Association of Urinary Biomarkers Noninvasively Forecasts The Extent of Renal Injury In The Early Diabetic Nephropathy Patients With Kidney Qi Deficiency Syndrome: A Retrospective Investigation 


 Background: Recently, diabetic nephropathy (DN) becomes a common health problem in China as one of the major microvascular complications of diabetes mellitus. Therefore, the incipient diagnosis and noninvasive detections in clinic by the association of urinary biomarkers are important for preventing the progress of DN. However, the clinical significance of urinary biomarkers is controversial nowadays. This study thereby aimed to further evaluate the clinical significance of the association of urinary biomarkers in noninvasively predicting the renal damaged extent of the early type 2 DN patients with kidney qi deficiency syndrome in an integrated traditional and western medical center, and preliminarily confirm the correlation between urinary tubular biomarkers and the biological bases of DN patients.Methods: Ninety-two patients in an integrated traditional and western medical center of China were categorized into 3 groups, 20 patients with normo-albuminuria, 50 patients with micro-albuminuria, and 22 patients with macro-albuminuria. In addition to urinary albumin (UAlb) and urinary albumin-to-creatinine ratio (UACR), serum creatinine, estimated glomerular filtration rate and various urinary tubular biomarkers were tested respectively. Besides, clinical characteristics and kidney asthenia syndrome distribution in all patients were observed. Results: In these 3 groups, 24-h UAlb and UACR showed stepwise and significant increases. Urinary cystatin C (UCysC), urinary N-acety1-β-D-glucosaminidase (UNAG) and urinary retinal binding protein (URBP) synchronously showed gradual increases consistent with albuminuria degree in 3 groups. Moreover, 24-h UAlb and UACR were positively correlated with UCysC, UNAG and URBP. In the 72 DN patients with albuminuria, there was a positive correlation between UNAG and URBP, and UCysC was also positively correlated with UNAG and URBP. Additionally, TCM syndrome distributional characteristics in all patients were consistent with the clinical manifestations of kidney qi deficiency syndrome. Conclusion: In this investigation, we ulteriorly demonstrated that the association within UCysC, UNAG, URBP and UAlb may be used as practical targets in noninvasively forecasting the extent of renal injury in the early type 2 DN patients with kidney qi deficiency syndrome. More importantly, we found that urinary tubular biomarkers may be one of the biological bases of DN patients with the specific traditional Chinese medicine syndrome.


