Zhonghua Qi

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.

Reduction of Renal Superoxide Dismutase in Progressive Diabetic Nephropathy

Superoxide excess plays a central role in tissue damage that results from diabetes, but the mechanisms of superoxide overproduction in diabetic nephropathy (DN) are incompletely understood. In the present study, we investigated the enzyme superoxide dismutase (SOD), a major defender against superoxide, in the kidneys during the development of murine DN. We assessed SOD activity and the expression of SOD isoforms in the kidneys of two diabetic mouse models (C57BL/6-Akita and KK/Ta-Akita) that exhibit comparable levels of hyperglycemia but different susceptibility to DN. We observed down-regulation of cytosolic CuZn-SOD (SOD1) and extracellular CuZn-SOD (SOD3), but not mitochondrial Mn-SOD (SOD2), in the kidney of KK/Ta-Akita mice which exhibit progressive DN. In contrast, we did not detect a change in renal SOD expression in DN-resistant C57BL/6-Akita mice. Consistent with these findings, there was a significant reduction in total SOD activity in the kidney of KK/Ta-Akita mice compared with C57BL/6-Akita mice. Finally, treatment of KK/Ta-Akita mice with a SOD mimetic, tempol, ameliorated the nephropathic changes in KK/Ta-Akita mice without altering the level of hyperglycemia. Collectively, these results indicate that down-regulation of renal SOD1 and SOD3 may play a key role in the pathogenesis of DN.

Differentiation of Cyclooxygenase 1- and 2-Derived Prostanoids in Mouse Kidney and Aorta

Accumulating evidence indicates cyclooxygenase (COX) 1 and COX2 differentially regulate cardiovascular and renal function. We have demonstrated previously in mice that COX2 inhibition enhances angiotensin II–induced hypertension, and COX1 inhibition attenuates the pressor effect of angiotensin II. To further elucidate the mechanism underlying the functional difference of COX1 versus COX2 inhibition, the present studies examined the prostaglandin (PG) profiles derived in COX1- or COX2-inhibited mouse kidney and aorta using gas chromatographic/mass spectrometric assays. PGE2 is the most abundant prostanoid in both renal cortex and medulla in normal C57BL/6J mice, followed by PGI2, PGF2&agr; and thromboxane A2. In contrast PGI2 was most abundant in aorta followed by thromboxane A2, PGE2, and PGF2&agr;. PGD2 was undetectable in control kidney or aorta. At baseline, inhibition of COX1 decreased total prostaglandins in renal cortex, medulla, and aorta, whereas COX2 inhibition decreased total prostaglandins only in renal medulla. Angiotensin II infusion significantly increased COX2-dependent/COX1-independent PGE2 and PGI2 in renal cortex and medulla. Angiotensin II also significantly increased renal PGF2&agr; in cortex, but not in medulla, through both COX1- and COX2-dependent mechanisms. These studies demonstrate that although COX1 primarily contributes to basal prostanoid production in the kidney and aorta, angiotensin II increases renal vasodilator prostanoids predominately via COX2 activity. These effects may contribute to the specific effect of COX2 inhibitors to increase blood pressure.

Cyclooxygenase-2 selective inhibitors and the kidney.

Cyclooxygenases (COX) are the target of non-steroidal anti-inflammatory drugs (NSAIDs) which exert their therapeutic effect by blocking COXs capacity to metabolize arachidonate to a series of biologically active fatty acids, designated prostaglandins. NSAID use is associated with two major tonicities: gastrointestinal bleeding and renal dysfunction. In the setting of significant physiologic stress, renal function becomes dependent upon prostaglandins and NSAID use may be associated with acute deterioration of renal function, including development of sodium retention, edema, hypertension, hyperkalemia, and or papillary necrosis. Two isoforms, COX1 and COX2, have been identified. They are products of distinct genes and their expression is under different regulatory control. Both COX1 and COX2 are highly expressed in the kidney and both are inhibited by conventional NSAIDs. Accumulating data using recently developed selective COX2 inhibitors suggest that while these agents spare the gastrointestinal tract they have similar renal effects as non-selective NSAIDs. Therefore, caution should be taken when prescribing selective COX2 inhibitor to patients, especially to patients with predisposed physiologic stress.

