Faculty Bibliography

BACKGROUND:Erythropoietin has recently been found to have cytoprotective effects in the central nervous system (CNS) and retina. The purpose of this study was to determine if darbepoetin alfa (DA) has cytoprotective properties in renal tissues. METHODS:DA was studied in LLC/PK1 and mesangial cells. Renal cellular injury was induced in different experiments by prostaglandin D2 synthase (PGDS), camptothecin, hydrogen peroxide, and hypoxia. Cellular proliferation and apoptosis were measured [apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay or by caspase-3 activity]. In a separate experiment, an inactive form of erythropoietin alfa was used to study receptor effects. RESULTS:DA protected against the antiproliferative effects of PGDS. In both LLC/PK1 (TUNEL and caspase-3) and mesangial cells (TUNEL), DA reduced the apoptotic stimulus of PGDS. Epoetin alfa was also found to reduce apoptosis. In LLC/PK1 cells, DA reduced apoptosis induced by camptothecin, but not hydrogen peroxide. DA reduced LLC/PK1 apoptosis induced by hypoxia when added 24 hours before hypoxia, but not when given concurrent with the hypoxic stimulus. Erythropoietin inactive did not protect against PGDS-induced apoptosis. CONCLUSION/CONCLUSIONS:DA has renal antiapoptotic effects for both toxic and hypoxic stimuli. The effect may be mediated via the Erythropoietin receptor.

Lipocalin-type prostaglandin D2 synthase (L-PGDS) has recently been linked to a variety of pathophysiological cardiovascular conditions including hypertension and diabetes. In this study, we report on the 50% increase in L-PGDS protein expression observed in vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR). L-PGDS expression also increased 50% upon the differentiation of normotensive control cells (WKY, from Wistar-Kyoto rats). In addition, we demonstrate differential effects of L-PGDS treatment on cell proliferation and apoptosis in VSMCs isolated from SHR versus WKY controls. L-PGDS (50 microg/ml) was able to significantly inhibit VSMC proliferation and DNA synthesis and induce the apoptotic genes bax, bcl-x, and ei24 in SHR but had no effect on WKY cells. Hyperglycemic conditions also had opposite effects, in which increased glucose concentrations (20 mm) resulted in decreased L-PGDS expression in control cells but actually stimulated L-PGDS expression in SHR. Furthermore, we examined the effect of L-PGDS incubation on insulin-stimulated Akt, glycogen synthase kinase-3beta (GSK-3beta), and ERK phosphorylation. Unexpectedly, we found that when WKY cells were pretreated with L-PGDS, insulin could actually induce apoptosis and failed to stimulate Akt/GSK-3beta phosphorylation. Insulin-stimulated ERK phosphorylation was unaffected by L-PGDS pretreatment in both cell lines. We propose that L-PGDS is involved in the balance of VSMC proliferation and apoptosis and in the increased expression observed in the hypertensive state is an attempt to maintain a proper equilibrium between the two processes via the induction of apoptosis and inhibition of cell proliferation.

Recently we demonstrated the induction of apoptosis by the addition of recombinant lipocalin-type prostaglandin D(2) synthase (L-PGDS) to the culture medium of LLC-PK(1) cells. Because protein kinase C (PKC) has been shown to be involved in the apoptotic process of various cell types, we examined the potential role of L-PGDS in phorbol 12-myristate 13-acetate (PMA)-induced apoptosis. We report here the enzymatic activation and phosphorylation of L-PGDS in response to phorbol ester in cell culture and the direct phosphorylation of recombinant L-PGDS by PKC in vitro. Treatment of cells with PMA or L-PGDS decreased phosphatidylinositol 3-kinase (PI3-K) activity and concomitantly inhibited protein kinase B (PKB/Akt) phosphorylation, which led to the hypophosphorylation and activation of Bad. In addition, hypophosphorylation of retinoblastoma protein was also observed in response to L-PGDS-induced apoptosis. Cellular depletion of L-PGDS levels by using an antisense RNA strategy prevented PI3-K inactivation by phorbol ester and inhibited caspase-3 activation and apoptosis. We conclude that phorbol ester-induced apoptosis is mediated by L-PGDS phosphorylation and activation by PKC and is accompanied by inhibition of the PI3-K/PKB anti-apoptotic signaling pathways.

This article reviews the possible role of prostaglandin D(2) synthase (PGD(2)S) in the progression of chronic renal failure and dialysis dementia. Such a proposal is based on our observation that PGD(2)S significantly increases the rate of apoptosis in cultured pig kidney proximal tubule LLC-PK1 and rat neuronal PC12 cells. Apoptosis was caspase mediated and inhibitable by PGE(1), PGE(2), PGF(2alpha), platelet-derived growth factor (PDGF), and by PGD(2)S inhibitors, selenium and anti-PGD(2)S antibody. Apoptosis was restored by the addition of downstream metabolic products, PGD(2) and 15 deoxy PG triangle up (12,14)J(2). The proposal that PGD(2)S contributes to progression of renal failure and dialysis dementia is based on: (1) the progressive creatinine-like increase in PGD(2)S levels in blood as renal function decreases, increased renal cyclooxygenase (COX) 2 in chronic renal failure, and reported increase in apoptosis noted in the remnant kidney model, and (2) a 35- to 150-fold increase in blood levels of PGD(2)S in dialysis patients. Both conditions appear to favor shifting the PG metabolic pathway to downstream apoptotic metabolites, PGD(2) and 15 deoxy PG triangle up (12,14)J(2). The diverse role that PGs, growth factors, and COX play in progression of chronic renal failure, their interactions with PGD(2)S, and the status of COX inhibitors in retarding the progression of renal failure are reviewed. In addition, the need for a more systematic longitudinal assessment of dementia in dialysis patients by standardized neuropsychologic testing, testing blood levels and glycosylated isoforms of PGD(2)S, and the effect of COX inhibition and erythropoietin administration on dialysis dementia are discussed.