Background
Nowadays, diabetic nephropathy (DN) becomes a common health problem in China as one of the major microvascular complications of diabetes mellitus (DM) [1]. 30-40% of the patients with DM develop to DN, which is the dominant cause of end-stage renal disease (ESRD) [2]. Therefore, the clinical incipient diagnosis and noninvasive detections are important for preventing the progress from DN to ESRD. It is reported that, albuminuria is one of the most typical clinical changes in the early stage of DN, and the level of albuminuria excretion can be determined as a way for DN patients screening [3]. However, growing evidences have recently suggested that the detection of albuminuria alone is not comprehensive and sensitive for DN patients, especially for those with inchoate and latent injuries in glomeruli and renal tubules [4]. On the other hand, although renal biopsy is thought to be the best method for the diagnosis of DN at the incipient stage [5], it is impossible to be performed in all cases because of its invasion. Given these shortcomings in clinic, more workable biomarkers in urine other than urinary albumin (UAlb) are crucially required to be explored for the earlier diagnosis and prediction of renal injurious extent in DN patients [6].
Generally speaking, glomerular dysfunction is a major cause of DN development [7]. Even though, the impaired absorption of the ltered proteins from renal tubular epithelium might also play many important roles in incipient DN [8]. Notably, the increasing evidences have demonstrated that, as biomarkers, some tubular injurious indicators in urine have clinical implications [9]. It includes urinary cystatin C (UCysC) [10,11], urinary N-acety1-β-D-glucosaminidase (UNAG) [12], urinary kidney injury molecule (UKim)-1 [12][13], urinary 1iver-type fatty acid-binding protein (UL-FABP) [14], urinary retinal binding protein (URBP), urinary neutrophil getatinase-associated lipocalin (UNGAL), urinary β2-microglobulin (Uβ2-MG) and so on [15,16]. Thereinto, UNAG and URBP have been especially reported to be associated with the progression of type 2 DM [15]. On the contrary, recently Macisaac et al. [17] considered there was neither a clinical assay nor an adequate study focuses on de ning urinary biomarkers' prognostic value in the DN progress. Besides, Hus et al. [18] lately reported the urine biomarkers of tubular injury did not improve on the clinical model predicting chronic kidney disease (CKD) progression. Therefore, so far, the clinical signi cance of urinary biomarkers in predicting renal injurious extent of DN is controversial.
In traditional Chinese medicine (TCM), DN is recognized as Xiaoke (a disease with symptomatic polydipsia)-related nephropathy. According to the fundamental principles of TCM theory, the main pathogenesis of DN lies in kidney asthenia [19][20]. More importantly, in clinic, Chinese herbal medicine (CHM) formulas focused on nourishing kidney such as Tangshen Formula (TSF) [21] and Liuwei Dihuang Pills (LDP) [22] have played many important roles in the treatment of Xiaoke and its related complications in the kidney. Yang et al. [21] reported that TSF combined with conventional therapy appear to be effective in reducing urinary protein and UL-FABP, which is considered to be associated with the severity of DN as a new urinary renal tubular biomarker. Correspondingly, our previous study based on 108 patients with stage III type 2 DN in China, a unicentral observation with a cross sectional design, indicated that 79 patients with the de ciency states of both kidney and spleen showed different increased levels of UCysC, UNAG and URBP, which as the speci c markers of renal tubular dysfunction rather than glomerular damage. Of note, the increase in UCysC has been closely related to UAlb in 30 patients with kidney de ciency syndrome [23]. These results strongly suggested that the association of urinary biomarkers is possibly used as practical targets in noninvasively forecasting the extent of renal injury in the type 2 DN patients with kidney asthenia syndrome. Hence, in this investigation, we aimed to further evaluate the clinical signi cance of the association of urinary biomarkers in noninvasively predicting the renal damaged extent of the early type 2 DN patients with kidney qi de ciency syndrome in an integrated traditional and western medical center, and preliminarily con rm the correlation between urinary tubular biomarkers and the biological bases of DN patients.

Study population
We retrospectively investigated the samples of urine from the 92 patients with type 2 DM who were consecutively enrolled at the Department of Endocrinology and TCM, Nanjing Drum Tower Hospital from March 2012 to April 2013. The proposal was approved by the Ethics Committee of The A liated Hospital of Nanjing University Medical School (Nanjing Drum Tower Hospital). All patients were categorized into 3 groups according to their albuminuria levels based on urinary albumin-to-creatinine ratio (UACR), including 20 patients with normo-albuminuria (< 30 mg/g creatinine), 50 patients with micro-albuminuria (30-300 mg/g creatinine), and 22 patients with macro-albuminuria (> 300 mg/g ceatinine) (Fig. 1).

Collection of kidney de ciency syndrome information
Signs and symptoms by kidney de ciency syndrome of TCM diagnostic methods mainly including kidney qi de ciency syndrome, kidney yang de ciency syndrome, kidney yin de ciency syndrome and kidney essence insu ciency syndrome were collected respectively [24,25,26]. The clinical manifestations include aching and weakness of loins and knees, dispiritedness and lassitude, frequent micturition, dripping urination, incontinence of urine, light-colored tongue with whitish fur and thin pulse; dizziness and tinnitus, insomnia and amnesia, ushed cheeks in the afternoon, bone-steaming tidal fever, night sweating, dry mouth and throat, emaciation, yellowish and scanty urine, reddish tongue with scanty fur and thin and rapid pulse; cold limbs and body, loose stool, early morning diarrhea, clear and profuse urine, profuse nocturnal urine, bright whitish complexion, light colored tongue with white fur as well as sinking and deep and weak pulse. All of kidney de ciency syndrome information was collected using a uni ed questionnaire.

