Международный эндокринологический журнал Том 19, №2, 2023
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Особливості іонорегулювальної функції нирок щурів у динаміці розвитку експериментального цукрового діабету
Авторы: О.А. Olenovych
Bukovinian State Medical University, Chernivtsi, Ukraine
Рубрики: Эндокринология
Разделы: Справочник специалиста
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Резюме
Метою дослідження було вивчити особливості іонорегулювальної функції нирок щурів у динаміці розвитку алоксан-індукованого експериментального цукрового діабету. Матеріали та методи. Дослідження проведені на 63 статевозрілих нелінійних самцях білих щурів, у 53 з яких викликали експериментальний цукровий діабет різної тривалості шляхом одноразового внутрішньоочеревинного введення розчину алоксану в дозі 160 мг/кг маси тіла, 10 інтактних щурів увійшли до контрольної групи. Через 10, 20, 25, 30, 40 та 45 діб після введення діабетогенної речовини тварин виводили з експерименту. Вивчення екскреторної функції нирок здійснювали кліренс-методом за умов водного індукованого 2-годинного діурезу з визначенням кліренсу ендогенного креатиніну, швидкості клубочкової фільтрації, концентрації іонів натрію та калію в пробах сечі та плазми крові й наступним розрахунком показників екскреції електролітів, інтенсивності їх фільтрації, абсолютної та відносної реабсорбції, концентраційного індексу, проксимального та дистального ниркового транспорту (у тому числі в перерахунку на 100 мкл клубочкового фільтрату). Результати. Іонорегулювальна функція нирок у щурів з алоксан-індукованим експериментальним діабетом характеризується інтенсифікацією натрій- та калійурезу на всіх стадіях експерименту. Збільшення втрат іонів натрію з кінцевою сечею на ранніх стадіях алоксан-індукованого експериментального діабету в першу чергу обумовлено клубочковою гіперфільтрацією з наступним зростанням фільтраційного навантаження нефрона натрієм. Втрата пропорційності між профільтрованою кількістю натрію та його проксимальною реабсорбцією викликає зменшення загального реабсорбційного потенціалу канальцевого відділу нефрона в динаміці розвитку алоксан-індукованого експериментального діабету. Це в першу чергу відображається на проксимальних канальцях, а в подальшому викликає функціональне послаблення сили канальцево-канальцевого зв’язку й відносну дисфункцію дистального відділу нефрона з наступним пригніченням альдостерон-залежних регуляторних механізмів. Висновки. Калійурична реакція діабетичної нирки може бути однією з ознак декомпенсації авторегуляції ниркового кровотоку за принципом канальцево-клубочкового зворотнього зв’язку, що є ініціюючим фактором дисфункції канальцевого апарату діабетичної нирки.
Background. The purpose of the research was to study the peculiarities of ionoregulatory renal function in the dynamics of alloxan-induced experimental diabetes mellitus. Materials and methods. The experiments were carried out on 63 white non-linear mature male rats, 53 with experimental diabetes mellitus of varying duration induced by intraperitoneal administration of alloxan in a dose of 160 mg/kg of body weight, and 10 intact rats (control group). Ten, 20, 25, 30, 40 and 45 days after administration of the diabetogenic substance, the animals were withdrawn from the experiment. Ionoregulatory function of the kidneys was studied by means of the clearance method under condition of water induced 2-hour diuresis to determine the clearance of endogenous creatinine, glomerular filtration rate, sodium and potassium content in the urine and blood plasma. It was followed by calculation of the electrolyte excretion, intensity of their filtration, absolute and relative reabsorption, their proximal and distal tubular transport (including standardized by glomerular filtrate volume). Results. The ionoregulatory function of the kidneys in rats with alloxan-induced experimental diabetes is characterized by the intensification of natriuresis and kaliuresis at all stages of the experiment. An increase in the urinary sodium loss in the early stages of alloxan-induced experimental diabetes is primarily stipulated by glomerular hyperfiltration, followed by an enhancement of filtration sodium load to the nephron. The loss of proportionality between the filtered amount of sodium and its proximal reabsorption causes a decrease in the total reabsorption potential of the tubular segment of the nephron in the dynamics of alloxan-induced experimental diabetes. It is reflected primarily on the proximal tubules, and subsequently induces a functional weakening of the tubule-tubular connection and relative dysfunction of the distal segment of the nephron with subsequent inhibition of aldosterone-dependent regulatory mechanisms. Conclusions. The kaliuric reaction of the diabetic kidney may serve as one of the signs of decompensation of the renal blood flow autoregulation by tubuloglomerular feedback, which is an initiating factor for the dysfunction of the tubular apparatus of diabetic kidney.
