Background: diabetes mellitus (DM) is characterized by an increased risk of developing chronic microvascular complications, including diabetic nephropathy (DN). Blood flow through the microvascular bed is largely determined by the red blood cells (RBC) rheological properties. The association and prognostic significance of impaired RBC rheological properties in the development of microvascular complications of type 1 diabetes mellitus (DM1) were the object of this study. Aim: to study the association and prognostic significance of impaired RBC rheological properties in patients with DM1 with a decrease in glomerular filtration rate (GFR) during the DN progression. Materials and Methods: 52 patients with DM1 were included in the study. The DN was determined based on the GFR value, which was calculated using the GFR-EPI formula, ml/min/1.73 m2. The blood rheological properties (RBC ability to aggregate and deform) were studied on venous blood samples of patients using a coaxial cylinder aggregometer-deformometer. Blood viscosity was determined using a capillary viscometer. Results: in DM1, hyperagregation of RBC is observed already in the early stages of chronic kidney disease (CKD). In GFR<90 ml/min/1.73 m2 versus GFR>90 ml/min/1.73 m2, there was a reduction in the time of the first aggregation stage of RBC (T1) to 7.6 [6.8; 9.1] and 6.9 [6; 8.4], respectively, (p=0.029) and an increase in the aggregation rate of kTb to 0.22 [0.18; 0.25] and 0.25 [0.19; 0.27], respectively (p=0.04). During an increase in albuminuria above 100 mg/day, there was an increase in the stability of RBC aggregates (I2,5) -22.4 [-24.7; -20.25] versus -20 [-21; -17] (p=0.02), and in albuminuria above 150 mg/day, there was a decrease in the RBC ability to deform ((.)begun) (s-1) 20.34 [14; 30] versus 37.7 [15.9; 97.2] (p=0.01). Conclusion: indicators of RBC hyperaggregation (the first stage time and speed of RBC aggregation, stability of the largest RBC aggregates) in DM1 are predictors of the early decrease in GFR and the development of grade 2–3a CKD. KEYWORDS: type 1 diabetes mellitus, RBC rheological properties, diabetic nephropathy, RBC aggregation, RBC deformity, chronic kidney disease. FOR CITATION: Fabrichnova A.A., Misnikova I.V., Kovaleva Yu.A., Glazkov A.A., Koshelev V.B. Disorders of the red blood cells rheological properties in diabetic nephropathy in patients with type 1 diabetes mellitus. Russian Medical Inquiry. 2024;8(9):513–517 (in Russ.). DOI: 10.32364/2587-6821-2024-8-9-2

