Abstract
Glomerular inflammation is a putative aggravation factor for type 2 diabetic nephropathy and urinary thrombin is a novel marker of glomerular inflammation. To clarify the relationship between glomerular inflammation and progression of the nephropathy, we measured urinary thrombin in 118 patients with type 2 diabetic nephropathy at different stages. To investigate the implications of urinary thrombin in the nephropathy, we compared urinary thrombin with expression of tissue factor, the trigger of blood coagulation activation, in glomeruli and with markers of renal injury (estimated glomerular filtration rate (eGFR) and proteinuria). Urinary thrombin was found in 4.9% (3/61), 0.0% (0/12), 29.6% (8/27) and 50.0% (9/18) of patient groups at stages 1, 2, 3 and 4, respectively. Thus, urinary thrombin was negligible in the patients at early stages (stages 1 and 2), but was present predominantly in the patients at advanced stages (stages 3 and 4). Tissue factor was expressed in accumulated macrophages in glomeruli, which indicates that thrombin may be generated in inflamed glomeruli presumably via inflammation-induced activation of the exudated coagulation factors into glomerular tissues and then be excreted in urine. Urinary thrombin was significantly associated with both decreased eGFR and increased proteinuria in type 2 diabetic nephropathy. Therefore, increased urinary thrombin in patients with advanced stages of type 2 diabetic nephropathy suggests that glomerular inflammation may injure the tissues, thereby impairing renal function. Monitoring an effect of anti-diabetic treatments on glomerular inflammation in the patients with type 2 diabetic nephropathy may be a possible application of urinary thrombin.
Introduction
Diabetic nephropathy is a major complication of type 2 diabetes, and is a world-wide leading cause of end-stage renal disease (Collins et al. 2015; Masakane et al. 2015; Kramer et al. 2019), which requires patients to receive chronic dialysis treatments for survival. Thus, to prevent the nephropathy from progressing to end-stage renal disease is an urgent issue.
Type 2 diabetic nephropathy often accompanies systemic inflammation (van Eeden et al. 1988) and systemic inflammation likely progresses the nephropathy according to recent studies (Pergola et al. 2011; Niewczas et al. 2012; Navarro-González et al. 2015). However, whether inflammation in glomerular tissue is involved in progression of the nephropathy is still not clear because no specific markers exist to detect glomerular inflammation in the patients with diabetic nephropathy (Moresco et al. 2013). Furthermore, type 2 diabetic nephropathy is usually diagnosed clinically, and histological examination to detect glomerular inflammation is not performed to avoid the risk of massive bleeding and pain accompanying renal biopsy. Therefore, non-invasive biomarkers specific for glomerular inflammation are necessary to determine whether glomerular inflammation exacerbates type 2 diabetic nephropathy in the patients.
We previously measured thrombin concentration in urine obtained from patients with glomerulonephritides (Kitamoto et al. 1998, 2004) and found high thrombin activity in patients with crescentic glomerulonephritis (Kitamoto et al. 2015). Crescentic glomerulonephritis is rare but severe with significant accumulation of macrophages and fibrin deposition in (extravascular) glomerular tissues (Mathew and Kincaid-Smith 1972). We assumed that thrombin was generated in inflamed glomeruli, presumably via activation of the extrinsic coagulation pathway initiated by tissue factor (TF) in macrophage (Tipping et al. 1995; Levi and van der Poll 2010), and was leaked into urine. Thrombin generated in the circulation (bloodstream) is promptly converted to thrombin-antithrombin (TAT) complex and is eliminated from the circulation (Lollar and Owen 1980); namely, thrombin does not appear in the urine. Indeed, urinary thrombin concentration was independent of plasma TAT concentration in the patients with glomerulonephritides (Kitamoto et al. 2015). Further, urinary thrombin was positive in 70.6% of patients with active crescentic glomerulonephritis but negative in all healed patients or healthy volunteers. Therefore, the sensitivity and specificity of urinary thrombin testing for glomerular inflammation (in crescentic glomerulonephritis) was 70.6% and 100%, respectively (Kitamoto et al. 2015). Although the number of patients was limited in that study, such high specificity and sensitivity indicated that urinary thrombin was a good marker of glomerular inflammation. Thus, urinary thrombin measurement may be helpful to detect glomerular inflammation associated with other diseases.