Ключевые слова
експериментальний цукровий діабет; алоксан; іонорегулювальна функція нирок
experimental diabetes mellitus; alloxan; ionoregulatory renal function
Introduction
Angiopathic complication of diabetes mellitus, diabetic kidney disease, is accompanied by damage of renal vessels in all parts of the nephron from glomerular capillaries and tubules to arterioles and arteries of all calibers. Therefore, it causes severe functional disorders including tubulopathies with the disturbance of reabsorption and osmoregulation processes, electrolyte disorders, and require a detailed study in the dynamics of diabetic renopathy progression [1].
These are disorders of the tubular transport of electrolytes affecting the processes of redistribution of tissue fluid, osmoregulation additionally leads to the changes in local hemodynamics in the kidneys, deterioration of nephron function and, subsequently, to structural changes in the kidney tissue, development of tubulointerstitial disorders and progressive impairment of kidney function [2, 3].
Therefore, the purpose of the research was to study the peculiarities of ionoregulatory renal function in the dynamics of alloxan-induced experimental diabetes mellitus (EDM).
Materials and methods
The experiments were carried out on 63 white non-linear mature male rats, 0.18–0.20 kg, kept under identical standard vivarium conditions. EDM was simulated by the intraperitoneal administration of alloxan monohydrate to 53 animals in diabetogenic dose of 160 mg/kg after the preceding 12-hour deprivation of food with preserved access to water ad libitum. Ten (group 1), 20 (group 2), 25 (group 3), 30 (group 4), 40 (group 5) and 45 days (group 6) after administration of the diabetogenic substance, the animals with EDM and 10 (intact) rats of the control group were withdrawn from the experiment. All the animals were loaded with water in the volume of 5 % of body weight, urine was collected for 2 hours, euthanasia was performed by decapitation under light ether anesthesia. The level of glucose in the blood samples, collected at the moment of animals’ decapitation, was determined considering only those with persistent hyperglycemia, exceeding 7.0 mmol/L.
Further analysis of urine samples, as well as blood plasma, enabled the assessment of the function of vascular-glomerular apparatus, proximal and distal tubular portions of the nephron by clearance method [4, 5]. Plasma and urine concentrations of sodium and potassium were detected by flame photometry with following calculation (considering water-induced 2-hour dieresis, clearance of endogenous creatinine and glomerular filtration rate) of excretory electrolytes fractions, intensity of their filtration, absolute and relative reabsorption, their proximal and distal tubular transport (including standardized by glomerular filtrate (GF) volume) [4, 5].
The data obtained were statistically processed with determination of the mean value and standard errors. The non-parametric Mann-Whitney rank test, provided by the software Statistica for Windows, v. 8.0 was used to assess the probability of difference between the studied groups [6].
The research was carried out in compliance with the provisions of the EU Directive No. 609 (1986) and the Order of the Ministry of Health of Ukraine No. 690 dated 09/23/2009 “On measures to further improve organizational norms for work with the use of experimental animals”.
Results
Analysis of the parameters of the ionoregulatory renal function in the dynamics of alloxan-induced experimental diabetes demonstrated the intensification of natriuresis already at the early stages of EDM (Table 1). On the 11th day of the experiment, the maximal urine loss of sodium was observed for the entire duration of the experiment. The urinary concentration of sodium ions in animals of this group exceeded the control index by 5.9 times, sodium excretion increased by 6.5 times, including standardized by GF (by 4.7 times), and the clearance of sodium ions in 11-day EDM 7-fold increased as compared with the control level. A significant increase in the filtration load of the nephron with sodium ions in the 11-day EDM group (by 1.3 times as compared with the control) caused an intensification of the absolute tubular transport of the electrolyte mainly in the initial segments of the nephrons. The proximal sodium transport in alloxan-diabetic rats 1.4 times increased in comparison with the control accompanied by practically unchanged distal transport of the cation. At the same time, a more detailed analysis of the transtubular transport of sodium ions in different sections of the renal tubules showed that scaled to the unit of the working nephron, its proximal reabsorption exceeded the control index by only 4.6 %, and standardized distal sodium reabsorption 27.8 % decreased as compared with the control level. A significant decline of relative sodium reabsorption in rats with 11-day EDM caused a reduction (by 6.1 %) of plasma sodium content and was accompanied by an almost 6-fold elevation of the sodium concentration index.