Clinical and laboratory measurement
All eligible subjects provided 20 mL second voided clean-catch urine samples in the early morning after an overnight fast. These samples were immediately frozen and stored at -80 °C to prevent protein degradation before the test. Urine samples were centrifuged at speed of 3000 r/min for 10 min. And the supernatants were collected for sample test in 2 hours. Twenty-four-hours UAlb (24-h UAlb) was tested using immunoturbidimetric assay (Yong Ye, ShangHai, China). Subsequently, baseline of albuminuria status was determined according to UACR. The levels of URBP, UNAG and UCysC were measured using immune turbidimetric method (KangTe, Zhejiang, China), colourimetry (Mei Kang, Zhejiang, China) and particle-enhanced nephelometric immunoassay (YanJing, ShangHai, China) respectively. All the relevant procedures were followed by the manufacturer's instructions.
In addition, all patients donated fasting venous blood (2 mL) in the morning. And serum was separated by centrifugation. The levels of estimated glomerular ltration rate (eGFR) were calculated using the

Statistical analysis
All analyses were performed using the SPSS software (Version 16.0, SPSS Inc, USA) and the Graph Pad Prism 5. The data were expressed as mean ± SD for normally distributed values and median (interquartile range) for nonparametric values. Qualitative data were described as frequencies and analyzed using the Chi-square test. Differences among groups were analyzed by the one-way ANOVA followed by the Bonferroni's test for normally distributed values, and by the Kruskal-Wallis test for nonparametric values. SNK (Student-Newman-Keuls) or LSD (Least signi cant difference) method was used for multiple comparisons. To test the correlations between different urinary markers, Pearson's correlation coe cient was employed for normally distributed values and Spearman's correlation coe cient for skew distributed values. To determine the association between UACR, 24-h UAlb and urinary markers respectively, with the exception of bivariate correlation analysis, we performed linear regression analysis with the urinary markers as independent variables and UACR, 24-h UAlb as dependent variables in order to investigate the urinary markers related to albuminuria. Clinical parameters related to the elevated urinary markers were analyzed using multivariate logistic regression according to the ''Enter'' procedure. The p-value report was two-sided and value of less than 0.05 was considered statistically signi cant.

