Reyadh Redha

Thiazolidinediones expand body fluid volume through PPARγ stimulation of ENaC-mediated renal salt absorption

Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-γ (PPARγ, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct–specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg flox/flox mice. Deletion of collecting duct Pparg decreased renal Na+ avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na+ absorption and Scnn1g mRNA (encoding the epithelial Na+ channel ENaCγ) expression through a PPARγ-dependent pathway. These studies identify Scnn1g as a PPARγ target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.

Opposite effects of cyclooxygenase-1 and -2 activity on the pressor response to angiotensin II

Therapeutic use of cyclooxygenase-inhibiting (COX-inhibiting) nonsteroidal antiinflammatory drugs (NSAIDs) is often complicated by renal side effects including hypertension and edema. The present studies were undertaken to elucidate the roles of COX1 and COX2 in regulating blood pressure and renal function. COX2 inhibitors or gene knockout dramatically augment the pressor effect of angiotensin II (Ang II). Unexpectedly, after a brief increase, the pressor effect of Ang II was abolished by COX1 deficiency (either inhibitor or knockout). Ang II infusion also reduced medullary blood flow in COX2-deficient but not in control or COX1-deficient animals, suggesting synthesis of COX2-dependent vasodilators in the renal medulla. Consistent with this, Ang II failed to stimulate renal medullary prostaglandin E(2) and prostaglandin I(2) production in COX2-deficient animals. Ang II infusion normally promotes natriuresis and diuresis, but COX2 deficiency blocked this effect. Thus, COX1 and COX2 exert opposite effects on systemic blood pressure and renal function. COX2 inhibitors reduce renal medullary blood flow, decrease urine flow, and enhance the pressor effect of Ang II. In contrast, the pressor effect of Ang II is blunted by COX1 inhibition. These results suggest that, rather than having similar cardiovascular effects, the activities of COX1 and COX2 are functionally antagonistic.

Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.

Macula densa (MD) cells express COX-2 and COX-2-derived PGs appear to signal the release of renin from the renal juxtaglomerular apparatus, especially during volume depletion. However, the synthetic machinery and identity of the specific prostanoid released from intact MD cells remains uncertain. In the present studies, a novel biosensor tool was engineered to directly determine whether MD cells release PGE2 in response to low luminal NaCl concentration ([NaCl]L). HEK293 cells were transfected with the Ca2+-coupled E-prostanoid receptor EP1 (HEK/EP1) and loaded with fura-2. HEK/EP1 cells produced a significant elevation in intracellular [Ca2+] ([Ca2+]i) by 29.6 +/- 12.8 nM (n = 6) when positioned at the basolateral surface of isolated perfused MD cells and [NaCl]L was reduced from 150 mM to zero. HEK/EP1 [Ca2+]i responses were observed mainly in preparations from rabbits on a low-salt diet and were completely inhibited by either a selective COX-2 inhibitor or an EP1 antagonist, and also by 100 microM luminal furosemide. Also, 20-mM graduated reductions in [NaCl]L between 80 and 0 mM caused step-by-step increases in HEK/EP1 [Ca2+]i. Low-salt diet greatly increased the expression of both COX-2 and microsome-associated PGE synthase (mPGES) in the MD. These studies provide the first direct evidence that intact MD cells synthesize and release PGE2 during reduced luminal salt content and suggest that this response is important in the control of renin release and renal vascular resistance during salt deprivation.

Prostaglandin E2-EP4 Receptor Promotes Endothelial Cell Migration via ERK Activation and Angiogenesis in Vivo

Prostaglandin E2 (PGE2), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1–4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4flox/flox mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE2 or the EP4 selective agonists PGE1-OH and ONO-AE1–329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4flox/flox cells, no effects were seen in adenoCre-transduced EP4flox/flox cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE2-induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE2, as well as PGE1-OH and ONO-AE1–329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.

