Tianxin Yang

Regulation of cyclooxygenase expression in the kidney by dietary salt intake

The present studies were undertaken to determine the effect of dietary salt intake on the renal expression of cyclooxygenase-1 (COX-1) and -2 COX-2). Protein levels were assessed by Western blotting, and mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) on cDNA prepared from kidney regions, dissected nephron segments, and cultured renal cells. Both isoforms were expressed at high levels in inner medulla (IM), with low levels detected in outer medulla and cortex. COX-1 mRNA was present in the glomerulus and all along the collecting duct, whereas COX-2 mRNA was restricted to the macula densa-containing segment (MD), cortical thick ascending limb (CTAL), and, at significantly lower levels, in the inner medullary collecting duct. Both isoforms were highly expressed at high levels in cultured medullary interstitial cells and at lower levels in primary mesangial cells and collecting duct cell lines. Maintaining rats on a low- or high-NaCl diet for 1 wk did not affect expression of COX-1. In IM of rats treated with a high-salt diet, COX-2 mRNA increased 4.5-fold, and protein levels increased 9.5-fold. In contrast, cortical COX-2 mRNA levels decreased 2.9-fold in rats on a high-salt diet and increased 3.3-fold in rats on a low-salt diet. A low-salt diet increased COX-2 mRNA 7.7-fold in MD and 3.3-fold in CTAL. Divergent regulation of COX-2 in cortex and medulla by dietary salt suggests that prostaglandins in different kidney regions serve different functions, with medullary production playing a role in promoting the excretion of salt and water in volume overload, whereas cortical prostaglandins may protect glomerular circulation in volume depletion.The present studies were undertaken to determine the effect of dietary salt intake on the renal expression of cyclooxygenase-1 (COX-1) and -2 (COX-2). Protein levels were assessed by Western blotting, and mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) on cDNA prepared from kidney regions, dissected nephron segments, and cultured renal cells. Both isoforms were expressed at high levels in inner medulla (IM), with low levels detected in outer medulla and cortex. COX-1 mRNA was present in the glomerulus and all along the collecting duct, whereas COX-2 mRNA was restricted to the macula densa-containing segment (MD), cortical thick ascending limb (CTAL), and, at significantly lower levels, in the inner medullary collecting duct. Both isoforms were highly expressed at high levels in cultured medullary interstitial cells and at lower levels in primary mesangial cells and collecting duct cell lines. Maintaining rats on a low- or high-NaCl diet for 1 wk did not affect expression of COX-1. In IM of rats treated with a high-salt diet, COX-2 mRNA increased 4.5-fold, and protein levels increased 9.5-fold. In contrast, cortical COX-2 mRNA levels decreased 2.9-fold in rats on a high-salt diet and increased 3.3-fold in rats on a low-salt diet. A low-salt diet increased COX-2 mRNA 7.7-fold in MD and 3.3-fold in CTAL. Divergent regulation of COX-2 in cortex and medulla by dietary salt suggests that prostaglandins in different kidney regions serve different functions, with medullary production playing a role in promoting the excretion of salt and water in volume overload, whereas cortical prostaglandins may protect glomerular circulation in volume depletion.

Expression of peroxisomal proliferator-activated receptors and retinoid X receptors in the kidney