Введение. Представление о патологической агрегации эритроцитов (АЭ) складывалось на основании ее изучения в отсутствие течения, способного влиять на этот процесс. Цель исследования: сопоставление процессов АЭ и дезагрегации эритроцитов (ДЭ) в покое и в потоке у больных сахарным диабетом (СД). Материалы и методы. Исследовали пробы венозной крови при СД 1-го типа и у здоровых лиц. Деформируемость эритроцитов контролировали методом эктацитометрии. Кинетику АЭ и ДЭ изучали путем измерения интенсивности обратного светорассеяния с помощью ротационного агрегометра двумя способами, определяя (1) стандартные значения АЭ в покое и сдвиговой ДЭ и (2) соотношение интенсивности АЭ и ДЭ в потоке при равном, но разнонаправленном изменении его скорости – скорость сдвига γ̇~ (4–80) с–1. Микроциркуляцию в коже руки пациентов тестировали методом лазерной доплеровской флоуметрии. Результаты. У здоровых лиц по мере ускорения потока доминирование АЭ сменялось доминированием ДЭ, в основном при пороговой скорости сдвига γ̇п≤ ~18 с–1; возрастное ухудшение деформируемости эритроцитов и усиление АЭ, выявляемое стандартным способом, наблюдали лишь в подгруппе с γ̇п> ~18 с–1. При СД имелись стандартные признаки усиленной АЭ, увеличенные значения γ̇пв потоке, и по мере ускорения потока нередко наблюдалось хаотическое чередование доминирования АЭ и ДЭ. Кроме пациентов с аномалиями АЭ, проявлявшимися при обоих способах ее тестирования, имелась группа больных (~20%), у которых интенсификация АЭ регистрировалась только при стандартном тестировании, но не в потоке. Эти пациенты характеризовалась улучшенными показателями микроциркуляции, особенно в условиях гипоперфузии. Заключение. Таким образом, при СД стандартные признаки усиленной АЭ могли сочетаться с нормальным соотношением АЭ и ДЭ в потоке. Introduction. The concept of abnormal erythrocyte aggregation (EA) was developed from studying EA in the absence of flow that may impact this process. Aim: to compare the processes of EA and erythrocyte disaggregation (ED) at rest and in flow in patients with diabetes mellitus (DM). Materials and Methods. The study analyzed venous blood samples from patients with type 1 DM and clinically healthy subjects. Erythrocyte deformability was monitored using ectacytometry. The kinetics of EA and ED was measured by backscattering utilizing laser-assisted rotational aggregometer in 2 ways to determine: (1) the standard characteristics of EA at rest and shear ED, and (2) the ratio of EA and ED intensities in flow with an equal but oppositely directed change in its speed – shear rate γ ~(4–80) s–1. Microcirculation in the skin of the patients’ hands was assessed using laser Doppler flowmetry. Results. During flow acceleration in healthy subjects, ED dominance replaced EA dominance, particularly at a γth≤ ~18 s–1 threshold; age-related deterioration of erythrocyte deformability and augmentation of EA were only observed in the subgroup with γth> ~18 s–1, as detected by standard methods. Patients with DM exhibited typical signs of augmented EA at rest and increased values of γthin flow. In addition to normal replacing of EA dominance by ED dominance, as the flow accelerated, their chaotic rotation was often observed. At the same time, in addition to the group of patients with the EA abnormalities regardless of the testing method, approximately 20% of the patients displayed EA intensification that was recorded only by standard test and not in flow. This group exhibited enhanced microcirculation, particularly under conditions of hypoperfusion. Conclusion. Thus, in DM, the typical signs of enhanced EA could be combined with a normal ratio of EA and ED in flow.

We have reviewed current understanding of ischemic brain damage and the main therapeutic approaches. Pathological factors affecting the survival of neurons and glial cells in the focus of ischemia are outlined: depolarization, cytotoxic and vasogenic edema, calcium overload, excitotoxicity, inflammation, free radical damage. Effective and rapid reperfusion significantly improves patient’s survival and functional outcomes, but other approaches to brain infarction treatment did not approve their effectiveness in large clinical trials. Dozens of drugs (neuroprotectors) are being studied in order to compensate isolated pathological brain ischemia pathways and to increase cellular survival, but they were ineffective in large clinical trials.The reason for the ineffectiveness of neuroprotective drugs may be a lack of understanding of the drug targets real importance. Many drugs that have shown promising results in preclinical studies have not been studied in large clinical trials until now. Additional pathogenetic mechanisms revealed in the last decade expand our knowledge about the brain infarction and may become promising directions for the development of new therapeutic approaches.