Clinical characteristics in all patients
The study population of type 2 DM is consisted of 20 patients with normo-albuminuria, 50 patients with micro-albuminuria and 22 patients with macro-albuminuria. All patients' baseline characteristics are summarized in Table 1. The mean age and DM duration of the patients were 63.12 ± 12.07 years and 12.62 ± 1.17 years respectively, and there were 47 males and 45 females. According to the baseline data, we did not nd a signi cant difference in sex, age, DM duration, systolic blood pressure (SBP), diastolic blood pressure (DBP), fast blood glucose (FBG), 2 hours postprandial blood glucose (2-h PBG), glycated hemoglobin (HbA1c), triglyceride (TG), blood urea nitrogen (BUN) and uric acid (UA) among normoalbuminuria, micro-albuminuria and macro-albuminuria groups. Although the levels of body mass index (BMI) and low-density lipoprotein cholesterol (LDL-C) in normo-albuminuria, micro-albuminuria and macro-albuminuria groups showed the stepwise increase with albuminuria level, there was no statistical signi cance among 3 groups. We also found that the level of total cholesterol (TC) in macro-albuminuria group was signi cantly higher than that in normo-albuminuria group (p = 0.017), and the levels of Scr and eGFR in macro-albuminuria group were signi cantly increased compared with those in micro-albuminuria group (p = 0.004, p = 0.022), whereas there were no signi cant differences between micro-albuminuria and normo-albuminuria groups. The data are expressed as mean ± SD for parametric variables and median (interquartile range) for nonparametric variables.
Distribution of kidney de ciency syndrome TCM syndrome could be validated by corresponding diagnostic standards of kidney asthenia [26]. Kidney de ciency syndrome of all patients was divided into the following types, including kidney qi de ciency syndrome, kidney yang de ciency syndrome, kidney yin de ciency syndrome and kidney essence insu ciency syndrome. As shown in Table 2, among these 92 patients with type 2 DM, the syndromes that occurred at least 30% included: aching and weakness of loins and knees (78.3%), dispiritedness and lassitude (78.3%), frequent micturition (47.8%), dripping urination (33.7%), dizziness and tinnitus (31.5%), light-colored tongue with whitish fur (86.9%) and thin pulse (30.4%). There is no doubt that these TCM syndrome distributional characteristics in all patients were consistent with the clinical manifestations of kidney qi de ciency syndrome. Differences of Scr, eGFR, 24-h UAlb and UACR Figure 2 shows the differences of Scr, eGFR, 24-h UAlb and UACR among normo-albuminuria, microalbuminuria and macro-albuminuria groups. We found that the levels of Scr and eGFR in macroalbuminuria group were signi cantly higher than micro-albuminuria group, but there were no signi cant differences between micro-albuminuria group and normo-albuminuria group. In addition, compared with the levels of Scr and eGFR, we concluded that the levels of 24-h UAlb and UACR showed a stepwise increase in normo-albuminuria, micro-albuminuria and macro-albuminuria groups (p = 0.000).
Changes of UCysC, UNAG and URBP Table 3 and Fig. 3 show the changes of urinary tubular biomarkers' levels in normo-albuminuria, microalbuminuria and macro-albuminuria groups according to the levels of albuminuria. We found that the levels of UNAG, URBP and UCysC synchronously showed a gradual and signi cant increase consistent with albuminuria degrees in 3 groups. The data are expressed as mean ± SD for continuous variables. p-values were obtained by ANOVA.

