Microrheology,microcirculation and structural compensatory mechanisms of a chronic kidney disease rat model. A preliminary study

Abstract

BACKGROUND:

Chronic kidney disease (CKD) models are known to study pathophysiology and various treatment methods. Renal dysfunction could influence erythrocytes through several pathways. However, hemorheological and microcirculatory relation of CKD models are not completely studied yet.

OBJECTIVE:

To evaluate erythrocyte micro-rheology, microcirculatory and structural compensatory mechanisms in a rat model of CKD.

METHODS:

Female Sprague-Dawley rats were subjected to nephrectomy group (NG, n = 6) or sham-operated group (SG, n = 6). NG rats were subjected to 5/6 nephrectomy in two stages. In SG no intervention was made on kidneys. Hemorheological and hematological measurements were carried out after each stage, and 5 weeks after the last operation. Histological and microcirculatory studies were done on the remaining kidney and compared with sham rats.

RESULTS:

Serum creatinine increased in NG (p = 0.008), accompanied with decrease of red blood cell count (p = 0.028) and hemoglobin (p = 0.015). Erythrocyte aggregation parameters slightly increased in NG, while the elongation index didn’t show significant changes. Microcirculation was intact in the remnant kidney of NG. However, in comparison with SG, the diameter of glomeruli increased significantly (p < 0.01).

CONCLUSIONS:

Erythrocyte mass was influenced more than micro-rheological properties in this model. The main compensation mechanism was rather structural than at microcirculatory level.

Keywords

Chronic renal failure hemorheology hematology erythrocytes rat model

1 Introduction

Chronic kidney disease (CKD) is a widespread systemic disease and the associated pathological changes have been the focus of many researchers to learn more about this disease and how to manage it. Many mechanisms accompanying CKD could influence endothelial function and the erythrocytes. Beside the homeostasis, acid-base balance and excretion of waste of metabolic processes, kidney functions as endocrine organ too, secrets hormones that are crucial like erythropoietin for hematopoiesis, renin to regulate blood pressure, and calcitriol to regulate mineralization of bones and calcium-phosphate homeostasis [1, 2]. Other mechanisms can be from uremic toxins which is retained and accumulated as a result of CKD in which it causes short life span of erythrocytes, iron deficiency, and inflammation [3 –5].

It is known that erythrocyte mass decreases significantly in patients suffering from CKD, while studies showed conflicting results about the behavior of erythrocytes as erythrocyte deformability [6]. These conflicts are not limited to the effects of CKD but also involve the effect of its management on the micro-rheological properties of the erythrocytes [6], and our study came as a part of one of our department’s projects to evaluate the effects of CKD and its management on the hemorheology and microcirculation.

Rodents are common CKD models [7]. The chemical models in rats gets higher intensity of cardiovascular lesion and calcium-phosphate homeostasis problems may be due to infiltration of parathyroid glands [8, 9], and the surgical models as well have surgical complication as bleeding and infection [9 –11].

The aim of this study was to study the hemorheological and hematological effects of a known 5/6 nephrectomy model. We also intended to evaluate the compensation of the remaining part of the kidney by studying the microcirculatory and structural changes, since the hormonal changes in kidney disease could lead to changes in the glomerular microcirculation, as well as changes in the function and mass of the intact glomerulus [12 –14].

2 Materials and methods

2.1 Experimental animals

The approved experiments were registered by the University of Debrecen Committee of Animal Welfare (Permission registration Nr.: 24/2016/UDCAW and 25/2016/UDCAW) in accordance with the Hungarian Animal Protection Act Law XXVIII/1998, EU Directive 2010/63/EU and the Ordinance 40/2013.

Twelve healthy female Sprague-Dawley rats were used in this study at 10–12 weeks of age, six in sham-operated group (SG, 265±25 g) and six in nephrectomy groups (NG, 257.57±7.23 g). The rats were acclimated for two weeks and housed in standard cages with water and food ad libitum. The rats were anesthetized using sodium-thiopenthal (40 mg/kg, i.p., Thiobarbital 0.5 g, B. Braun Medical S.A., Spain), Atropine (0.05 mg/kg, s.c.) was administered before the surgery and Flunixin (2.5 mg/kg, s.c.) was administered subcutaneous after each procedure.