Objective. Measurement and comparison of the aggregation and disaggregation forces of individual erythrocytes during the formation or breakdown of a paired aggregate in vitro in the blood of patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) using the optical tweezers method. Materials and methods. A total of 50 people were included in the study. Of these, 10 people with T1DM (aged 28±15.8), 26 people with T2DM (aged 66±13). The control group consisted of 14 apparently healthy volunteers (46±21 years old). Measurements of the forces of pair interaction of erythrocytes in a diluted suspension were carried out in vitro by the method of dual-channel optical tweezers. The force of aggregation of erythrocytes FA (pN) and the force of disaggregation FD (pN) were measured and their ratio FD/FA was calculated.Results. The erythrocyte aggregation forces in the T1DM group did not differ significantly from the control group. However, the forces of disaggregation in the T1DM group were significantly lower than in the control group (p<0,05). The ratio of the forces of disaggregation to the forces of aggregation was lower in the T1DM group compared to the control group (p<0.005). In T2DM group, erythrocyte aggregation forces were higher compared to the control group (p<0.005). At the same time, the ratios of the forces of disaggregation to the forces of aggregation in T1DM group and T2DM group did not differ.Conclusion. Both T1DM group and T2DM group are characterized by hyperaggregation of erythrocytes. However, given the data obtained, it can be assumed that the mechanisms of such hyperaggregation are different.

The influence of a hydrogen sulfide donor NaHS (2×10-5-10-3 M) on the rat erythrocyte deformability was analyzed by laser diffractometry. NaHS (6×10-5 M) increased, while NaHS (10-3 M) reduced erythrocyte deformability. The effect of NaHS (6×10-5 M) was similar to that of NO donor sodium nitroprusside (SNP, 10-7 M). However, simultaneous use of NaHS (6×10-5 M) and SNP induced less pronounced changes in erythrocyte deformability than their individual application. It is likely H2S, similar to NO, is involved in the regulation of erythrocyte deformability in the microvascular bed.

BACKGROUND: Diabetes mellitus leads to disruption of the skin repair processes, but the leading mechanisms of this pathology have not yet been identified. In this regard, in our work, we decided to check how hyperglycaemia affects the process of keratinocyte phenotype changes during wound healing. AIMS: To study the effect of hyperglycaemia on wound healing and differentiation of keratinocytes in a rat streptozotocin-induced diabetes model. MATERIALS AND METHODS: Diabetes mellitus was induced in rats by using streptozotocin, 65 mg / kg, intraperitoneally, once. The wound was applied in the supra-scapular region on the 42nd day, after which (after 8, 16, and 24 days) the repair process was evaluated using histological methods. Immunohistochemistry was used to evaluate the expression of cytokeratin-10 and cytokeratin-17. RESULTS: In rats with diabetes mellitus, wound healing slowed down in the later stages, compared with the control group. In general, wound healing was accompanied by an increase in the expression of cytokeratin-10 in its region compared with intact skin, and contractile keratinocytes activation was disrupted in diabetic rat wounds. CONCLUSIONS: Hyperglycaemia slightly slows wound healing in rats and impairs contractile keratinocytes activation.

Background : Diabetes mellitus (DM) has a negative impact on all organs. This is due to insufficiency of blood supply and the disruption of the trophic function of the nervous system. One of the most serious complication of DM is diabetic foot caused be vascular and neurological reasons. Correction of vascular disorders is effectively treated by modern therapeutic approaches, but the damage of nervous system has been studied insufficiently. Aims : To investigate the dynamics of damage to the vegetative nervous system on the laboratory model of DM. Materials and methods : DM in rats was induced by injection of streptozotocin at a dose of 65 mg/kg in citrate buffer (DM group). The control group of rats received a citrate buffer equivalent (CB group). Rats with DM were given a maintenance therapy with insulin in a dose of 2 units/kg/day. On 42 days of experience, a round wound with a diameter of 2 cm on the back of the animals was observed. Before the DM simulation, then on the 42, 50, 58 and 66 days of its development, an electrocardiogram (ECG) was recorded in the rats at a frequency of 2 kHz digitising in a state of calm wakefulness and after cold exposure. For 5 minutes ECG fragments, heart rate and heart rate variability (HRV) in the temporal domain were calculated, characterising: 1) the total heart rate variability (tHRV) according to SDRR, SDHR, KVRR and KVHR; 2) the effect of the parasympathetic department of the autonomic nervous system (aANS) for RMSSD and pNN3; 3) the contribution of the sympathetic department of the ANS (sANS) by SDAvgRR, SDAvgHR. The spectral parameters were estimated in the frequency domain: the total power of the spectrum is TR (range: 0–2.5 Hz), the powers in the low and high frequency ranges are LF (range: 0.2–0.8 Hz) and HF (range: 0.8–2.5 Hz) LF/HF. Weekly, the tail withdrawal time was measured in a temperature pain test (55°C). Results : During the development of diabetes, the level of glucose in the blood increased 4–7 times compared with the normal level. The reaction time of the pain test in rats with DM increased by 20%–30% at the end of the experiment. At 42 days, the development of bradycardia (267 beats/min) was observed in rats with DM. The indicators of tHRV decreased by a factor of 2 due to a decrease in the contribution of sANS. The reaction to CP in the SD group differs from the norm by the severity of the individual components of the HRV structure, which indicates functional denervation of the heart and the development of diabetic neuropathy. Conclusions : As the diabetes progressed, signs of neuropathy were observed. The overall HRV parameters decreased, the ratio of the contributions of sANS and pANS to the regulation of heart rate changed, and the temperature sensitivity decreased.