Discussion
Under the fundamental principles of TCM theory, the differentiation and treatment of DM and its complications have focused on the asthenia syndromes including the de ciency of lung, spleen (stomach) and kidney for a long time, of them, kidney asthenia is considered to be the key pathogenesis of DN, which is a well-known complication of long-standing DM [28,29,30]. However, there is no further observation on the distribution of kidney de ciency syndrome in the early DN patients. In the present study, rstly, we unexpectedly found that TCM syndrome distributional characteristics of DM without or with abnormal albuminuria wholly belong to kidney qi de ciency syndrome, which is different from the previous investigation about the 108 stage III type 2 DN patients with massive proteinuria [23]. Hence, we preliminarily suggest kidney qi de ciency is the potential pathogenesis of these 92 patients with type 2 DM.
In clinic, the incipient diagnosis and noninvasive detections of renal harmful risk of DM are undoubtedly important. In general, glomerular dysfunction is thought to be a main factor for DN at the early stage. The routine and classical evaluation of glomerular ltration dysfunction in DN patients includes the increased levels of UAlb and Scr, which is the basis of the calculation for eGFR [28]. Despite this, lamentedly, the overt increase in Scr might be lately found after the serious glomerular impairments [5], moreover, a decline of eGFR in the patients with DM is not always accompanied by an increase of UAlb [28].
Therefore, neither Scr nor eGFR is the perfect marker for the early detection of glomerular dysfunction in DN patients. Our results in the present study showed that 24-h UAlb and UACR, in comparison to Scr and eGFR, appeared a stepwise rise and a signi cant difference in the individuals with normo-albuminuria, micro-albuminuria and macro-albuminuria. In a nutshell, based on these 92 type 2 DM with kidney qi de ciency syndrome, we secondly con rmed that there is a really renal lesion characterized by the different levels of UAlb and UACR, the markers of injurious glomeruli.
The recent studies have demonstrated that renal tubulointersititial lesion plays a critical role along with glomerular injury in the pathogenesis of DN patients [30]. The several biomarkers indicating proximal tubular impairment have the potential to be the clinical markers for predicting renal damage extent of DN patients at the early stage. Thereupon, to evaluate the clinical implication of urinary tubular biomarkers in diagnosing renal lesion, we then investigated the changes of UCysC, UNAG and URBP in all type 2 DM patients with kidney qi de ciency syndrome.
It is well known that, the clinical biomarkers of renal tubular injury contain urinary enzymes (enzymuria) and urinary proteins with the low molecular weight (LW-protienuria), while LW-protienuria originates from the de cient reabsorption of plasma proteins by tubular epithelial cells [29]. CysC, as its low molecular weight, is freely ltered by glomeruli and metabolized by renal proximal tubule without the in uences of age or muscle mass in healthy conditions. It is reported that CysC in urine (UCysC) along with albuminuria could predict tubular impairment in the type 2 DN patients [31]. RBP, a low molecular weight protein, is synthesized mainly in hepatocytes, easily ltered by glomeruli and almost completely reabsorbed in renal proximal tubule. The level of urinary RBP (URBP) is thus very low in the nal urine.
Several researchers have demonstrated that the increased level of URBP was correlated with renal tubular dysfunction in DM patients [16,28]. In addition to these, it has already been reported that urinary NAG (UNAG), a proximal tubular brush border lysosomal enzyme, plays a crucial role in diagnosing tubular impairment in DM patients [12]. The data in this observation indicated that the above-mentioned urinary tubular biomarkers, UCysC, UNAG and URBP, were simultaneously increased in normo-albuminuria, microalbuminuria and macro-albuminuria groups accompanied by the rise of 24hUAlb and UACP, which are the earliest known clinical indexes for the diagnosis of DN. Furthermore, these biomarkers in urine were independently related to 24hUAlb and UACP. In brief, we also con rmed that UCysC, together with UNAG and URBP, as the acknowledged tubular dysfunctional biomarkers, have an independent association with renal injurious extent in the early type 2 DM patients with kidney qi de ciency syndrome.
Thirdly, we paid attention to the interrelation of UCysC, UNAG and URBP in the 72 type 2 DN patients with micro-albuminuria and macro-albuminuria. To our surprise, the results in this report unexpectedly displayed that UCysC was positively correlated with UNAG and URBP, moreover, there was also a positive relationship between UNAG and URBP in DN patients. Whereupon, we naturally speculated a possibility that which one is the more sensitive and speci c tubular dysfunctional biomarker among UCysC, UNAG and URBP based on the different levels of UAlb. To our knowledge, CysC is synthesized and secreted at a nearly constant rate by virtually all nucleated cells. Given its molecular weight is 13 kDa, in healthy subjects in human, CysC is almost freely ltered by glomeruli and entirely reabsorbed in renal proximal tubular epithelial cell like other urinary proteins with the low molecular weight. Therefore, it is not excreted in urine and the increased level of UCysC, an independent of serum CysC, is particularly useful for estimating renal tubular impairment [31]. Kim et al. [10] reported that UCysC was mainly increased in the type 2 DN patients with macro-albuminuria and not signi cantly different between those with microalbuminuria and normo-albuminuria. By contrast, being different from the above consequences, we amazedly found in the present study that UCysC was not only increased signi cantly in the type 2 DN patients with unnormal albuminuria but also closely related to a raise in UNAG and URBP. Additionally, we detected that UCysC was associated with BMI and FBG, the clinical baseline parameters of DM patients.
Consequently, it was further con rmed the clinical signi cance of assessing the levels of UCysC, UNAG and URBP in the type 2 DM patients on the basis of the different levels of albuminuria, especially in unnormal albuminuric individuals. More importantly, it is emphasized that UCysC, UNAG and URBP are all considered as sensitive and speci c tubular dysfunctional biomarkers.
Lastly, to be frank, there are several limitations in this report. First, except for the insu cient sample size in a signal-center, the study was di cult to illustrate the causal relationship between the risk factors and the natural course of normo-albuminuric renal insu ciency since it was conducted with a retrospective cross sectional design rather than based on a longitudinal observation. Second, due to the lack of healthy control group, we failed to clarify the relationship among the levels of UNAG, URBP and UCysC under healthy condition. Third, it is unknown whether the synchronized rise of UCysC, UNAG and URBP could assuredly indicate renal tubular impairment in the type 2 DN patients at the early stage without the histopathologic evidences in the kidney. Fourth, we only found kidney qi de ciency was the main TCM syndrome in these 92 DM patients, and could not demonstrate the relationships between urinary biomarkers and kidney yin or yang de ciency syndromes. Despite these, we have reasons to believe that the clinical implication of UCysC, UNAG and URBP in these 92 type 2 DM patients were clearly described, and the associated detection of urinary biomarkers could noninvasively forecast renal injurious extent in the incipient type 2 DN patients with kidney qi de ciency syndrome.