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Meanwhile, a kaliuric reaction developed in rats with 11-day EDM as well: urine concentration of potassium ions and its excretion increased significantly, 2.3 and 2.6 times exceeding those of the control animals. The ratio coefficient of sodium and potassium urine concentrations was found to be 2.5 times higher than that of the control level.
Sodium urinary loss on the 21st day of the experiment, despite a significant limitation in comparison with the level of natriuresis at the earlier stages of alloxan-induced diabetes, nevertheless, exceeded the control values. Sodium excretion including that standardized by the volume of GF, by the 21st day of the experiment decreased by 4.8 and 3 times, respectively, accompanied by a significant reduction of sodium urine level (by 2.2 times). However, the latter was found to be 2.7 times higher than that of the control level, and sodium excretion — absolute and standardized one — was 1.3 and 1.5 times higher, respectively.
Apparently, the mentioned sodium loss wasn’t caused by filtration overload of nephrons, which was reliably reduced (by 1.5 times) practically approaching to the control level with a corresponding decline of the absolute sodium tubular transport due to the proximal segment as well as due to distal tubular transport. On the 21st day of the experiment the proximal sodium reabsorption 1.5 times decreased coinciding with the level of control animals, and the distal transport of the cation was significantly reduced as compared with corresponding index of rats with 11-day alloxan diabetes as well as control level — 2.2 times. Regardless the fact that the relative sodium reabsorption increased significantly (by 0.13 %) to the control values from the 11th to the 21st day of the experiment, proximal and especially distal sodium reabsorption, standardized to the unit of the working nephron, reduced not only in comparison with the indices of the 11-day EDM (by 4.9 and 33.9 %, respectively) but with the control values (by 9.2 and 52.2 %, respectively) as well. Meanwhile, sodium clearance 4.5 times decreased, remaining 55.6 % higher than the control index. Due to the mentioned filtration-transport disorders, the concentration of sodium ions in the blood continued decreasing to the critical limit (by 6.5 % compared to the rate of rats with 11-day diabetes and by 12.2 % compared to the control level). It resulted in the decline of concentration sodium index by 2.1 times from the 11th to the 21st day of the experiment that remained almost three (2.8) times higher than the corresponding parameter of the control group.
The concentration of potassium ions in the urine of rats with 21-day EDM practically did not change from the 11th day of the experiment, while its excretion was found to be twice decreased, exceeding the control by only 22.4 %. This, in turn, almost brought the concentration coefficient of urine sodium and potassium ions to the level controls.
The ionoregulatory function of the kidneys of rats with 26-day diabetes was characterized by a sharp decline of the sodium urine excretion. The absolute sodium urine excretion on the 26th day of the experiment 1.3 times decreased as compared to that index on the 21st day of diabetes, practically reaching the control level. At the same time, cation excretion standardized by GF volume reached the lowest level for the entire period of the experiment on the 26th day after the administration of diabetogenic substance, decreasing almost six-fold in comparison to the index of 21-day diabetes and by 3.8 times compared to the controls. The lowest sodium urine concentration in rats with 26-day alloxan-induced diabetes (6.4 times less than the corresponding index at the early stages of EDM) balanced the concentration index of sodium ions, bringing it closer to the control values. Howe–ver, it did not lead to normalization of the content of sodium ions in the blood plasma, which, although slightly increased against the level of 21-day diabetes (by 6.6 %), but remained 11.4 % lower than the corresponding parameter of rats in the control group.
It was noteworthy that sodium filtration charge exceeded the control index by four times on the 26th day of the experiment, increasing by 4.7 times from the 21st day of alloxan diabetes with a corresponding enhancement of the absolute sodium tubular transport. The intensification of sodium tubular transport was mainly related to its proximal reabsorption, which exceeded the control values by 4.4 times, raising by 4.8 times from the 21st day of the experiment. Moreover, the standardization of proximal tubular reabsorption by the volume of GF also demonstrated a similar tendency: its increase by 8.7 % from the 21st to the 26th day after diabetes induction brought the mentioned index closer to the control level differing only by 6.6 % from the last one. At the same time, a significant intensification of sodium distal transport in the 26-day EDM (by 2.4 times as compared to the level the previous period of the experiment), calculated per unit of functioning nephron, revealed an opposite trend. A signi–ficant reduction of this parameter (by 1.7 times as compared to the level of rats with 21-day diabetes and by 3.9 times as compared to the control level) was registered. The level sodium relative reabsorption on the 26th day after alloxan administration turned out to be the highest for the entire period of the experiment, and sodium clearance, although slightly lowering concerning that index of 21-day alloxan-induced diabetes (by 26.7 %), remained, however, 22.2 % higher than the control parameter.