The discovery that 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is a ligand for the gamma-isoform of peroxisome proliferator-activated receptor (PPAR) suggests nuclear signaling by prostaglandins. Studies were undertaken to determine the nephron localization of PPAR isoforms and their heterodimer partners, retinoid X receptors (RXR), and to evaluate the function of this system in the kidney. PPARalpha mRNA, determined by RT-PCR, was found predominately in cortex and further localized to proximal convoluted tubule (PCT); PPARgamma was abundant in renal inner medulla, localized to inner medullary collecting duct (IMCD) and renal medullary interstitial cells (RMIC); PPARbeta, the ubiquitous form of PPAR, was abundant in all nephron segments examined. RXRalpha was localized to PCT and IMCD, whereas RXRbeta was expressed in almost all nephron segments examined. mRNA expression of acyl-CoA synthase (ACS), a known PPAR target gene, was stimulated in renal cortex of rats fed with fenofibrate, but the expression was not significantly altered in either cortex or inner medulla of rats fed with troglitazone. In cultured RMIC cells, both troglitazone and 15d-PGJ2 significantly inhibited cell proliferation and dramatically altered cell shape by induction of cell process formation. We conclude that PPAR and RXR isoforms are expressed in a nephron segment-specific manner, suggesting distinct functions, with PPARalpha being involved in energy metabolism through regulating ACS in PCT and with PPARgamma being involved in modulating RMIC growth and differentiation.The discovery that 15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2) is a ligand for the γ-isoform of peroxisome proliferator-activated receptor (PPAR) suggests nuclear signaling by prostaglandins. Studies were undertaken to determine the nephron localization of PPAR isoforms and their heterodimer partners, retinoid X receptors (RXR), and to evaluate the function of this system in the kidney. PPARα mRNA, determined by RT-PCR, was found predominately in cortex and further localized to proximal convoluted tubule (PCT); PPARγ was abundant in renal inner medulla, localized to inner medullary collecting duct (IMCD) and renal medullary interstitial cells (RMIC); PPARβ, the ubiquitous form of PPAR, was abundant in all nephron segments examined. RXRα was localized to PCT and IMCD, whereas RXRβ was expressed in almost all nephron segments examined. mRNA expression of acyl-CoA synthase (ACS), a known PPAR target gene, was stimulated in renal cortex of rats fed with fenofibrate, but the expression was not significantly altered in either cortex or inner medulla of rats fed with troglitazone. In cultured RMIC cells, both troglitazone and 15d-PGJ2 significantly inhibited cell proliferation and dramatically altered cell shape by induction of cell process formation. We conclude that PPAR and RXR isoforms are expressed in a nephron segment-specific manner, suggesting distinct functions, with PPARα being involved in energy metabolism through regulating ACS in PCT and with PPARγ being involved in modulating RMIC growth and differentiation.

MAPK Mediation of Hypertonicity-stimulated Cyclooxygenase-2 Expression in Renal Medullary Collecting Duct Cells

We have previously shown that hypertonicity stimulates cyclooxygenase-2 (COX-2) expression in cultured medullary epithelial cells. The aims of the present study were (i) to examine the role of cytoplasmic signaling through MAPK pathways in tonicity regulation of COX-2 expression in collecting duct cells and (ii) to assess the possible contribution of COX-2 to the survival of inner medullary collecting duct (IMCD) cells under hypertonic conditions. In mIMCD-K2 cells, a cell line derived from mouse IMCDs, hypertonicity induced a marked increase in COX-2 protein expression. The stimulation was reduced significantly by inhibition of MEK1 (PD-98059, 5–50 μm) and p38 (SB-203580, 5–100 μm) and was almost abolished by the combination of the two compounds. To study the role of JNK in tonicity-stimulated COX-2 expression, IMCD-3 cell lines stably transfected with dominant-negative mutants of three JNKs (JNK-1, -2, and -3) were used. Hypertonicity-stimulated COX-2 protein expression was significantly reduced in dominant-negative JNK-2-expressing cells and was unchanged in dominant-negative JNK-1- and JNK-3-expressing cells compared with controls. The reduction of COX-2 expression was associated with greatly reduced viability of dominant-negative JNK-2-expressing cells during hypertonicity treatment. 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (2–8 μm), an inhibitor of Src kinases, reduced the tonicity-stimulated COX-2 expression in a dose-dependent manner, whereas PP3, an inactive analog of PP2, had no effect. Inhibition of COX-2 activity by NS-398 (30–90 μm) and SC-58236 (10–20 μm) significantly reduced viability of mIMCD-K2 cells subjected to prolonged hypertonic treatment. We conclude that 1) all three members of the MAPK family (ERK, JNK-2, and p38) as well as Src kinases are required for tonicity-stimulated COX-2 expression in mouse collecting duct cells and that 2) COX-2 may play a role in cell survival of medullary cells under hypertonic conditions.