Цель исследования: изучение агрегации эритроцитов, реализующейся в сдвиговом потоке. Материалы и методы. В пробах крови здоровых добровольцев методом регистрации обратного светорассеяния с использованием коаксиально-цилиндрического агрегометра определяли стандартные гемореологические показатели, а также параметры, характеризующие «гистерезис» — разницу в суспензионном состоянии крови при данной скорости сдвига, но разной предыстории течения (его замедлении или ускорении). Результаты. Результаты позволили определить оптимальный показатель «гистерезиса» и охарактеризовать обратимую агрегацию эритроцитов в сдвиговом потоке комплексным образом. Заключение. Предложенный показатель «гистерезиса» позволяет выявлять ослабление и усиление агрегации эритроцитов в норме; однако требуется дополнительное исследование его эффективности при патологии, сопряженной с гемореологическими нарушениями. Aim: to study erythrocytes aggregation realized in a shear flow. Materials and methods. We registered standard hemorheological parameters in healthy volunteers under shear flow conditions using a light scattering concentric cylindrical aggregometer. Also characteristics of «hysteresis» were tested: erythrocytes rates. Results. This comparison revealed «hysteresis» parameters that correlated with low-grade erythrocytes aggregation or with «hyperaggregation» state. Conclusion. The revealed «hysteresis» parameters disclose a balance between erythrocytes aggregation and disaggregation in shear fl ow, but an extension clinical study is required to verify if such analysis would allow for detection of patients with anomalously high erythrocytes aggregation.

Objective. The role of ATP-dependent potassium (K+ ATP) channels in the neuroprotective effect of ischemic (IPre) and pharmacological (PPre) preconditioning and changes in blood levels of nitric oxide (NO) metabolites were studied in conditions of cerebral ischemia. Materials and methods. Ischemic stroke (IS) was modeled in male rats (n = 86) by electrocoagulation of a branch of the middle cerebral artery (MCA). The nonselective K+ ATP channel blocker glibenclamide and the K+ ATP channel activator diazoxide were used. IPre and PPre were performed one day before MCA occlusion. Blood concentrations of NO, nitrates (NO3 –) and nitrites (NO2 –) were determined in experimental animals at 5, 24, and 72 h after MCA occlusion. Results. IPre decreased the lesion zone by 37% (p < 0.05), while prior administration of glibenclamide countered the action of IPre. The protective effect of PPre was analogous to that of IPre. Decreases in blood levels of oxygenated R-conformers of hemoglobin-bound NO (Hb-NO) were seen 5 h after MCA occlusion, with an inversely proportional increase in the concentration of nonoxygenated T-conformers; there were also increases in NO3 – and NO2 – concentrations. NO3 – and NO2 – levels showed normalization by one day after MCA occlusion, along with changes in the concentrations of Hb-NO complexes – R-conformers dominated, while the blood level of T-conformers reached a minimum. Furthermore, by 24 h there was a correlation between blockade of K+ ATP channels and decreases in serum NO3 – and NO2 – levels (p < 0.03). Conclusions. The neuroprotective effect of preconditioning was due to activation of K+ ATP channels. Analysis of blood levels of NO metabolites in rats with IS showed that Hb-NO complexes in the R-conformation stored and carried NO to the tissues, releasing NO on occurrence of the R → T transition in ischemic conditions.