Markers of glycemic control in the mouse: comparisons of 6-h- and overnight-fasted blood glucoses to Hb A1c

The present studies examined the relationship between fasting blood glucose and Hb A(1c) in C57BL/6J, DBA/2J, and KK/HlJ mice with and without diabetes mellitus. Daily averaged blood glucose levels based on continuous glucose monitoring and effects of 6-h vs. overnight fasting on blood glucose were determined. Daily averaged blood glucose levels were highly correlated with Hb A(1c), as determined with a hand-held automated device using an immunodetection method. R(2) values were 0.90, 0.95, and 0.99 in KK/HIJ, C57BL/6J, and DBA/2J, respectively. Six-hour fasting blood glucose correlated more closely with the level of daily averaged blood glucose and with Hb A(1c) than did blood glucose following an overnight fast. To validate the immunoassay-determined Hb A(1c), we also measured total glycosylated hemoglobin using boronate HPLC. Hb A(1c) values correlated well with total glycosylated hemoglobin in all three strains but were relatively lower than total glycosylated hemoglobin in diabetic DBA/2J mice. These results show that 6-h fasting glucose provides a superior index of glycemic control and correlates more closely with Hb A(1c) than overnight-fasted blood glucose in these strains of mice.

Better nephrology for mice--and man.

The use of creatinine to estimate glomerular filtration rate in patients is prone to well-described artifacts that impact its interpretation. Eisner et al. now show that the impact of creatinine secretion on creatinine clearance is even larger in mice than in humans, raising questions regarding the utility of creatinine for measuring glomerular filtration rate in mice.

Measurement of glomerular filtration rate in conscious mice.

Glomerular filtration rate (GFR) is an important index of renal function and routinely used in patient care and basic research to evaluate progression of renal diseases or test the efficacy of novel therapeutic strategies. Determination of GFR in mouse models has been mostly practiced in anesthetized animals, which is not suitable for serial monitoring of GFR in the individual mouse. In this chapter, we outline two approaches for determining GFR in conscious mice including 1) determination of urinary excretion of fluorescein-labelled inulin (FITC-inulin), and 2) determination of plasma FITC-inulin decay following a single bolus injection. The GFR values determined using these two methods are comparable. The sensitivity of the methods in reflecting renal function was validated in nephrectomized mice and early stage diabetic mice. The effects of inbred mouse genetic background on GFR values are also discussed in this chapter.

Expression of Mediators of Renal Injury in the Remnant Kidney of ROP Mice Is Attenuated by Cyclooxygenase-2 Inhibition

To investigate the effects of cyclooxygenase-2 (COX-2) inhibition on renal injury of mice, ROP mice were subjected to subtotal ablation (‘remnant’). A subset of the remnant group was treated with a selective COX-2 inhibitor, SC58236, in the drinking water. At 12 weeks the remnant group developed significant albuminuria (181.3 ± 15.8 µg/24 h), which was blunted by SC58236 treatment (138.9 ± 17.1; p < 0.05 compared to remnant). SC58236 did not alter systemic blood pressure or GFR significantly. Immunoreactive COX-2 was upregulated in remnant (1.88 ± 0.35 fold sham, n = 8, p < 0.05), which was blunted by SC58236 (to 1.26 ± 0.31 fold sham). Collagen IV mRNA increased significantly in remnant kidneys (2.69 ± 0.34 fold sham, n = 8, p < 0.05), and this increase was inhibited by SC58236 treatment (to 1.84 ± 0.32 fold control). Immunoreactive TGF-β1, connective tissue growth factor, HGF receptor, c-Met, and fibronectin all increased in remnant (2.85 ± 0.51, 3.83 ± 0.55, 2.56 ± 0.31, and 2.80 ± 0.39 fold sham respectively, n = 4–8, p < 0.05), and SC58236 blunted the increases (to 1.45 ± 0.34, 1.85 ± 0.13, 1.75 ± 0.30, and 1.60 ± 0.32 fold sham). Immunohistochemistry indicated that the major localization for these progression factors was in the tubulointerstitium, especially in the scar area, which is in agreement with the expression of a macrophage marker, F4/80. Therefore, these results indicate that in a mouse model of subtotal renal ablation, COX-2 inhibition blocks expression of mediators of renal tubulointerstitial injury.

Diabetic nephropathy: leveraging mouse genetics.

Purpose of reviewAdvances in mouse genetics have made this species particularly useful as a model for human disease. This review will summarize recent advances regarding the pathogenesis of diabetic nephropathy discovered in mice. Recent findingsDiabetic nephropathy has been characterized in novel genetic models of murine diabetes including the Akita, Ove26, and ICER-Iγ mice. Mutagenesis resources targeting every gene of the genome and the importance of inbred genetic background are discussed. SummaryThrough the use of these resources mouse models should provide new insight into the pathogenesis of diabetic nephropathy, and complement human studies and validate the identity of candidate genes contributing to diabetic nephropathy.