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UDC 615.252.349.7:612.461.25:616.613-003.7
EFFECTS OF DRUGS INCREASING INSULIN SENSITIVITY ON PLASMA LEVEL OF URIC ACID AND URINE ACIDITY IN RATS DURING EXPERIMENTAL URATE NEPHROPLITHIASIS
Altai State Medical University, Barnaul
V.Yu. Perfilyev, Ya.F. Zverev, A.Yu. Zharikov, V.M. Bryukhanov, D.Yu. Lukyanenko, I.V. Lysenko
The purpose of this study is to evaluate the influence of metformin, dapagliflozin and pioglitazone on the urine pH level and plasma level of uric acid during preventive and therapeutic drug administration in experimental urate nephrolithiasis. Subjects and methods. To form urate nephrolithiasis we used earlier repeated classic model of uricase inhibition that causes hyperuricemia in rats. We used metformin, dapagliflozin and pioglitazone as drugs increasing insulin sensitivity. At the end of the experiment we measured the urine pH level and UA level in the blood of rats after their decapitation. Results. Long-term preventive administration of metformin and pioglitazone during experimental urate nephrolithiasis allows to increase the urine pH level. Dapagliflozin causes the reduction of the urine pH level regardless of the type of drug administration. Both types of metformin, pioglitazone and dapagliflozin administration to experimental animals significantly reduce plasma level of uric acid in.
Key words: metformin, dapagliflozin, pioglitazone, experimental urate nephrolithiasis, prevention, therapy.
Urate nephrolithiasis is a pathological process developing as a result of a purine metabolism disorder leading to hyperuricemia and hyperuricosuria with uric acid (UA) crystals depositing in the kidneys. At the same time the chemical nature of urate concrements allows them to fully dissolute under the influence of certain physicochemical factors [1]. Pharmacological modulation of such factors can contribute to effective and safe litholysis of urate calculi therefore it is of great current interest.
Today there is no denying the fact that urate nephrolithiasis is related to the metabolic syndrome (MS) [2]. According to the statistics urate nephrolithiasis develops in 21.9% of MS patients and in 4.1% of patients without MS [3]. When studying specific features of the pathogenesis of MS it was found that there is insulin-dependent urine acidification that triggers urate calculi formation. Moreover, it is known that one of the main factors contributing to the formation of urate microlites is urine acidification and hyperuricemia [4].
Taking the above into consideration, we suggested that administration of drugs increasing insulin sensitivity can lead to alleviation of factors being conductive to urate calculi formation.
Objective: the purpose of this study is to evaluate the influence of metformin, dapagliflozin and pioglitazone on the urine pH level and plasma level of uric acid during preventive and therapeutic drug administration in experimental urate nephrolithiasis.
Materials and methods
We studied 102 male Wistar rats weighing 220310 g. The animals were placed in single cages adapted for urine collection. Laboratory animal care was organized according to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1986).
To form urate nephrolithiasis we used earlier repeated classic model of uricase inhibition that causes hyperuricemia in rats [5].
We used metformin, dapagliflozin and pioglita-zone as drugs increasing insulin sensitivity.
Synthetic antidiabetic drug metformin (bigua-nide derivative) increases insulin sensitivity by increasing affinity of insulin receptors, by their conformational changes and by stimulation of the pathway at the receptor and post-receptor level [6, 7].
Dapagliflozin, a representative of a new class of oral sodium-glucose co-transporter 2 inhibitors, compensatively increases sensitivity of insulin receptors in association with reduction of renal glucose reabsorption by inhibiting SGLT2 in epithelial cells of renal tubules [7].
Pioglitazone, a thiazolidinedione derivative, increases the number of insulin receptors on the cell membrane by activating PPAR-y nuclear receptors [7, 8].
Experimental animals were provided with two types of drug administration: preventive and therapeutic. All the drugs were administered intragas-trically in the form of 2% starch mucilage.
The daily doses (Table 1) were calculated according to the interspecies scaling formula with reference to human therapeutic doses [9].
During studying of the potential of each of the drugs animals were divided into 3 groups. The control group was provided with 20 g of standard laboratory nutritional mixture including 500 mg/kg of oxonium acid and 1000 mg/kg of UA daily during 3 weeks. The second group («Prevention» group) was also provided with preventive oral drug administration during the same period of time. Animals from the third group («Therapy» group) were provided with the drug in the same dose from 11th to 21st day of the experiment.
At the end of the experiment we measured the urine pH level and UA level in the blood of rats after their decapitation.
Table 1
Doses of drugs changing insulin sensitivity
Substance Human daily dose, mg/kg Rat daily dose, mg/kg
Metformin 12.5 150.0
Dapagliflozin 0.125 0.7
Pioglitazone 0.375 2.2
For statistical analysis we used Microsoft Office Excel 2003 (Microsoft Corporation, USA) and Sig-ma-Stat 3.5 (Systat Software Inc., USA). The Krus-kal-Wallis test by ranks was employed for comparison between the three groups. The nonparametric Mann-Whitney U test was used for post hoc pair-wise comparison. All values are specified as a median and an interquartile range: Me (25%; 75%). A calculated difference of p < 0.05 was considered statistically significant [10].