Conclusion
In this investigation, we ulteriorly demonstrated that the association within UCysC, UNAG, URBP and UAlb may be used as practical targets in noninvasively forecasting the extent of renal injury in the early type 2 DN patients with kidney qi de ciency syndrome. More importantly, we found that urinary tubular biomarkers may be one of the biological bases of DN patients with the speci c TCM syndrome.

Figure 1
Trial ow diagram.

Figure 2
Changes of Scr, eGFR, 24hUAlb and UACR in all patients. The differences of Scr, eGFR, 24hUAlb and UACR among 3 groups were shown. The levels of Scr and eGFR were signi cantly different between micro-albuminuria group and macro-albuminuria group (p = 0.004, p = 0.022), but there were no signi cant difference between micro-albuminuria group and normo-albuminuria group. In addition, compared with Scr and eGFR, the levels of 24hUAlb and UACR showed the stepwise increase and the signi cant difference in 3 groups (P = 0.000). □ = normo-albuminuria group; ■ = micro-albuminuria group; ■ = macro-albuminuria group. Each value was expressed as mean ± SD. ap < 0.01 vs normoalbuminuria group; bp < 0.01 vs micro-albuminuria group.

Figure 2
Changes of Scr, eGFR, 24hUAlb and UACR in all patients. The differences of Scr, eGFR, 24hUAlb and UACR among 3 groups were shown. The levels of Scr and eGFR were signi cantly different between micro-albuminuria group and macro-albuminuria group (p = 0.004, p = 0.022), but there were no signi cant difference between micro-albuminuria group and normo-albuminuria group. In addition, compared with Scr and eGFR, the levels of 24hUAlb and UACR showed the stepwise increase and the signi cant difference in 3 groups (P = 0.000). □ = normo-albuminuria group; ■ = micro-albuminuria group; ■ = macro-albuminuria group. Each value was expressed as mean ± SD. ap < 0.01 vs normoalbuminuria group; bp < 0.01 vs micro-albuminuria group.

Figure 3
Levels of urinary tubular biomarkers in all patients. The changes of urinary tubular biomarker in 3 groups according to albuminuria were revealed. The levels of UNAG, URBP and UCysC synchronously showed the gradual increase along with albuminuria in 3 groups, and there was a signi cant difference between normo-albuminuria and micro-albuminuria groups (p = 0.000), as well as between micro-albuminuria and macro-albuminuria groups (p = 0.000). □ = normo-albuminuria group; ■ = micro-albuminuria group; ■ = macro-albuminuria group. Each value was expressed as mean ± SD. ap < 0.01 vs normo-albuminuria group; bp < 0.01 vs micro-albuminuria group.