Critical Shear Stress is Associated with Diabetic Kidney Disease in Patients with Type 2 Diabetes

Critical shear stress (CSS, mPa) is an index of red blood cell (RBC) aggregability, defined as the minimal shear stress required to disperse RBC aggregates. This study aimed to investigate the association between CSS and the risk of diabetic kidney disease (DKD). A total of 421 (mean age, 58.1 ± 11.5 years; male, 250) individuals with T2DM were enrolled and divided into three groups according to CSS level. CSS was measured using a transient microfluidic technique. DKD was defined as a glomerular filtration rate (GFR) <60 ml/min/1.73 m2 or a urine albumin-to-creatinine ratio (uACR) ≥30 mg/g. CSS was significantly higher in patients with DKD than in those without (317.43 ± 125.11 vs 385.22 ± 182.89, p < 0.001). Compared to the lowest CSS tertile, the highest CSS tertile was independently associated with the risk of DKD after adjusting for age, sex, duration of diabetes, presence of hypertension and haemoglobin. The cut-off value of CSS for DKD was approximately 310 mPa. These results suggest that haemorheologic changes may contribute to DKD, and further prospective studies are warranted to determine the role of CSS as a DKD screening tool.

The prevalence of type 2 diabetes mellitus (T2DM) is increasing worldwide. In 2015, 8.8% (415 million) of the global population aged between 20 and 79 years was estimated to have diabetes, and the disease is expected to increase 1.5 times to 2040 1 . In Korea, the prevalence of diabetes in those aged 30 years and over is estimated as 13.7% (4.8 million) and in those over 65 years of age as 30% 2 . Diabetic micro-and macro-vascular complications are a major cause of mortality in T2DM patients. Micro-vascular complications consist of diabetic retinopathy (DR), kidney disease (DKD), and peripheral neuropathy (DPN). Micro-vascular complications, especially DKD, can be a risk factor for macro-vascular complications such as atherosclerosis, myocardial infarction, stroke, and heart failure 3 . Therefore, the effort to screen for micro-vascular complications, including DKD, is essential to prevent the progression to macro-vascular complications and a deterioration in the quality of life 4 .
T2DM, hypertension, obesity and dyslipidaemia, also commonly referred to as metabolic syndrome, are status indicators of oxidative stress and chronic inflammation that alter haemorheology, decrease RBC deformability, alter RBC morphology, increase RBC aggregation, and increase plasma viscosity 5 . In T2DM, advanced glycation end products (AGE) play a critical role in haemorheologic change. RBCs produce AGEs, and many adhesion molecules are expressed in the vessels' endothelial cells, and their increased cohesion induces oxidant stress 6 . The correlation between haemorheological alterations and either diabetic micro-or macro-vascular complications has been recognised, and related studies have been reported. RBC deformability showed a significant decrement in DR 7 or DKD 8,9 while RBC aggregability and plasma viscosity showed a significant increment in acute coronary syndrome 10 , diabetic peripheral arterial occlusive disease 11 , or diabetic foot disease 12 .
RBC aggregability is shear-dependent, and increased aggregability affects the flow properties of RBCs in the microcirculation 13,14 . Critical shear stress (CSS, mPa), the minimal shear stress required to disperse RBC aggregates, has been recently suggested as an index of RBC aggregability 15 . Although reversible RBC aggregates can be easily observed in venules in a status of either stasis or high-to-low shear flow conditions, RBC aggregation in arteries is considered pathological, which may result in a worsening change in the clinical course 15,16 .
The association between diabetic micro-vascular complications and RBC deformability has been revealed in several previous studies; however, the association with CSS is little known. For this reason, this study was designed to investigate the association between CSS and the risk of diabetic micro-vascular complications.