Preconditioning of ischemic and hypoxic type was investigated as a method of protecting brain against acute ischemic injury. The preconditioning methods were applied to experimental rats 24 h before the time when local brain infarct was done by middle cerebral artery occlusion (MCAO). It was found that the ischemic and hypoxic preconditioning resulted in three general morphological changes: 1) the size of infarct zone was reduced by 2.2–3.8 times compared with rats that had not been treated with the preconditioning before MCAO; 2) the preconditioning treatment retained the number of living neurons in penumbra at the level of control rats, while without the preconditioning neuronal count in the penumbra after MCAO was 29% lower; 3) the number of glial cells in penumbra was increased after MCAO by 38% compared with the control level, and continued to increase under the preconditioning treatment up to 60%, that suggests an important role of neuroglia in neuroprotection. Selective blockers of ATP2dependant K+2channels (52hydroxydecanoate and glibenclamide) completely abolished the neuroprotective effects of the preconditioning.

The effect of peptide Semax on remodeling of cardiac sympathetic innervation was examined in rats with experimental myocardial infarction. In 28 days after ischemia/reperfusion injury, Semax diminished the growth of sympathetic innervation of ventricular septum, although it produced no effect on the density of β1 and β2 adrenoceptors.

Activation of the sympathetic nervous system aggravates the course of myocardial infarction. Semax peptide moderated the degree of this activation and prevented the increase in the density of sympathetic endings in rat caudal artery in 28 days after ischemia or ischemia/reperfusion. The peptide reduced the density of α-adrenoreceptors in the caudal artery of rats with myocardial infarction. Semax produced no effect on β-adrenoreceptors in both experimental models. The experiments on isolated segments of the caudal artery revealed reduced vascular responsiveness to electrical stimulation and norepinephrine infusion in rats treated with Semax after ischemia/reperfusion injury.

AIMTo study a role of ATP-dependent potassium channels (K+ATP) in the neuroprotective effect of ischemic (IP) and pharmacological (PP) preconditioning and evaluate the dynamics of blood nitric oxide (NO) metabolites in cerebral ischemia.MATERIAL AND METHODSA model of ischemic stroke induced by the electrocoagulation of a middle cerebral artery (MCA) branch was used in male rats (n=86). Glibenclamide, a selective inhibitor of ATP-sensitive K+ channels, and diazoxide, a potassium channel activator, were used. IP and PP were performed 24 h before MCA occlusion. Blood concentrations of NO, NO3- and NO2-were measured 5, 24 and 72 h after occlusion.RESULTSIP decreased a lesion area by 37% (p<0/05) and the preliminary introduction ofglibenclamide levelled the effect of IP. A protective effect of PP was similar to that of IP. A decrease in oxygenated R-conformers of Hb-NO and a reverse increase in non-oxygenated T-conformers as well as NO3- и NO2-were noted 5h after MCA occlusion. In the first 24 h after MCA occlusion, contents of NO3- and NO2- returned to normal values. There were changes in the concentrations of Hb-NO complexes as well, with the predominance of R-conformers and minimal contents of T-conformers. Moreover, the correlations between K+ATP channel blockade and the decrease in serum NO3- and NO2 were found (p<0/03).CONCLUSIONThe neuroprotective effect of preconditioning is caused by the activation of K+ATP channels. An analysis of NO metabolite concentrations in the blood of rats with IP suggests that Hb-NO complexes belonging to R-conformers deposit and carry NO in tissues releasing NO accumulated via R→T transfer in conditions of ischemia.