2.2 Operative techniques

The 5/6 nephrectomy was done in two stages, and the interval between the stages was 2 weeks. At the first stage 2/6 nephrectomy was performed through anterior intraperitoneal approach, and at the second stage the 5/6 nephrectomy was completed through retroperitoneal approach.

In detail, the anterior intraperitoneal approach at first stage was achieved using a midline incision for 2-3 cm under the xiphoid process. The left kidney was prepared without elevating it from its bed, and all the fat tissue around the kidney was cut beside the kidney using fine scissors with special attention to the tissue above the kidney, because the adrenal glands locate there. After the full dissection of the left kidney, the lower and upper poles of the left kidney were ligated perpendicular to the kidney using Vicryl thread 2/0. After a few minutes the color of the kidney was checked, to check the viability of the middle third of the kidney. Finally, the abdomen was closed in two layers using a 4-0 glycolide-ɛ-caprolactone (Monolac, Chirmax, Prague, Czech Republic).

After two weeks, and if the wound of the abdomen had healed well, the right retroperitoneal approach was done using a 1 cm incision parallel and right to the spine and directly below the ribs. The superficial muscle layer was cut, and the right kidney appeared here into the fat pad. At this time, the left kidney was pulled out and freed from the fat tissue, the renal hilum was ligated in block twice and the kidney excised.

The same procedures were applied on the sham rats, but without lost any part of the kidney, i.e., no ligations were done at the first stage and the right kidney wasn’t excised at the second stage.

2.3 Follow-up protocol

Each rat was followed-up for 5 weeks, the period was determined after literature review to determine the required period of serum creatinine to reach the value of the plateau, and it was 5 weeks approximately.

The measurements were carried out before the first stage (first stage measurements), before the second stage (second stage measurements) and at the end of the experiments (fifth week measurements). As described below, the measurements involved hemorheological and hematological test each time, the microcirculation of the kidney was assessed in the fifth week measurements. The histologic samples were taken at the end of the experiments.

2.4 Blood sampling

Blood samples were drawn at the first stage from the inferior vena cava through the midline incision and at the second stage through the lateral tail veins without using any tourniquet. At the end-point measurements, 5 weeks, the blood samples were drawn from the inferior vena cava.

About 300 μl of blood was drawn at each time using heparinized syringe, and the blood was put into BD Vacutainer^® tubes (5.4 g K₃-EDTA, 3 ml).

2.5 Laboratory measurements

Hematological parameters were studied using (Sysmex F-800 automate, Sysmex Co., Ltd. Japan). The required amount of blood for these measurements is 70 μl, and the studied parameters involved: white blood cell count (WBC, 10³/μl), red blood cell count (RBC, 10⁶/μl), hemoglobin (Hgb, g/dl), hematocrit (Hct, %).

Erythrocyte deformability was studied using a LoRRca MaxSis Osmoscan ektacytometer (Mechatronics BV, The Netherlands). The required amount of blood is 10 μl mixed in 2 ml of polyvinylpyrrolidone (PVP) – phosphate buffered saline (PBS) solution (viscosity: 27 mPas, osmolarity: 300 mOsm/kg, pH: ∼7.3). The erythrocyte deformability is studied by measuring the elongation under increasing shear stresses (SS) from 0.3 to 30 Pa. The elongation was measured by analyzing the laser diffraction pattern resulting from a laser beam transversing the suspension. The software calculates and presents the data as elongation index (EI) – shear stress curves (SS). The values of the elongation index are proportional to the erythrocyte deformability [15]. The maximal erythrocyte elongation index (EI_max) and the shear stress required for half-maximal deformation (SS_1/2 [Pa]) were calculated using Lineweaver-Burke analysis and used to present and compare erythrocyte deformability [16].