In the present study, we measured urinary thrombin activity in the patients with type 2 diabetic nephropathy at different clinical stages together with histological analysis of renal biopsied tissues to study the role of glomerular inflammation in the nephropathy.
Materials and Methods
Statement of ethics
The Ethics Committees of Sendai Shakaihoken Hospital (approval number: 2004003) and Kumamoto Chuo Hospital (approval number: H16003) approved this study in accordance with the Declaration of Helsinki, and all patients gave written informed consent for participation in our study.
Patients and sample collection
We conducted a prospective observational study of 118 consecutive patients with type 2 diabetic nephropathy who were admitted to Sendai Shakaihoken Hospital or Kumamoto Chuo Hospital or who visited the hospitals for an annual medical examination to check their health. Sixty-one patients at stage 1 nephropathy (pre-nephropathy), 12 patients at stage 2 nephropathy (incipient nephropathy), 27 patients at stage 3 nephropathy (overt nephropathy) and 18 patients at stage 4 nephropathy (kidney failure) were included (Table 1); specific classifications are according to recent criteria by the Joint Committee on Diabetic Nephropathy in Japan (Haneda et al. 2015).
Among the 118 patients studied, 37 patients (1 patient at stage 2, 25 patients at stage 3 and 11 patients at stage 4 disease), who presented persistent proteinuria according to test tape results (> 30 mg/dL) and were unable to be diagnosed by clinical examinations, underwent renal biopsies. During this study, none of the 118 patients had urological diseases that cause hematuria. Patients at stage 1 or stage 2 all had negative results for hematuria. All patients studied here received dietary prescriptions and/or anti-diabetic drugs including insulin, biguanides, glinides, sulfonylureas, α-glucosidase inhibitors and thiazolidine. Patients had neither clinical interventions nor control of drug administration regimens, and no patients at any stage showed a clear bias in the use of any anti-diabetic drugs.
Blood and urine samples were collected on the same day according to routine practice from patients who had no anticoagulant or antiplatelet treatments and had taken no oral anti-diabetic drugs for 12 h or longer at sampling; samples were collected from patients within 24 h before renal biopsy. Urine samples, obtained at the second morning void within 2 h after the first void, were centrifuged at 370 × g for 10 min at 4°C. Supernatants were immediately stored at −80°C and used within 2 weeks.
Assay of urinary thrombin activity
We measured urinary thrombin activity by determining the hydrolytic activity of t-butyloxycarbonyl-l-valyl-l-prolyl-l-arginine 4-methyl-coumaryl-7-amide (Peptide Research Institute, Minoh, Japan), as previously reported (Morita et al. 1977; Kitamoto et al. 2004). To exclude the activity of proteinases other than thrombin in urine, we used hirudin (Markwardt et al. 1994), a specific inhibitor of thrombin, in combination with the substrate. In brief, we assessed the hydrolytic activity for the substrate in urine with a fluorescence spectrophotometer (excitation at 380 nm, emission at 440 nm) for 5 min or longer to obtain a linear increase in fluorescence. We then added a molar excess of hirudin and measured fluorescence for 5 min. We determined the urinary thrombin activity as hirudin-inhibited hydrolytic activity, expressed as thrombin units calibrated by using 1 U/L human thrombin (Calbiochem, San Diego, CA, USA) as a standard. The lower detection limit of thrombin activity in urine was 0.2 U/L. To evaluate urinary thrombin excretion, we expressed the result as thrombin activity equivalent to 1 g of creatinine (U/g Cr).
Validation of the thrombin assay
The coefficient of variation (CV) of 10 measurements of standard thrombin activity (50 U/L) added to a sample of pooled urine from 10 healthy volunteers and the CV of 10 measurements of thrombin activity in a patient’s urine sample with thrombin activity of 1.5 U/L were 3.4% and 5.3%, respectively. The addition of either 10 mg/dL hemoglobin or 2 g/dL glucose to the pooled urine sample did not significantly affect urinary thrombin activity. All urine samples had negligible fluorescence at 440 nm in the assay buffer, which indicated that drugs administered to patients did not interfere with thrombin measurement.