To determine whether liquid ventilation (LV) causes less cell injury and improves lung function compared with conventional gas ventilation (GV), we analyzed pulmonary physiological profiles, lung histology, and cell death in 110- and 120-day preterm lambs, which were randomized to receive either ventilation modality on FI(O(2)) = 1. LV lungs were well expanded with adequate pulmonary function, whereas GV animals exhibited marked atelectasis, poor pulmonary function, and increased mortality. Both ventilatory strategies induced marked lung cell apoptosis, but with distinct patterns of distribution. Although GV induced apoptosis of epithelium primarily in the lining and within the lumina of bronchioles, LV induced significant apoptosis much more homogeneously throughout lung parenchyma including alveoli and interstitial spaces. These studies suggest that although both forms of ventilation cause regional apoptosis, LV more effectively delivers oxygen and recruits the lung more homogeneously than GV.

BACKGROUND:Prostaglandin D(2) synthase (PGD(2)S), a unique member of the lipocalin family, is found at elevated levels in the serum of patients with renal impairment and has recently been implicated as a new biochemical marker of renal insufficiency. The aim of this study was to investigate the apoptotic effects of PGD2S on a pig kidney epithelial cell line (LLC-PK1) and to investigate the effects of prostaglandins and growth factors on this process. METHODS:Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL), annexin V staining, and electron microscopy. RESULTS:A four- to fivefold increase in apoptosis was observed in PGD(2)S-treated cells as compared with controls and the apoptosis appeared to act via caspase-3. A cyclooxygenase-2 inhibitor, anti-PGD(2)S antibody, and selenium all significantly inhibited the apoptosis induced by PGD(2)S; however, none had any effect on the apoptosis induced by the known apoptotic inducer camptothecin. Furthermore, prostaglandins E(1) and E(2), known to induce mitogen-activated protein (MAP) kinase phosphorylation and exhibit cytoprotective effects, both inhibited PGD(2)S-induced apoptosis, while prostaglandin H(2) had no significant effect. Growth factors such as insulin, insulin-like growth factor-1, and platelet-derived growth factor also decreased PGD(2)S-induced apoptosis. In addition, PGD(2)S isolated from human serum seemed slightly more effective at inducing apoptosis than recombinantly expressed protein. CONCLUSIONS:We report on the induction of apoptosis by PGD(2)S in LLC-PK1 pig kidney epithelial cells, and speculate that the accumulation of PGD(2)S in the serum of kidney failure patients may further exacerbate renal problems and is most likely regulated by other prostaglandins and growth factors.

Apoptosis of neuronal cells is a proposed cause of certain neurological disorders. Here, we report on a 5- to 6-fold increase in apoptosis by exposure to prostaglandin D2 synthase (PGD2S) in PC12 neuronal cells. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, and appears to be mediated via caspase-3 activation. Neutralization with anti-PGD2S antibody or pre-treatment with selenium, which inhibits PGD2S enzymatic activity, both significantly inhibited the PGD2S-induced apoptosis, however, neither had any effect on the apoptosis induced by the known neuronal apoptotic inducer, glutamate. In addition, prostaglandins E1, E2, and F2alpha all inhibited the PGD2S-induced apoptosis while prostaglandin H2 had no significant effect. Furthermore, PGD2S isolated from human serum was more effective at inducing apoptosis then recombinantly expressed protein, presumably due to glycosylation. This novel role of PGD2S, as an inducer of apoptosis, may have implications in PC12 differentiation and possibly some neurological disorders.

Prairie dogs of both sexes were fed a semisynthetic diet containing 0.35% cholesterol for a period of 8 weeks. This lithogenic diet induced cholesterol gallstones in ten "lithogenic control animals", five males and five females. Three animals maintained with a high glucose, fat-free diet did not develop gallstones although the cholesterol saturation of their bile approached unity. The formation of gallstones was prevented in four out of five males and all five females fed the lithogenic diet plus 0.1% hyodeoxycholic acid (30 mg per kg body weight per day). The biles of the prairie dogs receiving hyodeoxycholic acid were abnormally colored, cloudy, and highly saturated with cholesterol but contained neither cholesterol crystals nor gallstones (with the exception of one male). Feeding the relatively hydrophilic bile acid, hyodeoxycholic acid, was associated with an increase in hepatic microsomal HMG-CoA reductase activity. Cholesterol 7 alpha-hydroxylase, on the other hand, was inhibited by the administered bile acid. The dietary hyodeoxycholic acid was transformed, in part, to 3 alpha, 6 beta-dihydroxy-5-beta-cholanoic acid and hyocholic acid. It is concluded that hyodeoxycholic acid and its metabolites did not prevent the induced cholelithiasis by causing a decrease in the concentration of biliary cholesterol. Instead, this hydrophilic bile acid apparently increases the amount of cholesterol in the bile, probably in the form of a liquid crystalline mesophase. Hyodeoxycholic acid apparently prevents gallstones by preventing the nucleation and aggregation of cholesterol crystals. The lithogenic diet induced moderate to marked bile duct proliferation together with portal fibrosis and inflammatory infiltration. The addition of hyodeoxycholic acid to the lithogenic diet reduced all of the portal tract changes.