Red cell deformability (RCD) is impaired in diabetic patients. It was thought to be more affected in those with microvascular complications such as neuropathy, nephropathy and retinopathy. This study aimed to find out the association between red cell deformability and severity of diabetic nephropathy. Forty-four type 2 diabetic (T2DM) patients participated in this study. The severity of diabetic nephropathy was categorized by the presence of albuminuria as well as estimated glomerular filtration rate (eGFR). The RCD was determined by whole blood filtration method and expressed as red cell deformability index (RCDI) and red cell flow rate (RCFR). Urine albumin excretion was determined by hemocue system and expressed as urine albumin creatinine ratio (UACR). Serum creatinine level was determined by Jaffe’s colourimetric test and eGFR was calculated.

Both RCDI and RCFR were significantly lower in the T2DM patients with macro-albuminuria than those with micro-albuminuria (p<0.05) and normo-albuminuria (p=0.001). Similarly, both RCDI and RCFR were significantly lower in the T2DM patients with very low eGFR than those with low eGFR (p<0.005) and normal eGFR ((p<0.005). The RCDI was significantly lower in patients with low eGFR than those with normal eGFR (p=0.027). However, there was no significant difference in RCDI and RCFR between those with micro-albuminuria and normo-albuminuria. There was also no significant difference in RCFR between those with low eGFR and normal eGFR. There was a significant negative correlation between RCD and UACR and a significant positive correlation between RCD and eGFR. However, after adjusting age and haemoglobin A1c (Hb A1c), there was no significant relationship between RCD, UACR and eGFR, indicating that age and HbA1c are confounders.

In conclusion, reduction in RCD was found in T2DM patients with macroalbuminuria and very low eGFR. However, the relationship between reduction in RCD and disease severity of diabetic nephropathy was not found. 

 •    Red cell deformability •    Estimated glomerular filtration rate •    Diabetic nephropathy •    Urine albumin creatinine ratio

Win HS, Htwe TN, Ohnmar (2018) Red Cell Deformability in Type 2 Diabetic Patients with Nephropathy. J Clin Nephrol Res 5(3): 1089.

Annually 1.1 million people died of diabetes and World Health Organization estimated that death due to diabetes will increase by more than 50% in the next 10 years [1]. Persons with diabetes mellitus are at high risk for microvascular damage leading to nephropathy [2]. Red blood cell deformability refers to the ability of red blood cells (RBCs) to change shape under a given level of applied stress, without hemolysing (rupturing). Erythrocytes change their shape while passing through capillaries [3]. Some studies demonstrated that peroxidative damage [4] and glycosylation of diabetic erythrocyte membrane protein spectrin [5] can cause membrane rigidity and thus reduction of red cell deformability (RCD). Several studies have reported the reduction of RCD in diabetic patients [6-9]. In addition, it was also found that there is association between reduced RCD and diabetic nephropathy with various levels of serum creatinine [8,9]. Urinary albumin creatinine ratio is used as an early marker of nephropathy and it also demarcates the progression of diabetic nephropathy [10]. Thus, the present study intended to know whether reduction in RCD is associated with disease severity in diabetic nephropathy or not.

This study was conducted in Diabetes Clinic of Yangon General Hospital and Renal Medical Ward of 500-Bedded Yangon Specialty Hospital from April 2015 to March 2016. Sample size calculation was obtained as follow:

where α = 0.05, Zα2 = 1.96, β = 0.2, Zβ = 0.842, σ = common standard deviation (SD), µ1 - µ2 = difference in mean, N = sample size per group. Acceptable sample size was obtained from previously published paper [9]. Calculated sample size for the present study is 3 for each group according to the formula. In the present study, a total of 44 T2DM patients with nephropathy (40-70 years old, both male and female) were recruited.

The patients were already diagnosed as diabetes by physicians from Yangon General Hospital and Yangon Specialty Hospital. Exclusion criteria were those with acute medical emergency such as diabetic ketoacidosis and those who are comatose or seriously ill. A written informed consent was obtained from the patients. This study was reviewed and approved by Post-graduate Board of Studies (Physiology), University of Medicine 1, Yangon.

Clinical history taking and physical examination including anthropometric measurement were done. Then, venous blood sample (5 ml) and spot midstream urine sample were taken. Urine albumin was determined by hemocue system. Serum and urine creatinine level was determined by Jaffe’s colourimetric test using CREP2 Reagent. Urine albumin creatinine ratio (UACR) was calculated. Then, estimated glomerular filtration rate (eGFR) was calculated from serum creatinine by using Modification of Diet in Renal Disease (MDRD) [12].