To determine the effect of storage at −80°C for 2 weeks on thrombin activity in urine samples, we added standard thrombin to thrombin-negative urine samples from 6 patients at final concentration of 10 U/L. These samples were then centrifuged at 370 × g for 10 min at 4°C, and supernatants were immediately stored at −80°C. We used these frozen samples to measure thrombin activity after 1 h and 2 weeks of storage periods. Thrombin activity measured after 2 weeks of storage decreased by 14.1% ± 11.8% (mean ± SD) compared with the activity after 1 h of storage (p < 0.05). Thrombin activity of urine samples was thus mostly stable under our storage condition.
Detection of nodular lesions and TF expression in glomeruli
We observed more than 20 glomeruli/a tissue section stained with periodic acid-Schiff, from 37 patients to detect glomerular nodular lesions, which are characteristic of type 2 diabetic nephropathy (Churg et al. 1995), with microscopy (BX51, Olympus, Tokyo, Japan). To investigate the activation of blood coagulation pathway (Davie et al. 1991) in glomeruli of the nephropathy, we examined TF expression in renal biopsy tissues. Because of small sizes of needle-biopsied tissues, only 10 patients (shown in Table 2) among 37 biopsied patients were studied for TF expression by immunostaining deparaffinized 3-µm-thick sections using anti-TF monoclonal antibody (Imamura et al. 1993) and the EnVision G|2 System/AP kit (Permanent Red) (Dako Japan, Tokyo, Japan) according to the manufacturer’s instructions. Normal mouse IgG (Dako) was used as a control. We counted the number of TF-positive cells in 15-16 glomeruli and showed an average number of TF-positive cells per glomerulus.
To investigate the inactivation of thrombin by antithrombin in urine, we measured urinary TAT levels in patients by using the enzyme-linked immunosorbent assay (Pelzer et al. 1988) and the latex photometric immunoassay LPIA-F·TAT test II (Mitsubishi/LSI Medience, Tokyo, Japan; CV 10%) with LPIA-A700 (Mitsubishi/LSI Medience).
Laboratory examinations of the patients’ samples
Concentrations of serum hemoglobin A1c, serum and urinary protein, serum and urinary albumin, and serum C-reactive protein (CRP) were measured using autoanalyzers in the laboratories of our hospitals.
Statistical analysis
StatView 7 from Abacus Concepts (Berkeley, CA, USA) was used for statistical analysis. The paired Student t-test was used to compare thrombin activities in urine samples of different storage periods. We used Fisher’s protected least significant difference test to compare patients’ characteristics and urinary thrombin excretion among patient groups at different stages of type 2 diabetic nephropathy, to compare the thrombin excretion between patient groups with and without nodular lesions, and to compare estimated glomerular filtration rate (eGFR) or proteinuria between patient groups with and without urinary thrombin (thrombinuria). We tested correlations and associations via regression analysis and Fisher’s z transformation, respectively. A p value of < 0.05 indicated statistical significance.
Results
Patients’ characteristics and thrombinuria in type 2 diabetic nephropathy at different stages
There were no differences in the age or HbA1c level among the patients at different stages of diabetic nephropathy (Table 1). Estimated glomerular filtration rate (eGFR) was calculated by using the formula (Matsuo et al. 2009): eGFR (mL/min/1.73 m2) = 194 × serum creatinine−1.094 × age−0.287 (× 0.739 if female). Patients with diabetic nephropathy at stage ١ had significantly (p < 0.001) higher eGFR than patients at stages 3 or 4. Patients at stage 2 had significantly (p < 0.001) higher eGFR than patients at stage 4. Patients at stage 3 had significantly (p < 0.001) higher eGFR than patients at stage 4. Patients at stage 4 manifested significantly more severer proteinuria than patients at stage 1 (p < 0.001), stage 2 (p < 0.001), or stage 3 (p < 0.05). Serum protein levels in patients at stages 1 or 2 were significantly (p < 0.001) higher than those in patients at stages 3 or 4. Serum albumin levels in patients at stages 1 or 2 were significantly (p < 0.01) higher than those in patients at stages 3 or 4.