A sharp reduction of kaliuresis on the 26th day after the induction of EDM (the concentration of potassium ions in the urine of rats decreased by 28.8 % from the 21st day of the experiment, exceeding the corresponding index of the control group by 89.7 %) was accompanied by a significant elevation of potassium excretion (by 1.8 times in comparison to that in rats with 21-day diabetes and by 2.2 times as compared to the control value). It resulted in the 2.3-fold decline of the ratio coefficient of urine concentrations of sodium and potassium concerning that on 21st day of the experiment and twice-fold — against the control value.
Analysis of monovalent cations transport in different segments of renal tubules on the 31st and 41st days of allo–xan-induced diabetes revealed, mostly, unidirectional chan–ges. Both 31- and 41-day diabetes were associated with the significant intensification of absolute and standardized sodium excretion in comparison with the corresponding values on the 26th day of the experiment (by 3.1 and 2.5 times as for the absolute excretion and by 2.9 and 3.1 times as for the standardized sodium excretion). It exceeded the level of electrolyte excretion in control rats by 3.3 and 2.7 times, respectively. However, remaining 24.6 and 19.7 % less than the level of control as for the standardized by the volume of GF sodium excretion. Meanwhile, the urinary sodium concentration increased markedly — by 4.1 times on the 31st day of the experiment and by 3.7 times in its 41-day duration. Moreover, the plasma content of sodium ions also increased significantly (by 33.3 % in the 31-day diabetes and by 8.8 % on the 41st day of the experiment), improving the concentration index of the cation by 3.0 and 3.3 times, respectively.
Mentioned changes developed against the background of an increase in the filtration charge and absolute sodium transport, which on the 31st day of EDM turned out to be the highest throughout the entire experiment (5.3 times higher than the index of control animals), 3.7-fold exceeding the control parameter on the 41st day of diabetes. Meantime, despite a significant elevation of relative sodium reabsorption in rats with 31-day diabetes, which developed due to a significant intensification of its proximal and distal tubular transport (5.6- and 1.2-fold more than the control parameters, respectively), sodium clearance raised twice, exceeding the control index by 2.8 times. A more detailed analysis of the tubular sodium transport revealed a 34.0% increase of the standardized equivalent of its proximal reabsorption, exceeding the control parameter by 25.2 % with an almost unchanged level of distal sodium transport, which, nevertheless, remained 4.3 times less than that of controls when scaled to the unit of the working nephron.
Mentioned changes in transtubular sodium transport, although expressed to a lesser extent, persisted until the 41st day of the experiment: its proximal reabsorption only by 3.9 times exceeded the control index, decreasing by 30.0 % from the 31st day of the experiment and practically not differing from control level when standardized by the volume of GF. Distal sodium transport in rats with 41-day EDM was 24.5 % higher than the control level, although reducing by 27.5 % versus the rate of 31-day diabetes. However, standardized by the volume of GF, its equivalent showed a tendency to decline after the 31st day of the experiment (by 14.3 %) and was the lowest during the entire study period (5 times lower than the index of control rats). At the same time, sodium clearance did not differ from the corresponding parameter of rats with 31-day EDM and exceeded the control index by 2.9 times.
The concentration of potassium ions in urine on the 31st and 41st days of the experiment continued to decrease versus the level of 26-day diabetes (by 26.5 and 6.7 %, respectively). It was significantly higher than that of the control level (by 20.5 and 76.9 %, correspondingly), accompanied by a reduction of potassium excretion after the 26th day of the experiment (by 2.1 and 1.6 times until the 31st and 41st days of diabetes, respectively) practically to the control level. At the same time, the ratio coefficient of urine concentrations of sodium and potassium significantly increased in both 31- and 41-day alloxan diabetes (was 3.2 and 2.2 times higher than the control level, correspondingly).
Changes in the tubular transport of sodium and potassium ions on the 46th day of alloxan-induced EDM significantly differed in features and severity from ionoregulatory renal function disturbances in the earlier periods of the experiment.
Despite the limitation of sodium urine loss, which was found to be markedly reduced as compared to the level of 41-day alloxan diabetes and, at the same time, still slightly exceeding the control level the intensity of natremia continued to decrease after the 41st day of alloxan-induced EDM (by 8.4 %). It remained 11.7 % less than control level, and sodium concentration index, although twice-reduced versus that index at the previous stage of alloxan-induced diabetes, still remained 66.7 % higher than that of the controls. Both absolute and standardized sodium excretion continued to reduce in the dynamics of alloxan diabetes, remaining 74.6 and 45.9 % higher than the values of animals of the control group, respectively.