Whole blood filtration method, developed by Reid et al. [13], was used for assessing erythrocyte deformability. Assessment of RCD was done within 2 hours after blood collection. Packed cell volume was determined before filtration. Then, the syringe containing one ml of whole blood was positioned in the apparatus set shown in Figure 1.

Figure 1 Model of whole blood filtration method used in the present study.

The blood passed through the pore of membrane filter (pore diameter 5 microns) with a negative pressure of 20 cm water. The volume of blood filtered at one minute was recorded. After filtration of one ml of blood, packed cell volume was determined again and red cell deformability index (RCDI) was calculated by the following formula. 

Red cell flow rate (RCFR) was also calculated as the volume of blood filtered per minute.

The patients were divided into three groups by UACR as well as eGFR. Those with UACR less than 30 mg/g in the spoturine were regarded as T2DM patients with normo-albuminuria (n=15), between 30-300 mg/g as T2DM patients with microalbuminuria (n=15), and more than 300mg/g as T2DM patients with macro-albuminuria (n=14) [14]. Then, the patients were regrouped by eGFR: those with eGFR value ≥ 90 mL/min as T2DM patients with normal eGFR (n=13), those with eGFR value 30-89 mL/min as T2DM patients with low eGFR (n=21) and those with eGFR value < 30 mL/min as T2DM patients with very low eGFR (n=10).

Data were analyzed by using the Statistical Package for Social Science (SPSS) software version 20. The results were expressed as mean ± SD. Comparison of variables among 3 different groups of T2DM patients was assessed by ANOVA with Post Hoc Bonferroni test. Data analysis was done using Log transformation in skewed data. Pearson’s correlation coefficient was calculated to assess the relationship between RCD, UACR, and eGFR. In the present study, p value < 0.05 was regarded as significant. Interaction between RCD and age, and RCD and HbA1c to the effect on eGFR and UACR were assessed by multifactorial analysis of covariances (ANCOVA).

Red cell deformability index (%) of the diabetic patients with normo-albuminuria, micro-albuminuria and macro-albuminuria were 95.53 ± 2.2, 93.07 ± 4.3 and 89.57 ± 3.8 respectively. Red cell flow rate (mL/min) of the diabetic patients with normo-albuminuria, micro-albuminuria and macro-albuminuria were 0.59 ± 0.07, 0.6 ± 0.08 and 0.44 ± 0.12respectively. Both RCDI and RCFR of the diabetic patients with macro-albuminuria were significantly lower than those of the diabetic patients with normo-albuminuria (p=0.001 and p=0.001 respectively) and micro-albuminuria (p=0.035 and p=0.001) respectively (Table 1).

Table 1: Baseline parameters of diabetic patients with normoalbuminuria, microalbuminuria and macroalbuminuria.

But there was no significant difference in RCD between the patients with normo-albuminuria and micro-albuminuria.

Red cell deformability index (%) of patients with normal eGFR, low eGFR, very low eGFR were 96.08 ± 2.25, 92.95 ± 3.63 and 88.2 ± 3.39 respectively. Red cell flow rate (mL/min) of these groups were 0.58 ± 0.1, 0.58 ± 0.06 and 0.42 ± 0.14respectively. Both RCDI and RCFR were significantly lower in the T2DM patients with very low eGFR than those with low eGFR (p<0.005) and normal eGFR ((p<0.005). The RCDI was significantly lower in patients with low eGFR than those with normal eGFR (p=0.027) (Table 2).

Table 2: Baseline parameters of diabetic patients with normal eGFR, low eGFR andvery low eGFR.

However, there was no significant difference in RCFR between those with low eGFR and normal eGFR.

There was a significant negative correlation between RCDI and log UACR (r = -0.673, n = 44, p = 0.000), and between RCFR and logUACR (r = -0.642, n = 44, p = 0.000). In addition, a significant positive correlation was found between RCDI and eGFR level (r = 0.705, n = 44, p = 0.017) and between RCFR and eGFR level (r = 0.544, n = 44, p = 0.09) (Figure 2).

Figure 2 Comparison of red cell deformability index (A) and red cell flow rate (B) in type 2 diabetic patients with normoalbuminuria, microalbuminuria and macroalbuminuria and comparison of red cell deformability index (C) and red cell flow rate (D) in diabetic patients with normal eGFR, low eGFR and very low eGFR * indicates significant difference, NS indicates no significant difference

However, there was no association between RCD and UACR as well as RCD and eGFR after adjusting age and HbA1c.