Participants and Methods
Study population. This cross-sectional and retrospective study enrolled 456 T2DM inpatients and outpatients who visited Yeungnam University Hospital (Daegu, Korea) between September 2014 and May 2017. Data and samples were collected at baseline visit. To exclude factors that can confound kidney function markers without kidney damage 17 , such as aging, liver disease and infection, or those that can affect statistical variance in analysis, the criteria for exclusion were as follows: over 85 years of age (n = 2); acute inflammation or infection (white blood cell ≥20,000/µL or high-sensitivity c-reactive protein (hs-CRP) ≥10 mg/dL, n = 8); anaemia (haemoglobin [Hb] ≤8 g/dL, n = 1); impaired liver function (aspartate aminotransferase ≥100 or alanine aminotransferase ≥100, n = 16); hypertriglyceridemia (triglyceride [TG] ≥1000 mg/dL, n = 2); and patients without blood urea nitrogen (BUN) or creatinine (n = 6) results. Finally, 421 participants were enrolled in this study.
All patients gave informed consent, and approval was obtained from the local ethics committee. The Institutional Review Board of Yeungnam University Hospital approved the study protocol. All experiments were performed in accordance with relevant guidelines and regulations.
Diabetic complications. All participants were examined for the presence of diabetic micro-vascular complications 18 . DR was defined as the presence of macular oedema, dilated veins, microaneurysm, haemorrhage, or vessel proliferation identified with retinal photography. DR was classified as normal, non-proliferative diabetic retinopathy (NPDR); proliferative diabetic retinopathy (PDR) was characterised by the growth of new blood vessels on the retina and on the posterior surface of the vitreous 19 . DKD was defined as the presence of albuminuria and/or a reduced estimated glomerular filtration rate (eGFR) in the absence of signs or symptoms of other primary causes of kidney damage, a urinary albumin-creatinine ratio (uACR) ≥30 mg/g, and/or an eGFR <60 mL/ min/1.73 m 2 . In detail, a uACR 30-300 mg/g was defined as moderately increased albuminuria and a uACR >300 mg/g as severely increased albuminuria 17  Measurements. Body mass index (BMI) was calculated as body weight in kilograms divided by height in meters squared (kg/m 2 ). Diabetes duration was measured in years. Blood pressure was measured twice in the sitting position, and a mean value was calculated.
All laboratory parameters were determined in the central laboratory of the Yeungnam University Hospital. Venous sampling was taken from the antecubital vein after an overnight fast. The levels of serum glucose, glycated haemoglobin (HbA1c), Hb, total cholesterol (T-cho), high-density lipoprotein cholesterol (HDL-cho), low-density lipoprotein cholesterol (LDL-cho), TG, BUN, creatinine, erythrocyte sedimentation rate (ESR), hs-CRP, fibrinogen were measured. A urine test was performed to measure uACR. uACR test was repeated three times within 12 months if elevated over 30 mg/g, and increased urinary albumin excretion was confirmed when at least two results were elevated over a 3-to 6-month period 18 .

Hemorheologic parameters. CSS was measured with a transient microfluidic haemorheometer
(Rheoscan-D, Sewon Meditech Inc., Seoul, Korea), using native whole blood without adjusting for haematocrit. Whole blood sample (500 µl) was stored in a reservoir chamber and sheared in the microchannel under continuously decreasing pressure differentials. As the pressure differential decreases, the RBC aggregates tend to disperse at high shear flows, and the corresponding backscattered light (BSL) intensity increases. However, as the pressure differential decreases further, the dispersed RBCs re-aggregate and the BSL intensity decreases. These time-varying BSL intensity and pressure data were simultaneously measured every 0.1 seconds and completed within 20 seconds. The time and shear stress corresponding to maximal BSL were determined as critical time and CSS, respectively 15,21,22 .
RBC deformability was measured with Rheoscan-D (Sewon meditech, Seoul, Korea) and expressed using the elongation index (EI) when RBC was exposed to shear stress of 3 Pascal (EI@3Pa, %) as reported previously 7 . Fibrinogen, EI@3Pa, and fibrinogen/EI@3Pa were considered as haemorheologic parameters to compare the efficacy with CSS. Statistical analysis. All statistical analyses were performed using SPSS (version 21.0, IBM Inc., Chicago, IL, USA). The baseline characteristics were presented as mean ± standard deviation (SD) values for continuous variables and as frequencies with percentages for categorical variables. The statistical significance of differences in continuous variables between two groups and among three groups were determined through an independent sample T-test and a one-way ANOVA, respectively. The statistical significance of differences in categorical variables was determined using Pearson's Chi-square test. Multiple logistic regression analysis was performed to assess the influence of CSS on diabetic micro-vascular complications after adjustment for covariates. A receiver operating characteristic (ROC) curve was performed to analyse the cut-off value, sensitivity, and specificity of CSS in predicting diabetes-related micro-vascular complications. A P-value less than 0.05 was considered statistically significant.

Results
Baseline characteristics. The mean age of all participants was 58.14 ± 11.50 years and the mean duration of diabetes was 8.27 ± 7.98 years. The percentage of males was 59.5%. The mean BMI was 24.76 ± 6.85 kg/m 2 and A comparison of CSS values, according to diabetic micro-vascular complications, is presented in Fig. 1. There was a significant relationship between CSS and DKD but not DR or DPN. CSS differed significantly among groups classified as either eGFR (≥60, <60 mL/min/1.73 m 2 ) or uACR (<30, 30-300, >300 mg/g) (p < 0.001, both). Within Pearson's correlation analysis, CSS showed a significant negative correlation with eGFR (r 2 = 0.023, p = 0.003) and a positive correlation with uACR (r 2 = 0.138, p < 0.001) (Supplementary 1).

