## 4 Discussion
These findings demonstrate that, although the studied miRNAs are all acceptable predictors of CKD, only hsa-miR-novel-chr13_13519 seems to offer an additional advantage. Contrary to our study, previous studies, which explored the predictive value of miRNAs analyzed for CKD in individuals with HTN or DM, quantified their expression profile in plasma [23; 34], platelet poor plasma [38], serum [37], and urine [34; 37], while in thecurrent study we used whole blood. As reviewed by [PERSON] in 2015 [40], the establishment of an accurate and reliable circulating miRNA biomarker for disease has proven to be quite challenging as it may be affected by pre-analytical factors, such as the starting material of biofluid, processing, and the type of normalization miRNA used. Although platelet-poor plasma is not widely biased by coagulation due to a lack of platelets [41], similarly to plasma and serum, the concentration of total miRNA is reduced after extraction [42] and may be affected by pre-analytical processes, such as sample handling and bias due hemolysis [13; 41]. Urine samples also present with some shortcomings, in particular with regard to the use of normalization control, with some studies showing that the inclusion of a normalization control might reduce the predictive value of urine miRNAs, whereas the exclusion of it might affect the accuracy of the results [43]. In the current study, we analyzed miRNA expression in whole blood samples, comprised of multiple different blood cell types with their own specific miRNA expression profile, which might have contributed to the profile of miRNAs observed. Individuals with CKD generally have lower levels of red blood cell (RBC) count, due to anemia, compared to their control counterparts [44]. It was reported that RBC-derived miRNAs constitute the majority of miRNAs expressed in whole blood [45]. However, we found high levels of miRNA expression in CKD individuals supposedly with low RBC count, therefore it is likely that our novel miRNAs are not highly expressed in RBCs and our results were not affected. Furthermore, [PERSON] et al. performed a statistical evaluation on the effect of different blood cell counts on a miRNA expression profile for different human diseases to test for disease-specific alterations. The authors found that different blood cell counts only partly affected the profile of miRNAs and that they did not significantly affect the feasibility of associating a miRNA profile and human disease, therefore supporting the use of whole blood for miRNA profile analysis as the basis for the detection of disease [46]. Moreover, whole blood has a high concentration of total miRNA after extraction and is not affected by pre-sample analysis and cell lysis [47]. The current study had some limitations, which need to be taken into consideration when interpreting the findings. The cross-sectional design of the study limited the exploration of a causal relationship between the whole blood miRNAs and CKD, therefore future longitudinal analysis is recommended to elucidate the causal relationship between studied miRNAs and the pathogenesis of CKD in individuals with HTN and/or DM. Although the present study had a large study sample, we had a small number of individuals with CKD in various stages of the disease, thereby not allowing for the exploration of miRNA profiles over the various stages of CKD. CKD was diagnosed based on eGFR estimated from a single time point creatinine measurement, which is not ideal. However, we included levels of ACR, which are important for interpretation when eGFR levels are above 60 mL/min/1.73 m\({}^{2}\) as recommended by the KDIGO guidelines [29]. HTN was diagnosed with BP measurements taken on one visit, although the International Society of Hypertension recommends that diagnosis should be made based on measurements taken at two or more visits separated by a period of one week [48].