In the present study, mean RCDI of the T2DM patients with normo-albuminuria (n=15) was 95.53 ± 2.2 %. Reid et al. (1976), stated that mean RCDI of normal subjects is 100 % [13]. In the present study, normal healthy group was not included. However, some previous Myanmar studies using same instruments and same methodology for determination of RCD reported that mean RCDI value of normal healthy control subjects were 99.94 ± 0.35 % [7] and 100% [15]. A significant reduction in RCDI was observed in the T2DM patients when compared with RCDI values of normal healthy control subjects participated in previous Myanmar studies. It was also in agreement with the findings of one Myanmar study [10] in which mean RCDI of 60 T2DM patients was 96.75 ± 4.57 %.

Red cell deformability depends not only on the RBCs membrane structure itself but also on the blood rheology changes. Accumulating body of evidence suggested that reduction in RCDI in diabetes might be due to elevated blood glucose concentration and hyperosmolarity [16], alterations in red blood cell membrane lipid composition [17], increased internal viscosity [18], and increased erythrocyte membrane rigidity caused by glycation of erythrocyte membrane [19]. Since we used whole blood for filtration, the observed reduction in RCD in diabetes mellitus might be due to changes in both RBC and blood properties.

The RCDI of the T2DM patients with nephropathy (n=29) was 91.38 ± 4.39% in the present study and it was significantly lower than that of T2DM patients without nephropathy (n=15) (i.e. 95.53 ± 2.2 %). In addition, RCDI of the T2DM patients with macro-albuminuria was significantly lower than that of the T2DM patients with normo- as well as micro-albuminuria whereas no significant difference was seen inRCDI between the T2DM patients with normo-albuminuria and micro-albuminuria (Table 3).

Table 3: Correlation analysis between RCD and severity of renal function in diabetic patients (n=44) with or without nephropathy.

These findings indicate that there is no appreciable reduction in RCDI in the early stage of diabetic nephropathy, but there might have been progressive reduction in RCD in T2DM patients with severe nephropathy. Brown et al. also reported that early reduction in RCD appears in diabetic patients with normal renal function and that as the renal function decreases (indicated as serum creatinine level), RCD declined more [9].

In the present study, RCFR was also determined to assess the RCD. Red cell flow rate was the amount of whole blood filtered in the first minute (ml/min). Schmid-Schonbein et al., stated that the filtration rate of red blood cell suspension in-vitro reflects the deformability of the red blood cell in the microcirculation in-vivo [20]. In the present study, the mean RCFR of T2DM patients with normo-albuminuria (n=15) was 0.59 ± 0.07 mL/min, which was comparable to that of normal healthy control subjects of previous Myanmar studies: 0.6 ml/min [15] and 0.63 ± 0.17 mL/min [21]. Moreover, mean RCFR of T2DM patients with nephropathy (n=29) was 0.52 ± 0.13 mL/min which was also significantly lower than that of T2DM patients without nephropathy. These findings also suggest the progressive reduction in RCD in diabetic nephropathy.

In the present study, T2DM patients with nephropathy were grouped not only by the urine albumin excretion but also by the estimated glomerular filtration rate. In fact, both parameters are used as the predictive state of the renal function loss, but there are some differences in clinical significance of these two parameters. Urine albumin excretion (UAE) is an indicator for microvascular involvement of renal functional impairment and the eGFR is an indicator for impaired glomerular filtration [22]. It was also found that the RCD of the T2DM patients with very low eGFR was significantly lower than those with low eGFR and normal eGFR. These findings also confirmed that there is a reduction of RCD when renal function impairment is very severe (i.e macro-albuminuria and very low eGFR). The findings in the present study were consistent with the findings of Shin and Ku [8] who reported that progressive impairment in RCD is associated with renal function loss in diabetic patients. In their study, RCD was assessed by Ektacytometer. Although the methods and the materials used were different from that used in the present study, findings were similar regarding RCD in various degree of renal function (Table 4).

Table 4: Effect of RCD on UACR and eGFR after adjusting age and HbA1c.

In the present study, both RCDI and RCFR showed a strong significant correlation with UACR as well as eGFR. Brown et al. [7], observed a significant correlation between serum creatinine concentration and impaired RCD in diabetic patients (r=0.43, p=0.02). The present study was a cross-sectional study, which limits the possibility of causative relationship between RCD and diabetic nephropathy. The presence of correlation between RCD and diabetic nephropathy observed in the present study indicates that the renal function decreases as RCD decreases. Yet, it does not mean that impaired RCD is either a cause or an effect of renal function impairment.

