Alvaro A. Reyes

The expression of mRNA for tumour necrosis factor-α increases in the obstructed kidney of rats soon after unilateral ureteral ligation

Summary: Cytokines, including transforming growth factor (TGF)‐β1, contribute to the tubulointerstitial fibrosis of ureteral obstruction. Tumour necrosis factor (TNF)‐α, a proinflammatory cytokine produced by multiple cells including macrophages and resident renal cells, has a role in inflammatory cell recruitment in glomerular injury. We measured TNF‐α mRNA in the renal cortex of rats at different times after the onset of unilateral ureteral obstruction (UUO) and determined whether angiotensin II (AngII) inhibition or total body irradiation affects the mRNA levels of TNF‐α. Rats were killed at 1, 2, 4, 24, 72 and 120h after UUO. Levels of TNF‐α mRNA increased significantly in the obstructed kidney at 1h (X 2), 2h (X 2.7), 4h (X 3.6), 24h (X 2.7), 72h (X 1.8) and 120h (X 2.8) after ureteral ligation when compared to the contralateral kidney of the same animals or to control (normal) kidneys. Tumour necrosis factor‐α mRNA increased in renal cortical tubules but not in glomeruli. Treatment with enalapril, an angiotensin‐converting enzyme (ACE) inhibitor, before and after UUO decreased TNF‐α mRNA levels in the obstructed kidney by about 40% at 4h after the onset of UUO, but at 120h there was no difference in TNF‐α levels in the obstructed kidney of treated and untreated animals. Total body irradiation, which depletes macrophages in the obstructed kidney, did not prevent the upregulation of TNF‐α mRNA expression at 4 h after UUO. Thus, TNF‐α may have a role in initiating tubulointerstitial injury in the obstructed kidney. Leucocytes infiltrating the renal interstitium of the obstructed kidney do not appear to contribute to the increased mRNA expression of TNF‐α. Angiotensin II may contribute, at least in part, to the early increased expression of TNF‐α mRNA in the obstructed kidney.

Osteopontin Expression in the Kidney during Unilateral Ureteral Obstruction

Osteopontin is a bone protein also expressed in other tissues. Increased osteopontin is thought to be associated with tissue inflammation. We used immunocytochemical analyses and polymerase chain reaction amplification of mRNA to examine osteopontin expression and regulation in unilateral ureteral obstruction (UUO) in rats, a model of inflammatory kidney disease. In the obstructed kidney, osteopontin mRNA and protein were significantly increased. The increase reached 4-fold after 1 day of UUO and persisted at this level for the 5-day duration of UUO. Immunocytochemical analyses showed increased osteopontin protein in tubular cells of the obstructed kidney cortex from days 1 through 5 of UUO. No such significant increase was apparent in the glomerulus or interstitium. Increased osteopontin mRNA and protein likewise occurred in the tubular cells of the obstructed kidney of rats that had undergone whole-body irradiation to eliminate macrophage infiltration into the experimental kidney. Purified osteopontin was found to be a chemoattractant for macrophages isolated from the rat peritoneum. Enalapril treatment, which decreases macrophage infiltration of the obstructed kidney, had no effect on the increase in osteopontin mRNA but significantly attenuated the increase in protein in tubular cells. Western blot analysis of whole cortical homogenates revealed that the osteopontin antibody recognized one protein of 67 kD. The amount of this protein was substantially decreased in kidney homogenates obtained from enalapril-treated compared to untreated animals. Increased osteopontin synthesis may, therefore, contribute in part to the inflammatory response that characterizes obstructive nephropathy.

Role of Vasopressin in Rats with Bilateral Ureteral Obstruction

Abstract After unilateral release of bilateral ureteral obstruction (BUO), there is a significant increase in renal vasoconstriction that accounts for the marked decrease in glomerular filtration rate and effective renal plasma flow seen in this setting. We examined the potential role of antidiuretic hormone (ADH), a vasoconstrictor of the renal circulation, on renal hemodynamics in female Sprague-Dawley rats with BUO of 24-hr duration. Rats with BUO had significantly higher plasma values of ADH (65.1 ± 12.2 vs 12.1 ± 4.1 pg/ml), sodium (145.4 ± 0.91 vs 138.6 ± 1.06 mEq/liter), and osmolality (375.6 ± 2.0 vs 310.1 ± 3.6 mOsm/kg) than sham-operated rats. Rats with BUO pretreated with enalapril, an angiotensin-converting enzyme inhibitor, before obstruction had somewhat higher, but not significantly different, plasma values for ADH (84.6 ± 20.8 pg/ml) than rats with BUO not given enalapril. Rats with unilateral ureteral obstruction of 24-hr duration had plasma levels of ADH (8.2 ± 1.3) not different from those in sham-operated rats. Rats with BUO pretreated with a specific antagonist of the V1-type receptor for ADH had significantly greater values for the glomerular filtration rate (2.31 ± 0.24 vs 1.44 ± 0.08 ml/min/kg body wt) and the effective renal plasma flow (8.95 ± 0.71 vs 3.81 ± 0.44 ml/min/kg body wt) and significantly lower values for mean arterial pressure (140.3 ± 2.0 vs 159.1 ± 5.5 mm Hg) than did BUO rats not given the antagonist. The results indicate that high levels of ADH play an important role in the decrease in the glomerular filtration rate and effective renal plasma flow observed in rats with BUO of 24 hr. The significant increase in ADH levels after BUO of 24-hr duration may be due to an increase in osmotic stimulation as a consequence of hypernatremia.