Obstruction stimulates COX-2 expression in bladder smooth muscle cells via increased mechanical stretch

Studies were performed to investigate the regulatory mechanism of bladder cyclooxygenase-2 (COX-2) expression after outlet obstruction. In situ hybridization of murine bladder tissues using COX-2-specific riboprobes demonstrated that COX-2 expression was induced predominantly in the bladder smooth muscle cells after outlet obstruction. To study the effect of increased mechanical stretch on COX isoform expression, cultured rat bladder smooth muscle cells were grown on silicone elastomer-bottomed plates coated with collagen type I and were subjected to continuous cycles of stretch/relaxation for variable duration. COX-1 mRNA levels did not change with stretch. COX-2 expression increased in a time-dependent manner after stretch, with maximal mRNA and protein levels occurring after 4 h. PGE2 levels increased more than 40-fold in the culture media after stretch, consistent with increased COX activity, and this was reduced to near completion in the presence of a COX-2 inhibitor, NS-398. Exposure to stretch over a 48-h period induced a 4.7 ± 0.6-fold increase in tritiated thymidine incorporation rate. This increase in DNA synthesis was markedly suppressed when the cells were stretched in the presence of NS-398. We conclude that in bladder obstruction COX-2 activation occurs predominantly in the smooth muscle cells in response to mechanical stretch. Our findings also suggest that stretch-activated COX-2 expression may participate in bladder smooth muscle cell proliferation and thereby play a role in pathological bladder wall thickening after obstruction.Studies were performed to investigate the regulatory mechanism of bladder cyclooxygenase-2 (COX-2) expression after outlet obstruction. In situ hybridization of murine bladder tissues using COX-2-specific riboprobes demonstrated that COX-2 expression was induced predominantly in the bladder smooth muscle cells after outlet obstruction. To study the effect of increased mechanical stretch on COX isoform expression, cultured rat bladder smooth muscle cells were grown on silicone elastomer-bottomed plates coated with collagen type I and were subjected to continuous cycles of stretch/relaxation for variable duration. COX-1 mRNA levels did not change with stretch. COX-2 expression increased in a time-dependent manner after stretch, with maximal mRNA and protein levels occurring after 4 h. PGE2 levels increased more than 40-fold in the culture media after stretch, consistent with increased COX activity, and this was reduced to near completion in the presence of a COX-2 inhibitor, NS-398. Exposure to stretch over a 48-h period induced a 4.7 +/- 0.6-fold increase in tritiated thymidine incorporation rate. This increase in DNA synthesis was markedly suppressed when the cells were stretched in the presence of NS-398. We conclude that in bladder obstruction COX-2 activation occurs predominantly in the smooth muscle cells in response to mechanical stretch. Our findings also suggest that stretch-activated COX-2 expression may participate in bladder smooth muscle cell proliferation and thereby play a role in pathological bladder wall thickening after obstruction.

Tubular localization and tissue distribution of peptide transporters in rat kidney

AbstractPurpose. To define the tubular localization and tissue distribution of PEPT1 (low-affinity, high-capacity transporter) and PEPT2 (high-affinity, low-capacity transporter) in rat kidney.nMethods. mRNA expression of PEPT1 and PEPT2 was assessed with reverse transcription-polymerase chain reaction (RT-PCR) methods using cDNA prepared from microdissected nephron segments in rat. Tissue localization of rat renal PEPT1 and PEPT2 mRNA was further assessed by in situ hybridization with radiolabeled probes.nResults. RT-PCR analysis of microdissected segments from rat nephron showed that both PEPT1 and PEPT2 are confined to the proximal tubule. While PEPT1 is specific for early regions of the proximal tubule (pars convoluta), PEPT2 is overwhelmingly but not exclusively expressed in latter regions of the proximal tubule (pars recta). All other segments along the nephron were negative for PEPT1 or PEPT2 mRNA transcripts. These findings were supported by in situ hybridization results in which PEPT1 was selectively expressed in kidney cortex and PEPT2 in the outer stripe of outer medulla.nConclusions. Contrary to current opinion, the data suggest that peptides are handled in a sequential manner in proximal regions of the nephron, first by the low-affinity, high-capacity transport system and second by the high-affinity, low-capacity transport system.