Age is one of the confounding factors for renal impairment. Some evidence reported that there is albuminuria [23] and decline in eGFR [24] with advanced in age. The present study also found a significant negative relationship between age, HbA1c and these renal parameters. Thus, multivariate analysis was done to assess the interaction of RCD and age, and RCD and HbA1c on the effect of eGFR and UACR and it was found that RCD was not significantly associated with UACR and eGFR after adjusting age and HbA1c.

The relationship among chronic hyperglycaemia, RCD reduction and renal function impairment is quite complex and diversified. Persistent hyperglycemia in diabetes causes viscoelastic changes in the erythrocyte membrane and its cytoplasm leading to alteration in the deformability [6,16]. Moreover, chronic hyperglycemia makes the erythrocytes a prime target for advanced glycation end products (AGEs) formation, i.e. HbA1c [25,26]. It has been reported that excessive accumulation of AGEs causes the alteration of rheologic properties of RBCs in patients with diabetes [22].

On the other hand,hyperglycaemia is the main metabolic perturbation causing irreversible kidney damage in diabetes. Greater elevation of tissue AGE peptide levels (indicated by skin autofluorescence) was seen in diabetic patients compared to non-diabetic hemodialysis patients [27]. The in vivo evidence stated that AGEs play a role in inducing glomerular hypertrophy and nephrosclerosis in normal kidneys which is associated with significant loss of protein and albumin [28]. The AGEs also interact with specific receptors and binding proteins and influence the growth and proliferation of the various renal cell types [29]. The AGEs can produce reactive oxygen species (ROS) [30]. Increased generation of ROS is thought to play a key role in the progression of diabetic nephropathy [31-33]. Taken together, chronic hyperglycaemia itself might be the key element for the RCD reduction and renal function impairment. Thus, changes in RCD and renal function might occur as a parallel event in consequence of accumulation of AGEs in tissue and red blood cell.

There were some limitations in the present study. The relevant information about duration of diabetes was not acquired in the study because most of the patients accidently recognized that they were suffered from diabetes only when they did investigations for other diseases. Medication history and smoking history were not asked in the present study. In addition, urine albumin excretion and serum creatinine were determined only one time. According to the literature, determination of UAE should be done at least two times with three months apart.

Another limitation of the study is that the urinary tract infection was not assessed by laboratory method. But, at the time of sample collection, all subjects have no signs and symptoms of overt urinary tract infection which is one of the causes of macroalbuminuria, and thus the patients with macro-albuminuria might be due to not only diabetic nephropathy but also urinary tract infection. But all the diabetic patients with macro-albuminuria have very low eGFR indicating that macro-albuminuria might probably be due to diabetic nephropathy rather than urinary tract infection. Despite of these limitations, relationship still existed between RCD and renal function impairment.

It was found that RCD was impaired in T2DM patients with normal renal function and that progressive reduction in RCD was found in T2DM patients with nephropathy. However, no true association was found between reduction in red cell deformability and disease severity of diabetic nephropathy in this study design with this sample size after adjusting age and HbA1c.

1. World Health Organization. Fact sheet N 312. 2008.

2. Fowler MJ. Microvascular and macrovascular complications of diabetes. Clinical Diabetes. 2011; 29: 116-122.

3. Chien S. Red cell deformability and its relevance to blood flow. Annu Rev Physiol. 1987; 49: 177-192.

4. Yang ZC, Xia K, Wang L, Jia SJ, Li D, Zhang Z, et al. Asymmetric dimethylarginine reduced erythrocyte deformability in streptozocininduced diabetic rats. Microvasc Res. 2007; 73: 131-136.

5. Schwartz RS, Madsen JW, Raybicky AC, Nagel RL. Oxidation of spectrin and deformability defects in diabetic erythrocytes. Diabetes. 1991; 40: 701-708.

6. Babu N, Singh M. Influence of hyperglycemia on aggregation, deformability and shape parameters of erythrocytes. Clin Hemorheol Microcirc. 2004; 31: 273-280.

7. Nang H. Relation between red cell deformability and cognitive function in type 2 diabetes mellitus. 2013.

8. Shin S, Ku Y. Hemorheology and clinical application: association of impairment of red blood cell deformability with diabetic nephropathy. KARJ. 2005; 17: 117-123.

9. Brown CD, Ghali HS, Zhao Z, Thomas LL, Friedman EA. Association of reduced red blood cell deformability and diabetic nephropathy. Kidney Int. 2005; 67: 295-300.