Filtration sodium fraction, as well as its absolute tubular transport, on the 46th day of EDM significantly differed from those values of animals with 41-day diabetes (decreased by 26.0 and 28.7 %, respectively), exceeding the control level by 2.7 and 2.6 times. However, in the absence of reliable changes in the relative sodium reabsorption after the 41st day of the experiment, its more detailed analysis revealed further limi–tation of the proximal sodium reabsorption, including that standardized by volume of GF (by 26.8 and 9.4 %, correspondingly). At the same time, the distal sodium transport in the 46-day EDM was found to be enhanced in absolute rates as well as standardized values as compared to its intensity on the 41st day of the experiment (by 29.7 and 61.1 %, respectively), however, not reaching the level of control animals. The level of sodium clearance while decreasing concerning that in 41-day diabetes, remained 2.1 times higher than the corresponding index of control animals. Urinary potassium excretion, its urine content and ratio coefficient of urine concentrations of sodium and potassium on the 46th day after induction of alloxan EDM continued to decrease versus the level of 41-day diabetes, approaching the control values.
Discussion
The revealed increase of sodium urine excretion in EDM of different duration results from both the hemodynamic-hyperperfusion nature of urination as well as local compensatory-adaptive mechanisms of autoregulation of renal activity. Thus, the intensification of urination and hyperfiltration, noted above for the 11th day of the experiment [7, 8], cause an augmentation of the filtration sodium load of the nephron with sodium ions, during which the charge on tubulocytes raises significantly due to the necessity of an enhanced reabsorption of the components of an excessive volume of ultrafiltrate [9, 10].
Overloading of the reabsorption transport systems in the proximal tubules, expected to reabsorb from 2/3 to 3/4 of filtrated fluid and equal amount of sodium [3], probably causes the total cation loss found in the early period of EDM. Scaled to the unit of the working nephron, the standardized sodium excretion in the 11- and 21-day EDM also maximally exceeds the control values, testifying to the structural integrity of the proximal tubules.
Weakening of the glomerular-tubular balance causes the flow of large volumes of hypernatrium intratubular fluid from the proximal tubules to the loop of Henle and distal segments of the nephron that is consequently expected to result in the augmentation of distal sodium reabsorption by tubular-tubular feedback mechanism. However, against the background of significant enhancement of sodium reabsorption in both proximal and distal tubules, scaled to the unit of the functioning nephron, it is found to be reduced, mainly in the distal segments. Therefore, under conditions of overload by the filtrate, despite the very strong reabsorption capabilities of the tubules for sodium ions, the proximal sodium reabsorption by a single nephron is limited. In its turn, it causes the maximal tension of the tubular-tubular connection and the relative inability of the distal tubules to provide the retention of those sodium ions that avoided proximal tubular reabsorption. As a result, hyponatremia develops. With preserved autoregulation of water-electrolyte homeostasis, the development of hyponatremia should cause sodium retention and, accordingly, reduce its excretion with urine [3]. Hyponatremia, detected on the 46th day of EDM, is accompanied, conversely, by an elevation of the urinary sodium concentration, which creates the impression of suppression of aldosterone-dependent regulatory mechanisms. An augmentation of kaliuria under these conditions can serve as a sign of decompensation of autoregulation of renal blood flow by tubuloglomerular feedback, contributing to the further progression of renal dysfunctions against the background of diabetes.
Conclusions
Ionoregulatory function of the kidneys in rats with alloxan-induced experimental diabetes is characterized by the intensification of natriuresis and kaliuresis at all stages of the experiment. The increase in urinary sodium loss in the early stages of alloxan-induced experimental diabetes is primarily stipulated by glomerular hyperfiltration, followed by an enhancement of filtration sodium load to the nephron. The loss of proportionality between the filtered amount of sodium and its proximal reabsorption causes a decrease in the total reabsorption potential of the tubular segment of the nephron in the dynamics of alloxan-induced experimental diabetes. It is reflected primarily on the proximal tubules, and subsequently induces a functional weakening of the tubule-tubular connection and relative dysfunction of the distal segment of the nephron with subsequent inhibition of aldosterone-dependent regulatory mechanisms. The kaliuric reaction of the diabetic kidney may serve as one of the signs of decompensation of the renal blood flow autoregulation by tubular-glomerular feedback, which is an initiating factor for the dysfunction of the tubular apparatus of the diabetic kidney.
Received 03.02.2023
Revised 10.03.2023
Accepted 14.03.2023
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