Cyclooxygenase-2 is expressed in bladder during fetal development and stimulated by outlet obstruction

Studies were undertaken to assess expression of inducible cyclooxygenase (COX)-2 in bladder during fetal development and COX-1 and COX-2 expression after outlet obstruction. Bladder tissue or bladder progenitor tissue was harvested from CD-1 murine embryos at embryonic days 11.5( E11.5), E14.5, E17.5, E20.5 (newborn), and from adult. Bladder obstruction was created in adult female mice by ligating the urethra, and bladders were harvested after 3-24 h of obstruction. Gene expression was assessed by semiquantitative reverse transcription-polymerase chain reaction and Western blotting. COX-2 was highly expressed at the early stages of bladder development and declined progressively throughout gestation. In adult bladder, both COX-1 and COX-2 were detectable at low levels under basal conditions. An ∼30-fold increase in COX-2 mRNA was seen after 24 h of obstruction. In contrast, COX-1 did not change with obstruction. COX-2 mRNA levels peaked at 6 h of obstruction. In regional bladder-distention models, COX-2 induction was confined to the area of distention. Bladder outlet obstruction stimulates COX-2 expression dramatically, reactivating a gene that is highly expressed during fetal development.Studies were undertaken to assess expression of inducible cyclooxygenase (COX)-2 in bladder during fetal development and COX-1 and COX-2 expression after outlet obstruction. Bladder tissue or bladder progenitor tissue was harvested from CD-1 murine embryos at embryonic days 11.5 (E11.5), E14.5, E17.5, E20.5 (newborn), and from adult. Bladder obstruction was created in adult female mice by ligating the urethra, and bladders were harvested after 3-24 h of obstruction. Gene expression was assessed by semiquantitative reverse transcription-polymerase chain reaction and Western blotting. COX-2 was highly expressed at the early stages of bladder development and declined progressively throughout gestation. In adult bladder, both COX-1 and COX-2 were detectable at low levels under basal conditions. An approximately 30-fold increase in COX-2 mRNA was seen after 24 h of obstruction. In contrast, COX-1 did not change with obstruction. COX-2 mRNA levels peaked at 6 h of obstruction. In regional bladder-distention models, COX-2 induction was confined to the area of distention. Bladder outlet obstruction stimulates COX-2 expression dramatically, reactivating a gene that is highly expressed during fetal development.

Inhibition of adenosine-1 receptor-mediated preglomerular vasoconstriction in AT1Areceptor-deficient mice

The effect of the adenosine type 1 receptor agonist N 6-cyclohexyladenosine (CHA) on glomerular vascular reactivity was studied in male angiotensin II type 1A (AT1A) receptor knockout mice (9). Vascular reactivity was assessed as the response of stop-flow pressure (PSF) to infusion of CHA into loops of Henle using micropuncture techniques. In AT1A +/+ mice at ambient arterial blood pressure (96.7 ± 2.8 mmHg), the presence of CHA (10-5 M) in the perfusate increased PSF responses from 6.8 ± 0.6 to 14.3 ± 0.9 mmHg when the loop of Henle of the index nephron was perfused and from 0.7 ± 0.3 to 12.3 ± 1.0 mmHg when the loop of an adjacent nephron was perfused. At reduced arterial blood pressure (82.8 ± 1.3 mmHg), index nephron perfusion with CHA increased PSF responses from 4.5 ± 0.3 to 9.4 ± 0.4 mmHg. In AT1A -/- mice with a mean arterial blood pressure of 80 ± 1.9 mmHg, CHA increased PSF responses only from 0.1 ± 0.3 to 3.6 ± 0.54 mmHg during index nephron perfusion and from 0.25 ± 0.2 to 2.7 ± 0.55 mmHg during adjacent nephron perfusion, significantly less than in wild-type animals ( P < 0.001). Responses to CHA were intermediate in AT1A +/- mice. Thus AT1A receptor knockout mice show a markedly reduced constrictor response to CHA both in the presence and absence of simultaneous activation of the tubuloglomerular feedback system. These data support the notion of a functional interaction between adenosine and angiotensin II in the regulation of afferent arteriolar tone.