Erythrocyte aggregation was measured using a Myrenne MA-1 erythrocyte aggregometer, (Myrenne GmbH, Germany), based on light-transmittance method. Measurements require about 20 μl of blood. It disperses the erythrocytes by applying high shear rate (600 s^–1) for 10 seconds, and then the transmitted light is integrated for 5 seconds (M 5 s) and 10 seconds (M 10 s) [15].

The serum creatinine was measured in this study by using an EPOC^® Blood Analysis System (Epocal Inc., Canada).

2.6 Microcirculatory measurements

After taking blood samples in the fifth week measurement, the abdominal cavity was approached through ventral midline incision, the adhesions on the anterior surface of the left kidney were partially lysed and the microcirculation was measured on this surface using laser Doppler (LD) tissue fluxmetry (LD-01, Experimetria Ltd., Hungary) and a standard pencil probe (Oxford Optronix Ltd., UK). S.P.E.L. Advanced Kymograph software (Experimetria Ltd., Hungary) was the used software. The laser Doppler recordings were analysed after stabilization of the waves, averaging the Blood Flux Units (BFU [au]) [17].

2.7 Histological examinations

After sacrifice of the rats, the left kidney in SG and the remaining part of left kidney in NG were excised. The right kidney samples, which were excised at the second stage, were sent also for histological studies. These sections were stained using Hematoxylin and Eosin (H&E) and Periodic acid–Schiff (PAS). Histology slides were scanned using (Pannoramic MIDI II, 3DHISTECH Ltd, Hungary) and this software (Pannoramic Viewer, 3DHISTECH Ltd, Hungary) was used to measure the diameter of the glomeruli. The diameter was measured only in glomerulus containing a vascular pole by measuring the distance between the vascular pole and the opposite side.

2.8 Statistics

The results were expressed as means±standard deviation (SD). For normally distributed data, the paired samples T test and the one-way ANOVA test were used to evaluate the difference between paired two samples and unpaired two samples, respectively. For non-normally distributed data, the Wilcoxon signed-rank test and the Mann-Whitney U test were used to evaluate the difference between paired two samples and unpaired two samples, respectively. P < 0.05 was considered statistically significant. IBM SPSS Statistics version 22 and Microsoft Excel 2016 were used for the Statistics.

3 Results

3.1 General observations

One rat died in NG seven days after the first stage without any known reason. Other rats survived the follow-up period, 5 weeks. Two complications occurred during the experiments in NG, one bleeding during the surgery and one abdominal wound infection. The creatinine value at the end-point measurement, fifth week post-operative, was significantly (p = 0.008) higher in NG (0.91±0.63) in comparison with SG (0.34±0.04).

3.2 Hemorheological parameters

Two describing parameters of erythrocyte deformability, SS_1/2 and EI_max/SS_1/2, were calculated from EI-SS curves and used to compare the groups, their values are shown in Fig. 1. No significant difference was observed between NG and SG based on these parameters in first stage, second stage and fifth week measurements. No significant differences were observed also between the first stage values and the values of the fifth week in FG or SG. In total, no significant differences were appreciated neither inter-group nor intra-group.

Fig.1

Red blood cell deformability parameters in sham-operated and 5/6 nephrectomy groups. (A) The shear stress required for half-maximal elongation (SS_1/2, [Pa]) and (B) the ratio of maximal elongation index and SS_1/2 (EI_max/SS_1/2 [Pa^–1]). means±S.D.

The measured aggregation indexes for 5 and 10 seconds didn’t show significant differences between NG and the parallel measurements in SG as shown in Fig. 2.

Fig.2

Red blood cell aggregation indexes M 5 s (A) and M 10 s (B). means±S.D.

3.3 Hematological parameters

In the fifth weeks postoperative measurements, the RBC count was significantly (p = 0.028) lower in NG in comparison with SG, although there was no significant difference in the first stage or second stage measurements. Hemoglobin was lower in the first stage measurements of NG in comparison with SG, and no difference was appreciated in the second stage measurements, but the reduction in hemoglobin was more significant (p = 0.015) in the fifth week measurements of NG. Hematocrit was markedly lower in fifth week’s measurements in NG comparing with SG but without reaching the significant level. No significant differences were observed in WBC count between the groups in all the measurements. Figure 3 summarizes these parameters.