A High Cholesterol Diet Ameliorates Renal Tubular Lesions in Diabetic Rats

Abstract These studies examine the effect of cholesterol feeding in normal rats and in rats with streptozotocin-induced diabetes mellitus. Four groups were studied: normal rats fed either a standard rat chow or a standard rat chow supplemented with cholesterol and diabetic rats fed standard chow or standard chow plus cholesterol. Diabetic rats fed a standard diet excreted more creatinine and urea in the urine, had higher levels of blood urea nitrogen, and lower serum albumin levels than rats fed standard diet plus cholesterol. Blood glucose levels were similar in the two groups; however, diabetic rats given cholesterol had a greater body weight at the end of the study than diabetic rats eating standard chow. Urine volumes and sodium and potassium excretion in the urine were greater in diabetic rats fed a standard diet than in those fed a high cholesterol diet. Diabetic rats fed a standard diet had distinctive renal lesions characterized by swelling of tubular epithelial cells with clearing of cytoplasm. The nephron segments involved by this striking vacuolar change were the distal convoluted tubule and the thick limbs of Henles loop. These lesions were identical to those described by Armanni-Ebstein in severely glycosuric patients. These lesions were not observed in any of the animals of the other three groups (including diabetic rats fed a high cholesterol diet). Glomeruli were normal in animals of all groups. Thus, cholesterol administration prevents the development of the Armanni-Ebstein lesions in diabetic rats despite persistent hyperglycemia. The mechanism by which cholesterol administration prevents the accumulation of glycogen in distal tubule cells has not been elucidated. It is suggested that glycogen accumulation in distal tubular segments may explain the greater urine volumes, natriuresis, kaliuresis, and proteinuria observed in diabetic animals fed a standard diet when compared with rats fed the same diet plus cholesterol.

DIETARY SUPPLEMENTATION OF L-ARGININE AMELIORATES RENAL HYPERTROPHY IN RATS FED A HIGH-PROTEIN DIET

Abstract Ingestion of a high-protein diet or intravenous administration of amino acids is associated with an increase in glomerular filtration rate (GFR). It can also lead to renal hypertrophy, and, if sustained, may cause glomerular sclerosis. L-Arginine administration ameliorates the progression of renal disease in rats with subtotal nephrectomy and prevents the increase in GFR observed in rats with experimental diabetes. The present study examines the potential effect(s) of L-arginine administration (1%) in the drinking water on the renal hypertrophy that occurs in rats fed a high-protein diet for 1 month. Four groups of female Sprague-Dawley rats, six in each group, were studied (95 ± 1 g). Groups 1 and 2 were fed a low-protein diet (12% casein, 0.504% L-arginine); Group 1 was given tap water, whereas Group 2 was given tap water supplemented with L-arginine. Groups 3 and 4 were fed a high-protein diet (40% casein, 1.68% L-arginine); Group 3 was given tap water, whereas Group 4 was given tap water supplemented with L-arglnine. The rats had free access to food and water during the study period. The kidney weight and the kidney to body weight ratio of rats of Group 3 were significantly greater than in the other groups of rats. Renal hypertrophy was prevented in the rats of Group 4. The excretion of orotic acid in the urine, an index of L-arginine deficiency, was significantly greater in rats of Group 3 than in rats of Group 4. Thus, the renal hypertrophy that occurs in rats fed a high-protein diet was decreased in rats given L-arginine supplementation in the drinking water. This effect was associated with less excretion of orotic acid in the urine in rats given L-arginine. A relative deficiency of L-arginine may occur during high-protein feeding that may shunt nitrogen metabolism from the urea cycle to the orotic acid pathway.

Role of platelet-activating factor in renal function in normal rats and rats with bilateral ureteral obstruction.

