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Alterations in renal cytosolic phospholipase A₂ and cyclooxygenases in polycystic kidney disease ¹

HAROLD M. AUKEMA^*,,,¶2, JENNIFER ADOLPHE^*, SUPARNA MISHRA^,¶, JIEYUAN JIANG^*, FRANCIS P. CUOZZO^,¶ and MALCOLM R. OGBORN^*,

^* Department of Human Nutritional Sciences and
Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada, R3T 2N2, and
Department of Nutrition and Food Sciences and
^¶ Department of Biology, Texas Woman’s University, Denton, Texas, USA

²Correspondence: H506 Duff Roblin Bldg., Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada, R3T 2N2. E-mail: Aukema{at}UManitoba.CA

SPECIFIC AIMS

We have previously reported that the level of the arachidonic acid rich phospholipid, phosphatidylinositol, is reduced in cystic kidneys from mice with polycystic kidney disease (PKD). Levels of lyso phosphatidylinositol after short-term isotope labeling are elevated in diseased kidneys, indicating that the lower levels of phosphatidylinositol observed in these kidneys may be due to increased phospholipase A (PLA) activity. We therefore hypothesized that levels of cytosolic PLA₂ (cPLA₂) would be elevated in PKD kidneys. When our initial findings in PKD mice supported this, we further hypothesized that the next enzymes in the eicosanoid metabolic pathway, cyclooxygenase-1 (COX-1) and COX-2, would be elevated. The aim of the current study was to test these hypotheses using the CD1-pcy/pcy (pcy) mouse in which decline in renal function begins in adulthood and the Han:SPRD-cy rat in which decline in renal function occurs during the growth phase.

PRINCIPAL FINDINGS

1. Levels of renal cPLA₂ in the diseased mice and rats were as much as 131% higher than normal in kidneys from animals with more advanced renal disease
The results of immunoblotting to determine cPLA₂ levels in polycystic kidneys demonstrate that this enzyme is present in cytosolic and particulate fractions in PKD mice and rats (Fig. 1 ). In the mouse kidney cytosolic fraction, there were significantly higher levels of cPLA₂ in the 180-day male (93% higher, P=0.0091) and female (106% higher, P=0.0050) mice with PKD. Levels of cPLA₂ were higher in the diseased female mice at 120 (87% higher, P=0.0012) and 60 (86% higher, P=0.0080; Fig. 1 ) days of age. In the mouse particulate fraction, there were significantly higher amounts of cPLA₂ in the diseased kidneys at 180 days of age in male (127% higher, P=0.0008) and female (131% higher, P=0.0115) mice, at 120 days of age in male (67% higher, P=0.0341) and female mice (98% higher, P=0.0197), and at 60 days in the female mice (38% higher, P=0.0461).

View larger version (37K): [in this window] [in a new window]   Figure 1. Representative immunoblots of normal and diseased a) 180-day-old male mouse and b) 70-day-old rat kidney particulate fractions. N, normal kidney; P, diseased kidney.

The alterations in cPLA₂ levels observed in pcy mice were detected in male but not female Han:SPRD-cy polycystic rats, consistent with the more rapid disease progression in male PKD rats. In the rat kidney cytosolic and particulate fractions, significant increases of cPLA₂ were found in kidneys of 70-day-old male animals vs. normals (39% higher, P=0.0031 and 110% higher, P<0.001, Fig. 1 , respectively).

Cytosolic PLA₂, is implicated in several pathophysiological processes in the kidney, as evidenced by alterations in renal disorders such as diabetic nephropathy, glomerulonephritis, and ischemic injury. Under resting conditions, cPLA₂ is present primarily in the cytosol and translocates to the perinuclear region when cells are activated. However, prolonged activation of cPLA₂ by increased [Ca²⁺] for an extended period results in continued membrane localization of the enzyme. If an equilibrium between cytosolic and membrane-bound forms of cPLA₂ is maintained in the cell, increased translocation may result in overall increased production and/or stabilization of this enzyme, resulting in higher levels in cytosolic and particulate fractions, as observed in the more diseased PKD kidneys in this study.

2. Similar to the cPLA₂ data, there were higher levels of COX-1 in the diseased mouse kidneys compared with the normals in older animals
PLA₂ catalyzes the hydrolysis of fatty acid from the sn-2 position of phospholipids, and COX enzymes convert 20-carbon fatty acids such as arachidonic acid into the prostaglandin precursor prostaglandin H₂. These enzymes are rate-limiting steps in the prostaglandin synthesis pathway. Kidney COX-1 was detected only in the particulate fractions of the kidney in both rodent models. In 180-day-old mice, COX-1 levels were 87% higher in males (P=0.0209, Fig. 1 ) and 86% higher in females (P=0.004). In Han:SPRD-cy rats, COX-1 levels were 120% higher (P=0.0080) in male 70-day-old diseased kidneys compared with normals (Fig. 1) .