Fig.3

Reduction of erythrocyte mass in nephrectomy group in comparison with sham-operated groups: red blood cell count (RBC [10⁶/μl]) (A), hemoglobin (Hgb [g/dl]) (B) and hematocrit (Hct [% ]) (C). Changes of white blood cell count (WBC [10³/μl]) (D). means±S.D, * p < 0.01 vs. base.

3.4 Microcirculation and histology of the kidney

The microcirculatory BFUs of the remaining part of the left kidney in NG were markedly lower than the BFUs of the left kidney in SG without reaching the significant level, as shown in Fig. 4.

Fig.4

Microcirculatory blood flux units (BFU) of the left kidney in sham and nephrectomy rats.

The histological study showed some adhesions between the kidney and the surrounding organs. The boundaries between the ligated and intact tissues are clear and infiltrated with chronic lymphocytes. No signs of infection or acute inflammation were observed in the ligated tissues. The mean glomerular diameter of the compensated left kidney in NG was 130±15 μm and significantly (p < 0.01) higher than the mean glomerular diameter of both left kidney in SG (112±17 μm) and excised right kidney in NG at the second stage (111±18 μm), as shown in Fig. 5.

Fig.5

Histological sections of glomeruli from left kidney of sham rats five weeks after surgery (A), right kidney of nephrectomy rats at the second stage (B), and from the remnant compensated left kidney of nephrectomy rats five weeks after surgery (C). PAS staining. Scale bar: 50 μm. Original magnification: ×600.

4 Discussion

The pathological changes in chronic kidney disease (CKD) are complex and differ according to the etiologies: hypertension, diabetes mellitus infections or drugs [7 , 18–20]. Hence it is not easy to obtain a model resembles these changes. Surgical animal models depend on the loss of 85% of the kidney mass to develop CKD [7]. Several surgical techniques were developed to achieve this result.

A pilot experiment was done at our department to evaluate the feasibility of nephrectomy by ligating branches of renal artery, but it was difficult to achieve the exact percentage due to anatomical variations of the renal arteries. The technique of cutting part of the kidney was associated with a high mortality rate postoperative despite the use of topical hemostatic agents. However, parenchymal ligation was a suitable technique in which we could precisely ligate the portion and it had a higher survival rate in comparison with others. It was fast and not particularly difficult method, using familiar cheap materials. Fife-sixths (5/6) nephrectomy model was used in this study to develop CKD. We tried to reduce the surgical complications in this model by performing two-stage surgery and waiting for sufficient recovery time between the stages, two weeks, as well as using the retroperitoneal approach to reduce the aggressiveness of the second stage.

In the current study, one nephrectomized rat died during the follow-up period without any obvious reason even after the dissection of the rat. Bleeding happened in one case after the excising of the right kidney through retroperitoneal approach, thus the incision had to be extended to control the bleeding from the stump of kidney vessels. One abdominal incision was infected after the first stage, the infection was limited to the superficial layers above the muscles and was treated locally and the second stage was postponed for more two weeks. No complications were observed in sham-operated group.

Reviewing the literature, the creatinine value increases significantly 2-3 weeks after the establishment of the 5/6 nephrectomy model and reaches a plateau after 5 weeks [21 –23], and Garrido et. al. reported another increase after the 9th week [24]. The creatine values in the current study approximately tripled at the fifth week measurements of NG in comparison with SG.

Some studies showed impairment of the erythrocyte deformability [25, 26], concluded that this impairment could contribute to short life-span of erythrocytes and considered these changes as an early sign of nephropathy in type 2 diabetes [27]. Others didn’t show significant changes of the erythrocyte deformability [6, 28], and our results coincides with these studies since the calculated parameters as SS_1/2 and EI_max/SS_1/2 in both groups in the fifth week postoperative measurements didn’t show any significant difference. Erythrocytes have higher aggregability in CKD even after the hemodialysis since the proteins responsible for aggregations are too large to pass through the membrane of the hemodialysis machines [6 , 28]. Our results showed a slight increase the aggregation indexes in NG in comparison with SG without rechaining the significance level.