Urinary thrombin was present at 4.9% (3/61), 0.0% (0/12), 29.6% (8/27) and 50.0% (9/18) of the patients at stage 1, stage 2, stage 3 and stage 4, respectively (Table 1). As shown in Fig. 1A, the 25th percentile, median and 75th percentile values for urinary thrombin excretion (U/g Cr) were (0, 0, 0) at stage 1, (0, 0, 0) at stage 2, (0, 0, 0.645) at stage 3 and (0, 0.095, 1.615) at stage 4, respectively. Therefore, both the occurrence and level of thrombinuria were the highest in patients at stage 4, followed by stage 3, and were negligible at stages 1 or 2. The occurrence of urinary thrombin in advanced nephropathy (stages 3 and 4) was 37.8% and was higher than that of early nephropathy (stages 1 and 2), which was 4.1% (Fig. 1B). The 25th percentile, median and 75th percentile values for urinary thrombin excretion (U/g Cr) were (0, 0, 1.08), respectively, for the advanced nephropathy and was significantly (p < 0.01) higher than those of the early nephropathy, which was (0, 0, 0), respectively. Thus, urinary thrombin was associated with the advanced diabetic nephropathy.
TF expression, a trigger of coagulation activation, in the glomerulus was compared with urinary thrombin. TF was expressed in the macrophages in the glomerular tissues irrespective of nodular lesions (Fig. 2A-C). Macrophages in the glomeruli expressed TF in all of the 10 patients examined, which was independent of thrombinuria (Table 2). In addition, the number of TF-positive macrophage per glomerulus did not correlate with the urinary thrombin excretion level of the patients (p = 0.1205).
Relationship between thrombinuria and glomerular nodular lesions
Glomerular nodular lesion was associated with histological features of inflammation (Zhao et al. 2004), thus, thrombinuria was compared with glomerular nodular lesions in the patients with diabetic nephropathy (Fig. 2D). Among the 37 patients with diabetic nephropathy who received renal biopsies, glomerular nodular lesions were present in 30 patients (1 patient at stage 2, 20 patients at stage 3 and 9 patients at stage 4) and absent in 7 patients (4 patients at stage 3 and 3 patients at stage 4). Thrombinuria was present in 11 patients (37%) (7 patients at stage 3 and 4 patients at stage 4) among the 30 nodular lesion-positive patients and present in 4 patients (57%) (1 patient at stage 3 and 3 patients at stage 4) among the 7 nodular lesion- negative patients. Twenty-fifth percentile, median, and 75th percentile values of urinary thrombin excretion (U/g Cr) were 0, 0 and 1.159 in patients with nodular lesions and 0, 0.190 and 0.402 in patients without nodular lesions, respectively. There was no difference in thrombin excretion level between the two groups.
Relationships between urinary thrombin and urinary TAT and between urinary TAT and proteinuria
Thrombin is converted to TAT by binding with antithrombin in the circulation, Thus, we compared urinary thrombin and urinary TAT concentrations in the patients with type 2 diabetic nephropathy. Urinary thrombin level did not correlate with urinary TAT in the patients with the nephropathy (Fig. 3A). Urinary TAT correlated strongly with urinary protein concentrations in the patients (Fig. 3B), which indicated that urinary TAT mostly derived from plasma TAT leaked from the circulation into urine together with other plasma proteins.
Relationship between urinary thrombin and serum CRP
To examine whether urinary thrombin activity was affected by systemic inflammation associated with the nephropathy patients, urinary thrombin was compared with serum CRP which increases by the inflammation. Urinary thrombin excretion levels did not correlate with serum CRP levels in the patients with diabetic nephropathy (Fig. 3C). Therefore, urinary thrombin may be independent of and unaffected by systemic inflammation in the patients.