Results and discussion
The experiment showed that both preventive and therapeutic drug administration of metformin significantly improves the course of urate nephro-lithiasis. It appeared that together with long-term drug administration there also took place the reduction of plasma level of UA: in the control group it was 1.4 (1.2; 1.8) mg/dl, in the "Prevention" group - 1.1 (0.8; 1.2) mg/dl (p = 0.009 in comparison with the control group), in the "Therapy" group -1.0 (0.7; 1.2) mg/dl (p = 0.013 in comparison with the control group).
As a result of long-term administration of dapagliflozin plasma level of uric acid significantly lowered in the "Prevention" group and in the "Therapy" group in comparison with the results in the control group by the end of the follow-up period: 1.3 (1.1; 1.8) mg/dl in the control group, 0.8 (0.7; 1.0) mg/dl (p = 0.006 in comparison with the control group) in the "Prevention" group, 0.7 (0.6; 0.8) mg/dl (p = 0.005 in comparison with the control group) in the "Therapy" group.
Both preventive and therapeutic administration of pioglitazone to animals with experimental urate nephrolithiasis caused the reduction of plasma level of UA in experimental animals in comparison with the control group: 1.3 (1.1; 1.5) mg/dl in the control group, 1.1 (0.9; 1.1) mg/dl (p = 0.007 in comparison with the control group) in the "Prevention" group, 0.9 (0.8; 1.1) mg/dl (p = 0.002 in comparison with the control group) in the "Therapy" group.
The obtained results prove that metformin, da-pagliflozin and pioglitazone do not only interfere into glycometabolism but also influence other types of metabolism including purine metabolism [11].
Table 2
Urine pH levels in experimental urate nephrolithiasis rats
Group Metformin Dapagliflozin Pioglitazone
Control 6.8 (6.5; 6.9) 6.8 (6.5; 6.9) 6.8 (6.6; 7.4)
Prevention 7.9 (7.5; 8.2) 6.1 (5.9; 6.2) 7.8 (7.5; 8.6)
Therapy 7.0 (6.6; 7.4) 5.9 (5.3; 6.6) 7.5 (7.2; 8.0)
Kruskal-Wallis test
P 0.003 0.003 0.011
Mann-Whitney U test
p, "C" and "P" 0.002 <0.001 0.013
p, "C" and "T" 0.102 0.037 0.058
p, "P" and "T" 0.030 0.732 0.064
Note: p - level of statistical significance. "C" and "P" - the control group in relation to the "Prevention" group; "C" and "T" - the control group in relation to the "Therapy" group; "P" and "T" - the "Prevention" group in relation to the "Therapy" group.
Taking into consideration the importance of pH level changes in the pathogenesis of urate nephrolithiasis it should be noted that metformin and pioglitazone caused the urine alkaline pH-shift in comparison with the control group, which was significant in the "Prevention" group and tended to appear in the "Therapy" group (Table 2).
As noted above, insulin resistance plays an important role in urinary acidification. It is known that by activating its own receptors in the renal tubules insulin stimulates 3 Na+/H+ transferase isoform which provides direct transportation of NH3 to the renal tubular lumen [12]. Thus, insulin resistance leads to the reduction of ammonium production and secretion - processes contributing
to urinary acidification. Apparently, by increasing insulin sensitivity of insulin receptors in the renal tubules metformin and pioglitazone make 3 Na+/ H+ transferase isoform active and consequently activate transportation of NH3 to the renal tubular lumen leading to the increasing of the urine pH level that must be admitted as a positive effect.
However, it should be noted that both preventive and therapeutic dapagliflozin administration (as it appears from Table 2) leaded to the certain urine acidic pH-shift in comparison with the control group: 10% pH-shift in the "Prevention" group, 13% pH-shift in the "Therapy" group. This result that seems to be unexpected can be explained in the following way. It is known that dapagliflozin administration results in reduction of sodium and glucose co-reabsorption in the renal tubules caused by direct renal inhibiting of glucose co-transporter SGLT2. It leads to the increase in the level of sodium ions in the proximal renal tubules and to the compensatory activation of mechanisms regulating their reabsorption in more distal parts of the renal tubules. It is known that a Na+/H+ transferase plays an important role in the regulation of urine acidity. It is located on the apical membrane of an epithelial cell in the descending portion of the proximal tubules and it provides reabsorption of sodium ions into the cell in exchange for protons, which appear in the urine as a result of carbonic acid disintegration [13]. When the level of sodium ions in the liquid from the upper portions inside the tubule increases the transferase starts to function more actively and in spite of the working ammonium buffer it leads to the urine acidic pH-shift. It is not improbable that the reduction of the urine pH level is the result of systemic euglycemic ketoacidosis that can develop in conditions of selective SGLT2 inhibition in response to dapagliflozin [14].
Conclusion
Long-term preventive administration of met-formin and pioglitazone during experimental urate nephrolithiasis allows to increase the urine pH level and this is an important positive effect. Da-pagliflozin causes the reduction of the urine pH level regardless of the type of drug administration. Both types of metformin, pioglitazone and dapagli-flozin administration to experimental animals significantly reduce plasma level of uric acid in rats and it is an important positive feature irrespective of the urine pH level.
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Contacts:
Corresponding author - Perfilyev Vyacheslav Yur-yevich, Lecturer of the Department of pharmacology of the FSBEI HE Altai State Medical University of the Ministry of Health of the Russian Federation, Barnaul.
656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 241859. Email: 1991PS@mail.ru