EPO is glycoprotein act as hormone produced by fibroblast-like cell in the kidney, thus CKD can cause EPO deficiency anemia. This anemia is a normochromic normocytic due to hypo-proliferation of erythroid cells in bone marrow [2 –4]. RBC count, Hgb and Hct were lower in NG in comparison with SG proving the reduction of erythrocyte mass in NG.

The change of erythrocyte mass alters blood viscosity and erythrocyte properties [29], and the standardization of hematocrit values between the groups is one of the common methods in the hemorheological studies [30]. It should be pointed out here that, although there were no significant changes of the erythrocyte rheological properties in the current study, the significant reduction of erythrocyte mass could mask some of these changes [31].

Microcirculation plays an emerging role in the pathologies of CKD, it is not easy to obtain a direct access to the kidney in human as in experimental animals [17, 32]. The microcirculatory status may differ according to the underlying etiologies, especially in the early stages of the disease [33]. CKD was induced in the current model depending on the reduction of kidney mass without any other pathologies such as diabetics and hypertension, so the remaining part of the kidney in this model could give us a real picture of the compensation microcirculatory changes in renal disease. Our results showed impairment of the BFU of the remaining part of the kidney in comparison with SG not as expected as a compensative mechanism. However, this impairment could be a result of the activation of renin-angiotensin system and the constrictor action of angiotensin II.

Many studies showed the compensation role of hypertrophy of intact glomerulus in chronic kidney disease even in systemic diseases [12, 34]. Comparing the mean glomerular diameter between the groups, no enlargement in glomerulus was observed in NG two weeks after 2/6 nephrectomy. Significant enlargement in glomerulus was detected in NG five weeks after 5/6 nephrectomy, the rate of increase of the glomerular diameter was 16% in comparison with the diameter either in NG at the second stage or in SG.

However, when extrapolating data, it is important to mention that rats have different autoregulatory reserve compared to other mammals [35], furthermore the unipapillate kidney of the rat also differs from the multipapillate kidney of other mammals [36]. Cortex to papilla height ratios differs in humans and rats [37, 38], which is a consequence of the human kidney development.

5 Conclusion

The results didn’t show significant changes of the hemorheological parameters in 5/6 nephrectomy model during the follow-up period. The main compensatory changes in the remaining intact kidney tissue were by increasing the diameter of the glomerulus without significant role of blood flow in the microcirculation. These findings would be helpful in further researches about the effects of arteriovenous fistula on erythrocytes as a suggested mechanism of fistula-related hypoperfusion.

Footnotes

Acknowledgments

Authors are grateful to the technical and laboratory staff of the Department of Operative Techniques and Surgical Research at University of Debrecen, and a special thanks to Györgyné Gödény for her dedicated work. The study was supported by the Stipendium Hungaricum program.

The authors comply with the Ethical Guidelines for Publication in Clinical Hemorheology and Microcirculation as published on the IOS Press website and in Volume 63, 2016, pp. 1-2. of this journal.

References

Kurt

, Kurtz

. Plasticity of renal endocrine function. Am J Physiol - Regul Integr Comp Physiol. 2015;308(6):R455–R466. DOI: 10.1152/ajpregu.00568.2013.

Hahn

, Hasselgren

, Björne

, Zdolsek

. Biomarkers of endothelial injury in plasma are dependent on kidney function. Clin Hemorheol Microcirc. 2019;72(2):161–168. DOI: 10.3233/CH-180444.

McGonigle

RJS

, Wallin

, Shadduck

. Erythropoietin deficiency and inhibition of erythropoiesis in renal insufficiency. Kidney Int. 1984;25(2):437–444. DOI: 10.1038/ki.1984.36.

Stauffer

, Fan

. Prevalence of anemia in chronic kidney disease in the United States. PLoS One. 2014;9(1):2–5. DOI: 10.1371/journal.pone.0084943.

Chateauvieux

, Grigorakaki

, Morceau

, Dicato

, Diederich

. Erythropoietin, erythropoiesis and beyond. Biochem Pharmacol. 2011;82(10):1291–1303. DOI: 10.1016/j.bc2011.06.045.

