Hui-Fang Cheng

Endothelial Nitric Oxide Synthase Deficiency Produces Accelerated Nephropathy in Diabetic Mice

Functionally significant polymorphisms in endothelial nitric oxide synthase (eNOS) and reduced vascular eNOS activity have been associated with increased human diabetic nephropathy (DN), but the pathogenic role of eNOS deficiency in the development of DN has not yet been confirmed. This study characterizes the severity of DN in eNOS(-/-) mice that were backcrossed to C57BLKS/J db/db mice. Although the severity of hyperglycemia was similar to C57BLKS/J db/db mice, by 26 wk, eNOS(-/-) C57BLKS/J db/db mice exhibited dramatic albuminuria, arteriolar hyalinosis, increased glomerular basement membrane thickness, mesangial expansion, mesangiolysis, and focal segmental and early nodular glomerulosclerosis. Even more remarkable, eNOS(-/-) C57BLKS db/db exhibited decreases in GFR to levels <50% of that in eNOS(+/+) C57BLKS db/db, as confirmed by increased serum creatinine. In summary, eNOS(-/-) db/db mice provide the most robust model of type II DN that has been described to date and support a role for deficient eNOS-derived NO production in the pathogenesis of DN.

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.

Cyclooxygenase-2-selective inhibitors impair glomerulogenesis and renal cortical development

BACKGROUND Antenatal exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) has been associated with renal dysgenesis in humans. METHODS These studies characterized cyclooxygenase-2 (COX-2) versus COX-1-selective inhibition on nephrogenesis in the rodent using histomorphometry, immunohistology, and in situ hybridization. RESULTS Administration of a COX-2-selective inhibitor (SC58236), started during pregnancy until weaning, significantly impaired development of the renal cortex and reduced glomerular diameter in both mice and rats. An identical phenotype was demonstrated in COX-2 -/- mice. In contrast to its effects on the developing kidney, a COX-2 inhibitor had no effect on glomerular volume in adult mice. This effect was specific for COX-2 because maternal administration of a COX-1-selective inhibitor (SC58560) did not affect renal development despite significantly inhibiting gastric mucosal prostaglandin E2 (PGE2) synthesis in pups. The expression of COX-2 immunoreactivity peaked in the first postnatal week and was localized to S-shaped bodies and the macula densa in the cortex. Treatment with a COX-2 inhibitor during this period (from postnatal day 0 to day 21) severely reduced glomerular diameter, whereas treatment limited to pregnancy did not affect glomerular size. CONCLUSION These data demonstrate an important role for COX-2 activity in nephrogenesis in the rodent, and define a specific time period of susceptibility to these effects.

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.

Angiotensin II upregulates type-1 angiotensin II receptors in renal proximal tubule.

Angiotensin II (Ang II) is an important regulator of proximal tubule salt and water reabsorption. Recent studies indicate that rabbit proximal tubule angiotensin II receptors are the type-1 (AT1R) subtype. We studied the effect of Ang II on proximal tubule receptor expression. Rabbits were treated with either angiotensin converting enzyme inhibitors or a low salt diet to modulate endogenous Ang II levels. In captopril-treated rabbits, liver and glomerular AT1R mRNA levels increased 242 +/- 125 and 141 +/- 60%, respectively (n = 6-7; P < 0.05), as determined by quantitative PCR. In contrast, proximal tubule AT1R mRNA levels decreased 40 +/- 11% (n = 6; P < 0.05). Binding of 125I Ang II to renal cortical basolateral membranes of captopril-treated rabbits decreased from 2.9 +/- 0.55 to 1.4 +/- 0.17 fmol/mg protein (n = 6; P < 0.025). In rabbits fed a sodium chloride-deficient diet for 4 wk, AT1R mRNA levels decreased 52 +/- 11% in liver and 43 +/- 7% in glomeruli (n = 4-5; P < 0.05), whereas they increased 141 +/- 85% (n = 5; P < 0.05) in proximal tubule. In basolateral membranes from rabbits on the sodium chloride-deficient diet, specific binding of 125I Ang II increased from 2.1 +/- 0.2 to 4.3 +/- 1.1 fmol/mg protein (n = 7; P < 0.05). To determine whether Ang II directly regulates expression of proximal tubule AT1 receptors, further studies were performed in cultured proximal tubule cells grown from microdissected S1 segments of rabbit proximal tubules and immortalized by transfection with a replication-defective SV40 vector. Incubation of these cells with Ang II (10(-11) to 10(-7) M) led to concentration-dependent increases in both AT1R mRNA levels and specific 125I Ang II binding. Pretreatment with pertussis toxin inhibited Ang II stimulation of AT1R mRNA. AT1R mRNA expression was decreased by either forskolin or a nonhydrolyzable cAMP analogue (dibutryl cAMP). Simultaneous Ang II administration overcame the inhibitory effect of forskolin but not dibutryl cAMP. These results indicate that proximal tubule AT1R expression is regulated by ambient Ang II levels, and Ang II increases AT1R mRNA at least in part by decreasing proximal tubule cAMP generation through a pertussis toxin-sensitive mechanism. Upregulation of proximal tubule AT1R by Ang II may be important in mediating enhanced proximal tubule sodium reabsorption in states of elevated systemic or intrarenal Ang II.

Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride

We have previously shown that in renal cortex, COX-2 expression is localized to macula densa and surrounding cortical thick ascending limb of Henle (cTALH). Dietary salt restriction increases local expression of COX-2, which mediates renin production and secretion. Given that decreased luminal chloride [Cl(-)] at the level of the macula densa increases renin production and secretion, we investigated the role of extracellular ion concentration on COX-2 expression. Quiescent rabbit cTALH cells were incubated in a physiological salt solution containing high or low levels of NaCl. Immunoreactive COX-2 expression increased significantly in the low NaCl solution. COX-2 expression also increased after administration of the Na(+)/K(+)/2Cl(-) cotransport inhibitor, bumetanide. Selective substitution of chloride led to increased COX-2 expression, whereas selective substitution of sodium had no effect. The p38 MAP kinase inhibitor PD169316 decreased low NaCl-induced COX-2 expression. Low-salt or low-chloride medium induced cultured cTALH to accumulate >/= 3-fold higher levels of pp38, the activated (phosphorylated) form of p38; low-salt medium also increased pJNK and pERK levels. Feeding rats a low-salt diet for 14 days induced a significant increase in renal cortical pp38 expression, predominantly in the macula densa and cTALH. These results suggest that reduced extracellular chloride leads to increased COX-2 expression, which may be mediated by activation of a p38-dependent signaling pathway.

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 Henles 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.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 Henles 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.

DOPAMINE DECREASES EXPRESSION OF TYPE-1 ANGIOTENSIN II RECEPTORS IN RENAL PROXIMAL TUBULE

Systemic and/or locally produced angiotensin II stimulates salt and water reabsorption in the renal proximal tubule. In vivo, dopamine (DA) may serve as a counterregulatory hormone to angiotensin IIs acute actions on the proximal tubule. We examined whether dopamine modulates AT1 receptor expression in cultured proximal tubule cells (RPTC) expressing DA1 receptors. Dopamine decreased basal RPTC AT1 receptor mRNA levels by 67 +/- 7% (n = 10; P < 0.005) and decreased 125I-angiotensin II binding by 41 +/- 7% (n = 4; P < 0.05). The DA1-specific agonist, SKF38393 decreased basal AT1 receptor mRNA levels (65 +/- 5% inhibition; n = 5; P < 0.025), and the DA1-specific antagonist, SCH23390 reversed dopamines inhibition of AT1 receptor mRNA expression (24 +/- 10% inhibition; n = 8; NS) and angiotensin II binding (5 +/- 15%; n = 4; NS). DA2-specific antagonists were ineffective. In rats given L-DOPA in the drinking water for 5 d, there were decreases in both proximal tubule AT1 receptor mRNA expression (80 +/- 5%; n = 6; P < 0.005) and specific [125I] Ang II binding (control: 0.74 +/- 0.13 fmol/mg pro vs. 0.40 +/- 0.63 fmol/mg pro; n = 5; P < 0.05). In summary, dopamine, acting through DA1 receptors, decreased AT1 receptor expression in proximal tubule, an effect likely mediated by increased intracellular cAMP levels. Local dopamine production also led to decreased AT1 receptor expression, suggesting dopamine may reset sensitivity of the proximal tubule to angiotensin II.

Overexpression of Cyclooxygenase-2 Predisposes to Podocyte Injury

Increased podocyte cyclooxygenase-2 (COX-2) expression is seen in rats after renal ablation and Thy-1 nephritis and in cultured murine podocytes in response to mechanical stress. For investigation of whether COX-2 overexpression plays a role in podocyte injury, transgenic B6/D2 mice in which COX-2 expression was driven by a nephrin promoter were established. Selective upregulation of COX-2 expression in podocytes of transgenic mouse kidneys was confirmed by immunoblotting and immunohistochemistry. Whether upregulation of podocyte-specific COX-2 expression enhanced sensitivity to the development of Adriamycin nephropathy was examined. Adriamycin administration induced dramatically more albuminuria and foot process effacement and reduced glomerular nephrin mRNA and immunoreactivity in transgenic mice compared with wild-type littermates. Adriamycin also markedly increased immunoreactive COX-2 expression in podocytes from transgenic mice compared with the wild-type mice. Reverse transcriptase-PCR indicated that this increase represented a stimulation of endogenous COX-2 mRNA expression rather than COX-2 mRNA driven by the nephrin promoter. Balb/C mice, which are susceptible to renal injury by Adriamycin, also increased podocyte COX-2 expression and reduced nephrin expression in response to administration of the drug. Long-term treatment with the COX-2-specific inhibitor SC58236 ameliorated the albuminuria that was induced by Adriamycin in the transgenic mice. SC58236 also reduced Adriamycin-induced foot process effacement in both the COX-2 transgenic mice and Balb/C mice. Therefore, overexpression of COX-2 may predispose podocytes to further injury.

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.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 β-actin × 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 β-actin × 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 chronicl-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 β-actin × 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 orl-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.