation in LLC-PK1 cells. They also raise the possibility that activation of protein kinase C may act as an important mediator in this process.

Signal transduction events whereby PGF2 alpha inhibits the ammoniagenic response to acute acidosis.

Subconfluent cultures of LLC-PK1 cells were incubated for 1 h in Krebs-Henseleit buffer (KHB) of pH 7.4 or 6.8 to investigate the signal transduction events associated with prostaglandin F2 alpha (PGF2 alpha) inhibition of ammonia metabolism. Exposure of these cultures to PGF2 alpha (0.1 ng/ml) inhibited the acute low pH stimulation of ammonia production and to a lesser degree alanine formation in a manner analogous to the response exhibited with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment with an inhibitor of protein kinase C [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, i.e., H-7] or utilization of cultures with downregulated protein kinase C activity abolished the inhibitory response to PGF2 alpha. Exposure to PGF2 alpha for 10 min in KHB of pH 6.8 resulted in an activation of protein kinase C, as demonstrated by a significant increase in membrane-bound enzyme activity. Incubation of the cells with PGF2 alpha in KHB of pH 6.8 also resulted in a significant increase in inositol trisphosphate formation. Treatment of the cultures with verapamil in calcium-containing medium or removal of calcium from the incubating medium resulted in a significant loss of the PGF2 alpha inhibitory response on both ammonia and alanine production. Furthermore, under conditions of calcium-free incubation, PGF2 alpha had no significant effect on protein kinase C activity. Because both PGF2 alpha- and TPA-induced inhibition of ammoniagenic response to acute acidosis was prevented by amiloride, the underlying mechanism may involve protein kinase C-mediated changes in intracellular pH. These results indicate that the activation of protein kinase C plays a key role in mediating PGF2 alpha inhibition of ammoniagenesis.

Pathways of acute pH regulation of ammoniagenesis in LLC-PK1 cells: study with [15N]glutamine.

The present study utilized [15N]glutamine labeled at amide (5-N) and amino (2-N) groups to analyze the metabolic fate of glutamine nitrogen in basal and in acute pH regulation of ammoniagenesis. One-hour incubation of LLC-PK1 cultures in a media of pH 7.4, 7.0, or 7.6 containing either [5-15N]glutamine or [2-15N]glutamine resulted in parallel alterations in glutamine consumption in response to acute acid-base maneuvers. Incubation with [5-15N]glutamine resulted in substantial enrichment and production of ammonia at pH 7.4, which was unaffected by the changes in media pH, and in no significant enrichment of alanine, aspartate, and glutamate. In contrast, significant enrichment and production of 15N-labeled ammonia, alanine, aspartate, and glutamate were detected from cultures incubated with [2-15N]glutamine. Ammonia formation, from incubation with [2-15N]glutamine, was stimulated significantly by a low pH and inhibited by high pH. Alanine production was altered in a fashion similar to ammonia formation, whereas aspartate production was unaltered and glutamate formation significantly decreased by a low pH. Furthermore, a low pH significantly increased the production of alpha-ketoglutaramate in a fashion qualitatively similar to alanine production. In contrast to our prior conclusions based on total metabolite production, these studies indicate that although ammonia formation at pH 7.4 is predominantly generated from the mitochondrial phosphate-dependent glutaminase pathway, the increased ammonia formation in acute acidosis is a result of increased flux through glutamate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin F2 alpha inhibits the ammoniagenic response to acute acidosis in LLC-PK1 cells.

A kidney epithelial cell line, LLC-PK1, which does not synthesize prostaglandins, provides an ideal in vitro model system to investigate the effect of prostaglandins in the regulation of renal ammoniagenesis. Previous studies from our laboratory have demonstrated significant increases in glutamine-dependent ammonia and alanine production by rocked cultures of LLC-PK1 cells subjected to either acute metabolic or respiratory acidosis. In the study presented here, experiments were conducted to investigate the role of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) in the response of ammonia metabolism to acute metabolic acidosis by LLC-PK1 cells. A low dose of PGF2 alpha (0.1 ng/mL) dramatically inhibited the stimulatory effect of a low pH (pH 6.8) on ammonia production. In contrast, the inhibition of cytosolically generated alanine was less dramatic and averaged only 20% of the effect on ammonia production. Furthermore, PGF2 alpha increased cellular alpha-ketoglutarate concentration, suggesting an increase in intramitochondrial pH. Thus, the cellular mechanism of PGF2 alpha action appears to involve either interference with the cytosolic pH signal or its translation to the intramitochondrial compartment. The inhibitory response of PGF2 alpha on pH-stimulated ammoniagenesis was progressively lost at higher concentrations. Both low-dose (0.1 ng/mL) and high-dose (10 ng/mL) PGF2 alpha had no significant effect on the basal rates of ammonia and alanine production at pH 7.4. PGE2, on the other hand, did not exhibit any significant response on ammonia or alanine production at either pH 6.8 or 7.4 when given in a wide range of doses.(ABSTRACT TRUNCATED AT 250 WORDS)