Tubuloglomerular feedback in ACE-deficient mice

In these experiments, we used a strain of angiotensin converting enzyme (ACE) germline null mutant mice, generated by J. H. Krege and co-workers (J. H. Krege, S. W. M. John, L. L. Langenbach, J. B. Hodgin, J. R. Hagaman, E. S. Bachman, J. C. Jennette, D. A. OBrien, and O. Smithies. Nature 375: 146-148, 1995), to examine the effect of chronic ACE deficiency on the magnitude of tubuloglomerular feedback (TGF) responses. The genotype was determined by PCR on DNA extracted from the tail and was verified after each experiment by assessment of the blood pressure response to an injection of ANG I. To assess TGF responsiveness, we determined the change in stop-flow pressure (PSF) caused by increasing NaCl concentration at the macula densa by using micropuncture techniques. When loop of Henle flow rate was increased from 0 to 40 nl/min, PSF fell from a mean of 42.3 +/- 1.95 to 33.6 +/- 2.09 mmHg (n = 6, P = 0.005) in wild-type mice (+/+), fell from 40.6 +/- 2.35 to 38.6 +/- 1.93 mmHg in heterozygous (+/-) mice (n = 7, P = 0.014), and did not change in homozygous ACE (-/-) mice [36.7 +/- 2.02 mmHg vs. 36.4 +/- 2.01 mmHg; n = 4, P = not significant (NS)]. During an infusion of ANG II at a dose that did not significantly elevate blood pressure (70 ng. kg-1. min-1), TGF response magnitude (PSF 0 - PSF 40) increased from 6.5 +/- 1.4 to 9.8 +/- 1.19 mmHg in +/+ (P = 0.006), from 1.14 +/- 0.42 to 4.6 +/- 1.3 mmHg in +/- (P = 0.016), and from 0.42 +/- 0.25 to 4.02 +/- 1.06 in -/- mice (P = 0.05). Absence of TGF responses in ACE null mutant mice and restoration of near-normal responses during an acute infusion of ANG II supports previous conclusions that ANG II is an essential component in the signal transmission pathway that links the macula densa with the glomerular vascular pole.In these experiments, we used a strain of angiotensin converting enzyme (ACE) germline null mutant mice, generated by J. H. Krege and co-workers (J. H. Krege, S. W. M. John, L. L. Langenbach, J. B. Hodgin, J. R. Hagaman, E. S. Bachman, J. C. Jennette, D. A. OBrien, and O. Smithies. Nature 375: 146-148, 1995), to examine the effect of chronic ACE deficiency on the magnitude of tubuloglomerular feedback (TGF) responses. The genotype was determined by PCR on DNA extracted from the tail and was verified after each experiment by assessment of the blood pressure response to an injection of ANG I. To assess TGF responsiveness, we determined the change in stop-flow pressure (PSF) caused by increasing NaCl concentration at the macula densa by using micropuncture techniques. When loop of Henle flow rate was increased from 0 to 40 nl/min, PSF fell from a mean of 42.3 ± 1.95 to 33.6 ± 2.09 mmHg ( n = 6, P = 0.005) in wild-type mice (+/+), fell from 40.6 ± 2.35 to 38.6 ± 1.93 mmHg in heterozygous (+/-) mice ( n = 7, P = 0.014), and did not change in homozygous ACE (-/-) mice [36.7 ± 2.02 mmHg vs. 36.4 ± 2.01 mmHg; n = 4, P = not significant (NS)]. During an infusion of ANG II at a dose that did not significantly elevate blood pressure (70 ng ⋅ kg-1 ⋅ min-1), TGF response magnitude (PSF 0 - PSF 40) increased from 6.5 ± 1.4 to 9.8 ± 1.19 mmHg in +/+ ( P = 0.006), from 1.14 ± 0.42 to 4.6 ± 1.3 mmHg in +/- ( P = 0.016), and from 0.42 ± 0.25 to 4.02 ± 1.06 in -/- mice ( P = 0.05). Absence of TGF responses in ACE null mutant mice and restoration of near-normal responses during an acute infusion of ANG II supports previous conclusions that ANG II is an essential component in the signal transmission pathway that links the macula densa with the glomerular vascular pole.

