REDUCED ACTIVITIES OF DIVALENT CATION ACTIVATED ATP-ASE AND 5′ -NUCLEOTIDASE IN GLYCEROL INDUCED ACUTE RENAL FAILURE

• 5′-Nucleotidase
• ATPase
• Acute renal failure

Reduced renal blood flow and glomerular filtration rate are the common findings in mioglobinuric acute renal failure (ARF). On the other side, adenosine plays an important role in regulation of renal blood flow, glomerular filtration rate and activity of 5′-nucleotidase. Ecto-ATPases (Ca²⁺ and Mg²⁺) hydrolyzing extracellular nucleotide tri-and/or diphosphates with 5′-nucleotidase seem to represent the major source of extracellular adenosine in the kidney [[1]]. Experimentally glycerol-induced ARF is also followed by generation of reactive oxygen species, especially with release of hydrogen peroxide [[2]]. Phenolic plant flavonoid – quercetin (3,3′,4′,5,7-pentahydroxyflavon) has been established as a strong antioxidative agent that have protective effects on kidney in ischemic renal injury [[3]] and hydrogen peroxide induced apoptosis [[4]]. However, it is well known, that quercetine inhibit activity of many enzymes including ATPases. The present study was undertaken to evaluate changes of divalent cation (Ca²⁺ and Mg²⁺) activated ATPase and 5′-nucleotidase during the course of experimentally glycerol-induced ARF. At the same time, protective effects of quercetine were tested.

Acute renal failure was induced in male Sprague–Dawley rats (weighing 200–280 g.) with injection of glycerol (8 mL/kg 50% solution) in hindlimb muscle. The animals were dehydrated 24 h prior to injection, and sacrificed 48h later. Quercetine was injected intraperitoneally the day before (15 mg), and on the day of receiving glycerol (10 mg). ARF was characterized by elevated plasma levels of urea and creatinine.

Enzyme activities were determined in 10% kidney cortex homogenates upon addition of 3 mM of ATP in the medium containing 30 mM Tris-HCl, 130 mM NaCl, 3 mM MgCl₂, 5.5 mM glucose for Mg²⁺-ATPase. Ca²⁺-ATPase was measured in the same medium containing 5 mM CaCl₂ instead MgCl₂. Activity of 5′-nucleotidase was determined upon addition of 3 mM AMP in the same medium as for Mg²⁺-ATPase. The amount of liberated phosphate was measured according to the method of Gomory [[5]]. Tissue protein was estimated by the method of Lowry [[6]].

These results indicated reduced activity of all ectoenzymes that degrade adenine nucleotides to adenosine. Activity of Ca²⁺-ATPase was reduced for 41.51percnt;, Mg²⁺ for 39.68percnt; and 5′-nucleotidase for 19.4percnt; (Table 1). This is in accordance with decreased Mg²⁺-ATPase activity, demonstrated in isolated nephron segments (medullary thick ascending limb), in model of severe acute tubular necrosis induced by glycerol [[7]].

Table 1. Activities of Investigated Enzymes

Group	N	5′-Nucleotidase nmol min^−1 mg prot^−1	Ca^2+-ATPase nmol min^−1 mg prot^−1	Mg^2+-ATPase nmol min^−1 mg prot^−1
Control	6	45.95 ± 5.29	64.71 ± 12.65	72.01 ± 3.72
Glycerol	7	37.04 ± 6.95^a	37.85 ± 10.62^b	43.43 ± 5.86^c
Quercetin	7	40.01 ± 4.05	61.62 ± 6.76	69.03 ± 5.45
Quercetin + Glycerol	7	39.26 ± 5.03	40.16 ± 7.21^b	47.05 ± 7.25^c

Values are means ± SD.

^avs control p < 0.05

^bvs control p < 0.001

^cvs control p < 0.01

During the initiation phase of glycerol induced ARF, Trifillis [[8]] reported decrease in renal adenine nucleotide pools, as a consequence of intracellular phosphate trapping. Furthermore, Zager [[9]] clearly demonstrated, that myoglobinuria itself promote a time dependent reduction in total adenine nucleotide pool (65 minutes after infusion of myoglobin), without affecting renal blood flow. Considering these observations, it could be concluded that reduced activities of Ca²⁺, Mg²⁺-ATPase and 5′-nucleotidase are the consequence of decreased renal adenine nucleotides pool.

Beneficial effect of quercetin on impaired renal function was demonstrated by attenuated level of urea and creatinine in the group receiving quercetin together with glycerol (Table 2).

Table 2. Urine and Blood Creatinine in Control and Treated Rats

Group	N	Urea (mmol/l)	Creatinine (μmol/l)
Control	6	7.7 ± 1.18	63.23 ± 20.78
Glycerol	7	88.82 ± 21.034-5^a, b	625.08 ± 183.044-5^a, b
Quercetin	7	6.74 ± 0.44	46.95 ± 10.51
Quercetin + Glycerol	7	61.00 ± 6.644-6^a, b, c	402.85 ± 108.344-6^a, b, c

Values are means ± SD.

^avs control p < 0.001

^bvs quercetin p < 0.001

^cvs glycerol p < 0.01

At low concentration, quercetin was reported to inhibit mitochondria ATPase [[10]] and decrease sperm motility through inhibition of Ca²⁺-ATPase [[11]] In this model of myoglobinuric acute renal failure we did not find any inhibition of divalent cation activated ATPases by this flavonoid.