Abstract Platelet-activating factor (PAF) is a powerful vasodilator with important effects on kidney function. It has been suggested that the renal effects of PAF are mediated by thromboxane A2 (TxA2). We examined the effect of PAF on renal function in sham-operated rats and rats that had undergone unilateral release of bilateral ureteral obstruction (BUO) of 24-hr duration, a condition in which the synthesis of TxA2 is increased. To eliminate systemic hemodynamic changes, PAF was infused directly into the left renal artery using the lowest dose that affected renal function (2.3 × 10–13 moles/min). Infusion of PAF significantly decreased the glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) in normal rats and rats with BUO. Normal (shamoperated) rats pretreated with an inhibitor of TxA2 synthesis also had a significant decrease in GFR after administration of PAF (ERPF also decreased, but not significantly). Rats with BUO pretreated with an inhibitor of TxA2 synthesis had significantly greater basal GFR and ERPF (increases of 72 and 171%, respectively) than untreated BUO rats. Administration of PAF to the former group further increased GFR and ERPF (by 37 and 39%, respectively; P < 0.001). The role of endogenous PAF was evaluated by administering a specific PAF receptor antagonist. Sham-operated rats pretreated with high doses of the PAF receptor antagonist had significantly higher mean arterial pressure values than normal untreated rats, and had no decrease in GFR and ERPF during PAF infusion. Rats with BUO pretreated with the PAF antagonist had a significant, dose-dependent decrease in basal GFR and ERPF. These data suggest that endogenous PAF has a vasodilatory role in obstructive nephropathy. No significant differences in eicos-anoid excretion in the urine corrected per GFR were observed during infusion of PAF in any of the groups examined. In BUO rats with intact TxA2 synthesis, exogenous administration of PAF decreased renal function, presumably through further increases in the production of TxA2. However, when TxA2 production was inhibited, PAF administration increased GFR and ERPF, presumably due to its unopposed vasodilatory properties. The data suggest an important role of PAF in the hemodynamic changes seen in obstructive nephropathy.

Cytochrome P-450 pathway in renal function of normal rats and rats with bilateral ureteral obstruction.

Abstract Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) are decreased and mean arterial pressure (MAP) and renal vascular resistance (RVR) are increased after unilateral release of bilateral ureteral obstruction (BUO) of 24 hr duration. An imbalance between vasoconstrictor and vasodilator substances may explain these hemodynamic changes. We examined the role of the cytochrome P-450 pathway in this setting. After unilateral release of BUO, GFR and ERPF (ml/min/kg body wt) were significantly lower in these rats than in sham-operated rats (SOR) 1.14 ± 0.09 vs 6.7 ± 0.5 and 3.09 ± 0.2 vs 23.5 ± 3.4, respectively). BUO rats had significantly higher MAP (mm Hg) and RVR (mm Hg/ml/min/kg body wt) than SOR (155 ± 5 vs 120 ± 1 and 29.1 ± 1.7 vs 3.2 ± 0.4, respectively). SOR given 3-methylcholanthrene and β-naphthoflavone to induce the cytochrome P-450 system had no significant changes in renal function, RVR, or MAP. SOR given ketoconazole to inhibit the cytochrome P-450 system had significantly lower GFR (4.8 ± 0.5) than temporal control rats without significant changes in ERPF (21.2 ± 4.6), MAP (127 ± 6), or RVR (4.2 ± 0.9). Rats with BUO given ketoconazole had lower but not significantly different GFR (0.84 ± .1) and ERPF (2.61 ± .4) than BUO controls. Values for MAP did not differ in BUO rats given ketoconazole versus BUO temporal controls. BUO rats given 3-methylcholanthrene and β-naphthoflavone had significantly higher GFR and ERPF (2.01 ± 0.24 and 6.66 ± 1.36, respectively) and significantly lower RVR (14.7 ± 3.9) than control rats with BUO; MAP was unchanged. Microsomal preparations from indomethacin-treated isolated kidneys obtained from BUO rats when compared with preparations obtained from SOR had significantly less activity of the P-450 cytochrome-dependent ω/ω-1 hydroxylase (103 ± 6 vs 130 ± 7 pmol hydroxyeicosatetraenoic acids produced per mg of protein/min, P < 0.02) and the P-450 cytochrome-dependent epoxygenase (11 ± 0.3 vs 30 ± 4 pmol lipoxyeicosatrienoic acids produced per mg of protein/min, P < 0.04). Indomethacin-treated microsomes prepared from kidneys of BUO rats converted significantly less 14C-arachidonic acid through the P-450-dependent hydroxylases (13.5 ± 0.8 vs 17.0 ± 0.1% of 14C-arachidonic acid converted to 19- and 20-hydroxyeicosatetraenoic acids, P < 0.02), and significantly less through the epoxygenases (1.4 ± 0.4 vs. 3.8 ± 0.5% of 14C-arachidonic acid converted to epoxyeicosatrienoic acids). The data suggest that a metabolite(s) of the cytochrome P-450 pathway modifies renal function in vivo in such a way that inhibition or activation of the cytochrome P-450 system is reflected in decreases or increases in GFR and ERPF in BUO rats without affecting mean arterial pressure. We conclude that the cytochrome P-450 pathway plays an important role in renal hemodynamics of normal rats and rats with BUO of 24 hr duration. [P.S.E.B.M. 1992, Vol 201]