Post-translational Processing and Renal Expression of Mouse Indian Hedgehog

The full-length mouse Indian hedgehog (Ihh) cDNA was cloned from an embryonic 17.5-day kidney library and was used to study the post-translational processing of the peptide and temporal and spatial expression of the transcript. Sequence analysis predicted two putative translation initiation sites. Ihh translation was initiated at both initiation sites when expressed in an in vitro transcription/translation system. Expression of an Ihh mutant demonstrated that the internal translation initiation site was sufficient to produce the mature forms of Ihh. Ihh post-translational processing proceeded in a fashion similar to Sonic and Drosophila hedgehog; the unprocessed form underwent signal peptide cleavage as well as internal proteolytic processing to form a 19-kDa amino-terminal peptide and a 26-kDa carboxyl-terminal peptide. This processing required His313 present in a conserved serine protease motif. Ihh transcript was detected by in situ RNA hybridization as early as 10 days postcoitum (dpc) in developing gut, as early as 14.5 dpc in the cartilage primordium, and in the developing urogenital sinus. In semiquantitative reverse transcription-polymerase chain reaction experiments, Indian hedgehog transcript was first detected in the mouse metanephros at 14.5 dpc; transcript abundance increased with gestational age, becoming maximal in adulthood. In adult kidney, Ihh transcript was detected only in the proximal convoluted tubule and proximal straight tubule.

SA Gene Expression in the Proximal Tubule of Normotensive and Hypertensive Rats

Previous studies have shown that the SA gene is expressed at higher levels in the kidney of genetically hypertensive rats than in control strains and that in hybrid crosses of genetically hypertensive rats and normotensive controls, markers in or close to the SA gene cosegregate with blood pressure. The present studies examine the localization of the SA gene product in the kidney by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). cDNA was prepared from microdissected nephron segments from Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHRs), and Wistar-Kyoto (WKY) rats, and RT-PCR was performed using specific primers. In all three strains, SA gene mRNA was found to be abundantly expressed in proximal tubules. SA PCR product was occasionally detected at approximately 100-fold lower abundance in glomeruli, while no signal was obtained from the collecting duct, thick ascending limb of the loop of Henle, or arcuate artery. Within the proximal tubule of normotensive rats, distribution of SA mRNA was found to be strain dependent: in SD rats it was expressed at high levels in the proximal convoluted tubule, whereas in WKY rats it was restricted to the proximal straight tubule. In SHRs, SA PCR product was detected along the entire proximal tubule. Induction of hypertension by renal artery clamping (two-kidney, one-clamp Goldblatt model) did not alter the pattern of expression observed in the SD rat. These results indicate that an extension of SA gene expression to the full length of the proximal tubule accompanies spontaneous hypertension and that in nonhypertensive animals the pattern of gene product expression is more restricted but shows substantial strain variability.