Martínez

, Vayá

, Alvariño

, et al Hemorheological alterations in patients with chronic renal failure. Effect of hemodialysis. Clin Hemorheol Microcirc. 1999;21(1):1–6.

Nogueira

, Pires

, Oliveira

. Pathophysiological mechanisms of renal fibrosis: A review of animal models and therapeutic strategies. In Vivo.. 2017;31(1):1–22. DOI: 10.21873/invivo.11019.

Jia

, Olauson

, Lindberg

, et al A novel model of adenine-induced tubulointerstitial nephropathy in mice. BMC Nephrol. 2013;14(1). DOI: 10.1186/1471-2369-14-116.

Claramunt

, Gil-Peña

, Fuente

, Hernández-Frías

, Santos

. Animal models of pediatric chronic kidney disease. Is adenine intake an appropriate model? Nefrologia. 2015;35(6):517–522. DOI: 10.1016/j.nefroe.2015.08.003.

10.

Perez-Ruiz

, Ros-Lopez

, Cardús

, Fernandez

, Valdivielso

. A forgotten method to induce experimental chronic renal failure in the rat by ligation of the renal parenchyma. Nephron - Exp Nephrol. 2006;103(3):126–131. DOI: 10.1159/000092198.

11.

Togoe

, Schettert

, Ii

, et al Animal model of chronic kidney disease using a unilateral technique of renal ischemia and reperfusion in White New Zealand rabbits1. Acta Cirúrgica Bras. 2014;29(2910):651–657. DOI: 10.1590/S0102-8650201400160005.

12.

Schnaper

. Remnant nephron physiology and the progression of chronic kidney disease. Pediatr Nephrol. 2014;29(2):193–202. DOI: 10.1007/s00467-013-2494-8.

13.

Ichikawi

, Harris

. Angiotensin actions in the kidney: renewed insight into the old hormone. Kidney Int. 1991;40(4):583–596.

14.

Fisher

. Overview of the renin-angiotensin system - UpToDate. https://www.uptodate.com/contents/overview-of-the-renin-angiotensin-system. Published 2017.

15.

Hardeman

, Goedhart

, Shin

. Methods in hemorheology. In: Baskurt

, Hardeman

, Rampling

, Meiselman

, editors. Handbook of Hemorheology and Hemodynamics, Amsterdam, IOS Press; 2007, p. 242–66.

16.

Baskurt

, Hardeman

, Uyuklu

, et al Parameterization of red blood cell elongation index – shear stress curves obtained by ektacytometry. Scand J Clin Lab Invest. 2009;69(7):777–788. DOI: 10.3109/00365510903266069.

17.

Nemeth

, Szabo

. Microcirculation. In: Chen

, Martins

, eds. Advances in Experimental Surgery, Volume 2. Nova Science Publishers; 2017:317–357.

18.

Wouters

, O’Donoghue

, Ritchie

, Kanavos

, Narva

. Early chronic kidney disease: Diagnosis, management and models of care. Nat Rev Nephrol. 2015;11(8):491–502. DOI: 10.1038/nrneph.2015.85.

19.

Chen

, Lee

, Chen

, Hwu

, Petibois

. 3D digital pathology for a chemical-functional analysis of glomeruli in health and pathology. Anal Chem. 2018:acs.analchem.7b04265. DOI: 10.1021/acs.analchem.7b04265.

20.

Sandsmark

, Messé

, Zhang

, et al Proteinuria, but not eGFR, predicts stroke risk in chronic kidney disease: chronic renal insufficiency cohort study. Stroke. 2015;46(8):2075–2080. DOI: 10.1161/STROKEAHA.115.009861.

21.

Chang

, Tsai

, Chen

, et al Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial-to-mesenchymal transition and immune modulation. J Cell Mol Med. 2012;16(12):2935–2949. DOI: 10.1111/j.1582-4934.2012.01610.x.

22.

Freundlich

, Quiroz

, Zhang

, et al Suppression of renin-angiotensin gene expression in the kidney by paricalcitol. Kidney Int. 2008;74(11):1394–1402. DOI: 10.1038/ki.2008.408.