10. Hoy WE, Wang Z, Vanbuynder P, Baker PR, Mcdonald SM, Mathews JD. The natural history of renal disease in Australian Aborigines; Albuminuria predicts natural death and renal failure. Kidney Int. 2001; 60: 249-256.

11. Allen JC. A sample size calculation for two independent groups: A useful rule of thumb. Proc Sing Healthcare. 2011; 20: 138-140.

12. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999; 130: 461-470.

13. Reid HL, Barnes AJ, Lock PJ, Dormandy JA, Dormandy TL. A simple method for measuring erythrocyte deformability. J Clin Pathol. 1976; 29: 855-858.

14. National Kidney Foundation. KDOQI clinical practice guidelines and clinical practice recommendations diabetes and chronic kidney disease. Am J Kidney Dis. 2007; 39: 15-154.

15. Win-Yu-Aung. Effect of zinc supplementation on red cell deformability in beta-thalassemic patients. 2010.

16. Schmid-Schönbein H, Volger E. Red-cell aggregation and red-cell deformability in diabetes. Diabetes. 1976; 25: 897-902.

17. Baba Y, Kai M, Kamada T, Setoyama S, Otsuji S. Higher levels of erythrocyte membrane microviscosity in diabetes. Diabetes. 1979; 28: 1138-1140.

18. McMillan DE, Utterback NG, La Puma J. Reduced erythrocyte deformability in diabetes. Diabetes. 1978; 27: 895-901.

19. Maeda N, Kon K, Imaizumi K, Sekiya M, Shiga T. Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins. Biochim Biophy Acta. 1983; 735: 104-112.

20. Schmid-Schonbein H, Gosen J, Heinich L, Klose HJ, Volger E. A counter-rotating “rheoscope chamber” for the study of the microrheology of blood cells aggregation by microscopic observation and microphotometry. Microvasc Res. 1973; 6: 366-376.

21. Su-Su-Htwe. Plasma malondialdehyde level and red cell deformability in normal and preeclamptic pregnant women. 2010.

22. Johnson D. Diagnosis, classification and staging of chronic kidney disease. Kidney Health Australia. 2012; 4: 1-32.

23. Chowta NK, Pant P, Chowta MN. Microalbuminuria in diabetes mellitus: Association with age, sex, weight, and creatinine clearance. Indian J Nephrol. 2009; 19: 53-56.

24. Rossing K, Christensen PK, Hovind P, Tarnow L, Rossing P, Parving HH. Progression of nephropathy in type 2 diabetic patients. Kidney Int. 2004; 66: 1596-1605.

25. Miller JA, Gravallese E, Bunn H. Nonenzymatic glycosylation of erythrocyte membrane proteins: Relevance to diabetes. J Clin Invest. 1980; 65: 896-901.

26. Makita Z, Vlassara H, Rayfield E, Cartwright K, Friedman E, Rodby R, et al. Hemoglobin-AGE: a circulating marker of advanced glycosylation. Science. 1992; 258: 651-653.

27. Vlassara H, Striker LJ, Teichberg S, Fuh H, Li Y, Steffes M. Advanced glycosylation end products induced glomerulosclerosis and albuminuria in normal rats. Proc Natl Acad Sci. 1994; 91: 11704- 11708.

28. Yoshika K. Skin autofluorescence is a noninvasive surrogate marker for diabetic microvascular complications and carotid intima-media  thickness in Japanese patients with type 2 diabetes: A cross-sectional study. Diabetes Ther. 2018; 9: 75-85.

29. Forbes JM, Cooper ME, Oldfield MD, Thomas MC. Role of advanced glycation end products in diabetic nephropathy. J Am Soc Nephrol. 2003; 14: 254-258.

30. Schleicher ED, Wagner E, Nerlich AG. Increased accumulation of the glycoxidation product N(epsilon)-(carboxymethyl) lysine in human tissues in diabetes and aging. J Clin Invest. 1997; 99: 457-468.

31. Forbes JM, Coughlan MT, Cooper ME. Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes. 2008; 57: 1446-1454.

32. Ha H, Hwang IA, Park JH, Lee HB. Role of reactive oxygen species in the pathogenesis of diabetic nephropathy. Diabetes Res Clin Pract. 2008; 82: 42-45.

33. Kashihara N, Haruna Y, Kondeti VK, Kanwar YS. Oxidative stress in diabetic nephropathy. Curr Med Chem. 2010; 17: 4256-4269.