Selective targeting of cyclooxygenase-2 reveals its role in renal medullary interstitial cell survival

Renal medullary interstitial cells (MICs) are a major site of cyclooxygenase (COX)-mediated PG synthesis. These studies examined the role of COX in MIC survival. Immunoblot and nuclease protection demonstrate that cultured MICs constitutively express COX2, with little constitutive COX1 expression. SC-58236, a COX2-selective inhibitor, but not SC-58560, a COX1 inhibitor, preferentially blocks PGE2 synthesis in MICs. Transduction with a COX2 antisense adenovirus reduced MIC COX2 protein expression and also decreased PGE2production. Antisense downregulation of COX2 was associated with MIC death, whereas a control adenovirus was without effect. Similarly, the COX2-selective inhibitor SC-58236 (30 μM) and several nonselective COX-inhibiting nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac, ibuprofen, and indomethacin, all caused MIC death. In contrast, SC-58560, a COX1-selective inhibitor, was 100-fold less potent for inducing MIC death than its structural congener SC-58236. NSAID-induced MIC death was associated with DNA laddering and nuclear fragmentation, consistent with apoptosis. These results suggest that COX2 plays an important role in MIC survival. COX2 inhibition may contribute to NSAID-associated injury of the renal medulla.

Biotin transport in the human intestine: Site of maximum transport and effect of pH*

Previous studies from our laboratory have characterized the transport process of biotin across the brush border membrane and the basolateral membrane of the human intestine. In this study we further characterized biotin transport in the human intestine by examining the vitamins transport process in different areas of the small intestine (duodenum, jejunum, and ileum) and the effect of pH on the transport process using a brush border membrane vesicle technique. In all areas examined, the transport of biotin as a function of concentration was saturable in the presence of a Na+ gradient (out greater than in) but was linear and lower in the presence of a choline gradient (out greater than in). Transport of biotin by the Na+-dependent process (i.e., the carrier-mediated process) was found to be higher in the duodenum than the jejunum, which was in turn higher than that in the ileum. This decrease in biotin transport distally was found to be due to a decrease in the Vmax of the transport process of the vitamin with no changes in the apparent Km. This indicates that the number (i.e., the density) of transport carriers for biotin decreases distally. In the presence of a Na+ gradient (out greater than in), decreasing incubation buffer pH from 8.0 to 5.5 (intravesicular pH was 7.4) was found to cause an increase in biotin transport. This increase was found to be due to the acidic buffer pH (i.e., not due to the pH gradient imposed across the membrane) and occurred through an increase in the transport of the vitamin by the nonmediated process. These results demonstrate that the proximal part of the small intestine is the site of maximum transport of biotin in humans. Furthermore, variation in incubation medium pH affects biotin transport through changes in the substrate transport by the nonmediated process.

Biotin transport in basolateral membrane vesicles of human intestine

The characteristics of the exit process of biotin from the enterocyte, i.e., transport across the basolateral membrane, was determined using an enriched basolateral membrane vesicle preparation of human intestine. Purity and suitability of basolateral membrane vesicles for transport studies was confirmed by enzymatic and functional criteria. Orientation of human basolateral membrane vesicles was determined by [3H]ouabain binding studies and was found to be 64% inside-out vesicles and the rest right-side-out vesicles and membrane sheets. Osmolarity studies indicated that the uptake of biotin by these vesicles represents transport into the intravesicular compartment, with little binding to membrane surfaces. The rate of biotin transport was linear for approximately 40 s but decreased thereafter. Transport of biotin was (a) Na+-independent, (b) saturable as a function of concentration, with an apparent KM of 1.1 microM and Vmax of 0.9 pmol/mg protein.15 s, (c) inhibited by structural analogues (desthiobiotin and biotin methyl ester) and related compounds (thioctic acid and thioctic amide), and (d) stimulated by inducing a positive intravesicular electrical potential. These studies are the first to demonstrate the existence of a carrier-mediated transport system for biotin in the basolateral membrane of human intestine.