23.

Rodríguez-Iturbe

, Ferrebuz

, Vanegas

, et al Early treatment with cGMP phosphodiesterase inhibitor ameliorates progression of renal damage. Kidney Int. 2005;68(5):2131–2142. DOI: 10.1111/j.1523-1755.2005.00669.x.

24.

Garrido

, Ribeiro

, Fernandes

, et al Iron-hepcidin dysmetabolism, anemia and renal hypoxia, inflammation and fibrosis in the remnant kidney rat model. PLoS One. 2015;10(4):1–24. DOI: 10.1371/journal.pone.0124048.

25.

Bareford

, Lucas

, Stone

PCW

, Caldwell

, McGonigle

, Stuart

. Erythrocyte deformability in chronic renal failure. Clin Hemorheol Microcirc. 1986;6(6):501–510. DOI: 10.3233/CH-1986-6602.

26.

Brimble

, McFarlane

, Winegard

, Crowther

, Churchill

. Effect of chronic kidney disease on red blood cell rheology. Clin Hemorheol Microcirc. 2006;34(3):411–420.

27.

Lee

, Lee

, Nam

, et al Hemorheological Approach for early detection of chronic kidney disease and diabetic nephropathy in type 2 diabetes. Diabetes Technol Ther. 2015;17(11):808–815. DOI: 10.1089/dia.2014.0295.

28.

Ertan

, Bozfakioglu

, Ugurel

, Sinan

, Yalcin

. Alterations of erythrocyte rheology and cellular susceptibility in end stage renal disease: Effects of peritoneal dialysis. PLoS One. 2017;12(2):e0171371. DOI: 10.1371/journal.pone.0171371.

29.

Rampling

. Compositional properties of blood. In: Baskurt

, Hardeman

, Rampling

, Meiselman

, editors. Handbook of Hemorheology and Hemodynamics, Amsterdam, IOS Press; 2007, p. 34–44.

30.

Baskurt

, Boynard

, Cokelet

, et al New guidelines for hemorheological laboratory techniques. Clin Hemorheol Microcirc. 2009;42(2):75–97. DOI: 10.3233/CH-2009-1202.

31.

Nemeth

, Deak

, Szentkereszty

, Peto

. Effects and influencing factors on hemorheological variables taken into consideration in surgical pathophysiology research. Clin Hemorheol Microcirc. 2018;69(1-2):133–140. DOI: 10.3233/CH-189105.

32.

Zafrani

, Ince

. Microcirculation in acute and chronic kidney diseases. Am J Kidney Dis. 2015;66(6):1083–1094. DOI: 10.1053/j.ajkd.2015.06.019.

33.

Maric-Bilkan

, Flynn

, Chade

. Microvascular disease precedes the decline in renal function in the streptozotocin-induced diabetic rat. Am J Physiol Renal Physiol. 2012;302(3):F308–F315. DOI: 10.1152/ajprenal.00421.2011.

34.

Gundersen

, Osterby

. Glomerular size and structure in diabetes mellitus. II. Late abnormalities. Diabetologia. 1977;13(1):43–48.

35.

Krinke

. The laboratory rat. Academic Press, 2000. pp. 386–394.

36.

Franke

, Jung

. Pathophysiology of the contrastmedia-induced nephropathy (CIN) in patientsundergoing coronary interventions. Clin Hemorheol Microcirc. 2013;53(1-2):143–153. DOI: 10.3233/CH-2012-1582.

37.

Kriz

. Nieren und harnableitende Organe. In: Drenckhahn

, Zenker

(eds.): Benninghoff Anatomie Band 2. Urban & Schwarzenberg, Munchen – Baltimore. 1994. p. 37.

38.

Wang

, Zhang

, Yang

, Wang

, Zhang

, Fang

, Jiang

. Time course study on the effects of iodinatedcontrast medium on intrarenal water transport function using diffusion-weighted MRI. J Magn Reson Imaging. 2012;35(5):1139–1144. DOI: 10.1002/jmri.23511.