3. In contrast to cPLA₂ and COX-1, the level of COX-2 immunoreactive protein was lower in diseased rat kidneys compared with controls
In rats with PKD, COX-2 levels were 58% lower (P=0.0028) in diseased kidneys compared with normals in 70-day-old males (Fig. 1) . COX-2 null mice develop several renal abnormalities, including severely underdeveloped kidneys with few functional nephrons, immature small glomeruli, and an abundance of underdeveloped mesenchymal tissue. Tissue-specific and time-dependent expression of COX-2 is required for normal postnatal kidney development and maintaining normal renal architecture and function. Higher levels of COX-2 are found in the embryonic kidney and peak again shortly after birth, with adult levels being very low. Our studies were consistent with these findings, as COX-2 was detected in the particulate fraction in kidneys of growing rats, but not in the mice that have almost reached adult body weight by 60 days of age.

Lower COX-2 levels may be related to some of the earliest manifestations of renal disease in PKD, namely, the inability to concentrate urine and the development of hypertension associated with altered renin-angiotensin activity. Hypertonicity results in increased COX-2 in the medullary interstitial cells, so an inability to concentrate urine is consistent with lower COX-2 levels. COX-2 protein is also found in cells of the thick ascending limb and macula densa in the cortex of rat kidneys and is involved in renin secretion as well as the response to dietary salt intake. COX-2 inhibition is associated with decreased renin levels and activity and there is evidence of negative regulation of cortical COX-2 by angiotensin II, which is elevated in the Han:SPRD-cy rat.

4. Cytosolic PLA₂ appears to be associated with COX-1, but not COX-2 in rat kidneys
cPLA₂ and COX-1 were higher in similar amounts in diseased kidneys in rats and mice. Hence, the cPLA₂/COX-1 ratio was not significantly different in normal vs. diseased kidneys in any of the animals in any age group or gender. On the other hand, COX-1/COX-2 and cPLA₂/COX-2 ratios were four- to fivefold lower in diseased kidneys compared with normals in 70-day-old male of rats; there was no change in these ratios in rats with lesser disease progression: females and 28-day-old rats (Fig. 2 ).

View larger version (38K): [in this window] [in a new window]   Figure 2. Enzyme ratios in normal and polycystic 70-day-old male Han:SPRD-cy rat kidney particulate fractions. **P < 0.01, significantly different from normals.

Coordinatedregulation of specific COX isoforms with cPLA₂ suggests that fatty acid metabolism by these pathways can occur independently. Cytosolic PLA₂ is functionally coupled to COX-1 and COX-2 in COS-1 cells and embryonic kidney 293 cells, specifically coordinated with COX-2 in human synovial cells and in macrophages, and coordinated with COX-1 in glomeruli isolated from rats with anti-Thy-1 glomerulonephropathy. The results of the present study indicate a coupling between cPLA₂ and COX-1 in PKD, as COX-1 and cPLA₂ levels are both higher in diseased kidneys from older models whereas COX-2 levels are lower.

CONCLUSIONS

These studies demonstrate that the levels of cPLA₂ and COX-1 are elevated in diseased kidneys in two rodent models of PKD, whereas COX-2 levels are lower in diseased rat kidneys compared with normals. In general, these alterations are greater as the disease progresses, with few changes being detected in the early stages of the disease. The kidney is a relatively rich source of eicosanoids that regulate renal processes such as hemodynamics, water and solute transport, and renin secretion in the normal kidney. In the diseased kidney, eicosanoids appear to play a role in maintaining GFR and are involved in inflammatory processes in response to renal injury. In some types of renal disease, a reduction in eicosanoid formation is associated with amelioration of the disease process; in others, eicosanoids appear to have a protective effect.

The finding that animals with PKD have altered levels of COX-1 and COX-2 in the latter stages of the disease leads to questions regarding treatment with nonsteroidal anti-inflammatory drugs and selective COX inhibitors. Much interest in COX-2 selective inhibitors stems from their putative ability to relieve pain and inflammation while sparing the gastrointestinal tract. Further reducing the lowered COX-2 levels in PKD may reduce the elevated renin levels and hypertension in PKD and could be beneficial. However, the effect of blocking only COX-2 on the PKD kidney has not yet been tested, and results from studies of other renal disorders and in renal cells provide conflicting results on the benefit of this treatment, warranting caution in the use of these drugs at this time. COX-2 inhibitors can impair renal development and may be associated with neonatal end-stage renal failure when the fetus is exposed to these inhibitors. Because the PKD kidney has some immature characteristics and lower COX-2 levels than normal, further reduction of COX-2 may worsen the disease.

The differences in these models with respect to COX-2 levels may help explain why diets enriched in n-3 fatty acids have a more favorable effect on disease progression in the Han:SPRD-cy rat than in the pcy mouse, as COX-2 metabolizes a wider range of fatty acids as substrates and oxygenates eicosapentaenoic acid and -linolenic acid more efficiently than COX-1. The effects of specific dietary fatty acid therapy or selective COX inhibitors in PKD, therefore, need to be elucidated before either can be routinely used in individuals with this disorder.

View larger version (9K): [in this window] [in a new window]   Figure 3. Renal cPLA2 and cyclooxygenase levels in PKD models of early, rapid (Han:SPRD-cy) and late, slow (CD1-pcy/pcy) progression of renal injury. cPLA2 appears to be coupled to COX-1 in these kidneys. Diet and specific COX inhibitors potentially alter the level and type of specific eicosanoids produced in the kidney.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0460fje; to cite this article, use FASEB J. (December 17, 2002) 10.1096/fj.02-0460fje

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