Studies on the Intestinal Surface Acid Microclimate: Developmental Aspects

ABSTRACT. The existence and general characteristics of the intestinal surface acid microclimate (ISAM) in the developing intestine of suckling and weanling rats were examined. ISAM pH measurements were performed in vitro using a sensitive glass pH-microelectrode. The results showed that the ISAM does exist in both suckling and weanling rat intestine. In both suckling and weanling rats, ISAM pH was significantly (p < 0.01) lower in the jejunum than in the ileum, an observation similar to that previously reported in the small intestine of adult rats. In the colon, however, ISAM pH of suckling rats was significantly (p < 0.01) lower than that of weanling and adult rats. Studies on the relationship between jejunal ISAM pH of weanling rats and incubation buffer pH showed that the two are not in equilibrium. Jejunal ISAM pH of weanling rats was significantly inhibited by: 1) the mucolytic agent N-acetyl- L-cysteine, 2) stirring of the incubation medium, 3) Na+ removal, 4) glucose removal (or substitution by the unmetabolizable galactose), and 5) metabolic inhibitors (iodoacetate and dinitrophenol). These results demonstrate the existence of the ISAM in the developing intestine of suckling and weanling rats and shows the dependence of the ISAM on Na+, metabolizable substrate(s) and normal intracellular metabolism. Furthermore, surface mucus appears to play a role in maintaining the ISAM, most probably through retaining the H+ at the intestinal surface.

Intestinal Uptake of Retinol in Suckling Rats: Characteristics and Ontogeny

ABSTRACT: Uptake of retinol in the developing intestine of suckling rats (14–15 day old) and its maturation in adult rats (90 day old) was examined using intestinal everted sacs. Uptake of retinol (0.06 μM) in the jejunum of suckling and adult rats was linear for 5 min incubation and occurred at a rate of 31.20 and 6.98 pmol/g tissue/min, respectively. In both age groups, uptake of retinol (0.06 μM) was significantly higher (p < 0.01) in the jejunum than the ileum. Uptake of retinol was significantly higher (p < 0.01) in suckling rats as compared to adult rats both in the jejunum and the ileum. In both suckling and adult rats, the uptake of retinol in the jejunum was 1) saturable with a Vmax value of 19.78 and 6.24 nmol/g tissue/5 min and an apparent Km value of 16.20 and 8.19 μM, respectively, 2) not affected by metabolic inhibitors, and 3) partially temperature dependent (Q10 = 2.51 and 1.92, respectively). The structural analogues retinal (50 μM) and retinoic acid (50 μM) did not affect the uptake of [3H] retinol (0.06 μM) whereas unlabeled retinol (50 μM) caused significant (p < 0.01) inhibition. No difference in retinol metabolism by intestinal tissue was observed in the two age groups. These results demonstrate that retinol uptake in suckling rats is similar to that of adult rats in being a passive carrier-mediated process. The results also suggest that a decrease in the number and/or activity and an increase in the affinity of the uptake system of retinol occurs with maturation.

Transport of glycyl-L-proline in intestinal brush-border membrane vesicles of the suckling rat: characteristics and maturation

Transport of the dipeptide glycine-L-proline (Gly-L-Pro) in the developing intestine of suckling rats and its subsequent maturation in adult rats was examined using the brush-border membrane vesicles (BBMV) technique. Uptake of Gly-L-Pro by BBMV was mainly the result of transport into the intravesicular space with little binding to membrane surfaces. Transport of Gly-L-Pro in BBMV of suckling rats was: (1) Na+ independent; (2) pH dependent with maximum uptake at an incubation buffer pH of 5.0; (3) saturable as a function of concentration (apparent Km = 21.5 +/- 7.9 mM, Vmax = 8.6 +/- 1.5 nmol/mg protein per 10 s); (4) inhibited by other di- and tripeptides; and (5) stimulated and inhibited by inducing a negative and positive intravesicular membrane electrical potential, respectively. Similarly, transport of Gly-L-Pro in intestinal BBMV of adult rats was saturable as a function of concentration (apparent Km = 17.4 +/- 8.6 mM, Vmax = 9.1 +/- 2.1 nmol/mg protein per 10 s) and was stimulated and inhibited by inducing a relatively negative and positive intravesicular membrane potential, respectively. No difference in the transport kinetic parameters of Gly-L-Pro was observed in suckling and adult rats, indicating a similar activity (and/or number) and affinity of the transport carrier in the two age groups. These results demonstrate that the transport of Gly-L-Pro is by a carrier-mediated process which is fully developed at the suckling period. Furthermore, the process is H+-dependent but not Na+-dependent, electrogenic and most probably occurs by a Gly-L-Pro/H+ cotransport mechanism.