Discussion
In our present study, the patients with higher CSS levels were more likely to have DKD. In addition to CSS, the common risk factors for DKD in both eGFR and uACR standards were the duration of diabetes, presence of hypertension, Hb value, ESR, fibrinogen, and the presence of DR. After adjusting for age, sex, duration of diabetes, presence of hypertension, and Hb, the risk of developing DKD was approximately 2.5-3.0 times greater in the highest CCS tertile than in the lowest, with statistical significance.
Currently, the uACR measured in a fresh, first morning, spot sample is preferred as a screening tool for DKD. Compared to urinary total protein, urine albumin measurement provides a more specific and sensitive measure of changes in glomerular permeability 17 . However, there is a disadvantage in that uACR is recommended for repeated testing because the results vary according to the patient's exercise, upright posture, and condition of infection or the sample's storage temperature 17,23 . Additionally, approximately 20% to 63% of patients with low eGFR(<60 mL/min/1.73 m 2 ) were reported to be normoalbuminuric 24 . For this reason, new potential novel biomarkers for early detection of DKD have been suggested, targeting several pathogeneses of DKD, including hyperfiltration, inflammation, and renal remodelling 25 . Recently, oxidative stress has emerged as a new pathophysiology of DKD which eventually alters haemodynamics 26,27 . Among the alterations in haemodynamics, reduced RBC deformability and increased RBC aggregation have been strongly featured and implicated in the pathogenesis of diabetic micro-and macro-vascular complications 28,29 .
There have been several studies reporting the relationship between haemorheologic markers and diabetic micro-vascular complications, and the comparison with the present study is as follows. First, DR was associated eGFR (mL/min/1.73 m 2 ) P-value ≥60 (n = 345) <60 (n = 76) with impairment of RBC deformability 7 , plasma fibrinogen, or conventional aggregation indices 30 ; however, no significant differences in haemorheologic markers between NPDR and PDR were noted 30 . In our study, there was no significant difference in CSS among normal, NPDR, and PDR numbers. This appears to be due to the small number of patients diagnosed with PDR in our study. In addition, since impairment of RBC deformability precedes RBC aggregation 5 and DR precedes DKD 17 , the association of RBC deformability might be strong in DR, whereas the association of CSS is strong in DKD. Second, moderately increased albuminuria (uACR 30-300 mg/g) was significantly associated with impairment of RBC deformability 9 or fibrinogen divided by RBC uACR (mg/g) P for trend <30 (n = 259) 30-300 (n = 100) >300 (n = 31)   deformability 8 , compared with uACR <30 mg/g, but CSS showed only a significant difference between severely increased albuminuria (uACR >300 mg/g) and uACR <30 mg/g 8 . Our study provides additional evidence of CSS as a significant differential marker for moderately increased albuminuria (uACR 30-300 mg/g). Besides, CSS was the most significant indicator for DKD among the haemorheologic indices; we confirmed again that CSS is an independent hemorheologic index reflecting the synergistic effect of reduced RBC deformability and increased fibrinogen 31 . Third, the increased impairment of RBC deformability was noted in DPN without statistical significance 7 . In our study, CSS also increased without statistical significance. CSS is one of several indices that represents RBC aggregation. CSS has an advantage in that it does not require haematocrit adjustments, unlike the conventional aggregation indices 15 , and fibrinogen does not affect the value of CSS if it is measured from the BSL of a transient microfluidic aggregometer 22 . Additionally, it has a similar trend to changes in whole blood viscosity with temperature variations 32 . Moreover, the new role of RBCs in coagulation has been recognised. A recent study reported that increments in CSS significantly increased platelet activation while RBC deformability was not associated 33 . The enhanced aggregation and the induced central compaction of RBC favours the migration of platelets to the marginal flow zone and modulates the possibility of platelet activation 16 , which causes vascular occlusion. Therefore, CSS may be used as a novel biomarker of hemorheological risk in both diabetic microcirculation and seasonal ischemic macro-vascular diseases.
The cut-off value of CSS for detecting DKD was approximately 310 mPa in our study (data was not shown). In previous studies, the mean CSS in the channel flow has been reported as 200.5 mPa 15 . Interestingly, an approximately 30% increase in CSS in acute coronary syndrome was noted: 265 mPa in stable angina, 338 mPa in unstable angina, and 324 mPa in acute myocardial infarction 34 . Further studies to establish the relationship between increased CSS values and diabetic vascular complications, and the study of each cut-off value as a screening tool, may be worthwhile.
To the best of our knowledge, this is the first study that has revealed the relationship between CSS and early stage DKD and presented the CSS cut-off values for DKD. However, there were some limitations to our study. This study was designed retrospectively and was a cross-sectional study; therefore, a causal relationship was hard to determine. In addition, omitted variable bias might have occurred due to the lack of important confounding variables in the regression analysis, such as medication. Further research with consideration to usage of cardiovascular medication is needed. With regard to hemorheologic parameters, we used the measured value once and did not use the average value from the repeated measurement. Although the mean value may more accurately reflect the hemorheologic change, single measurement could be acceptable based on previous studies [7][8][9][10] . Well-designed, prospective studies with a larger sample size are warranted in the future.
In conclusion, the elevation of CSS was closely associated with an increased risk of DKD. These results reinforce the possibility that RBC aggregability might contribute to DKD development. We anticipate that if additional studies and reference ranges are accumulated, haemorheologic parameters, including CSS, may have a role as screening tools for diabetic micro-vascular complications.