Relationships of thrombinuria with eGFR and proteinuria
To study a relationship between glomerular inflammation and renal injury, urinary thrombin was compared with renal injury markers; eGFR and proteinuria. The 25th percentile, median and 75th percentile of eGFR of patients with thrombinuria were 24.5, 31.9 and 69.7 mL/min/1.73 m2, respectively, and was significantly lower than eGFR of patients without thrombinuria; the 25th percentile, median and 75th percentile were 52.7, 70.9 and 84.9 mL/min/1.73 m2, respectively (Fig. 4A, upper panel). Thrombin excretion correlated negatively with eGFR weakly (Fig. 4A, lower panel). These results indicated a significant association between increased urinary thrombin and decreased eGFR. The 25th percentile, median and 75th percentile of urinary protein concentration of patients with thrombinuria were 0.67, 3.91 and 5.54 g/g Cr, respectively, and was significantly higher than those of patients without thrombinuria; the 25th percentile, median and 75th percentile were 0.02, 0.04 and 1.57 g/g Cr, respectively (Fig. 4B, upper panel). Urinary thrombin excretion showed a weak correlation with urinary protein excretion (Fig. 4B, lower panel). These results indicated a significant association between increased urinary thrombin and increased urinary protein. Taken together, thrombinuria was associated with renal injury markers.
Discussion
We reported that thrombinuria was a novel marker specific for glomerular inflammation (Kitamoto et al. 2015). In the present study, we found thrombinuria in the patients with type 2 diabetic nephropathy. The increased occurrence and level of thrombinuria were associated with the progression of the nephropathy (Table 1, Fig. 1A, B), which indicated the presence of glomerular inflammation in the advanced nephropathy and suggested a possible involvement of glomerular inflammation in glomerular injuries and exacerbation of the nephropathy.
TF expression on the cell membrane of the extravasated macrophages (Fig. 2A-C) suggests that TF initiated the sequential activation of latent blood coagulation factors (Tipping et al. 1995; Levi and van der Poll 2010), which exudated into glomerular tissues (Perretti and Ahluwalia 2000). Then, thrombin generated in the glomerular tissues leaked into urine as thrombinuria. No thrombinuria in some patients in spite of TF expression in glomeruli (Table 2) may be explained by that plasma exudation, which supplies the plasma coagulation factors into the inflammatory glomerular tissues, may occur transiently. Subsequently, thrombin generation in the glomerular tissues may be transient, thus thrombinuria was not always found in the patients even under TF expression in glomeruli in type 2 diabetic nephropathy.
Thrombin may enhance glomerular inflammation as an inflammatory mediator (Bar-Shavit et al. 1983; Cunningham et al. 2000) and promote glomerular remodeling as a growth factor (Shultz et al. 1989), thereby participating in glomerular injuries in diabetic nephropathy. Glomerular nodular lesions lack capillaries, thereby do not provide urine, which may explain no association of thrombinuria with the presence of glomerular nodular lesions (Fig. 2D).
Elevated plasma TAT levels (Shimizu et al. 1995) in type 2 diabetic nephropathy due to increased thrombin generation in the bloodstream were reported. Under the regulation of blood coagulation, almost all the thrombin in the blood stream is promptly inactivated by antithrombin, which forms TAT (Lollar and Owen 1980) before leakage into glomeruli. Therefore, thrombin generated in the circulation does not appear in urine (Kitamoto et al. 2015). Urinary TAT levels did not correlate with urinary thrombin levels (Fig. 3A) but strongly correlated with proteinuria levels (Fig. 3B), which indicated that TAT in the circulation leaked into urine together with other plasma proteins through injured glomerular capillaries and accounted for most of the urinary TAT in the patients. Therefore, increased urinary TAT likely reflects increased TAT formation in the circulation caused by hypercoagulable state in rype 2 diabetic nephropathy.
No correlation between thrombinuria and serum CRP (Du Clos 2000) in Fig. 3C may be explained by that serum CRP is not much sensitive for glomerular inflammation (glomerulonephritis) (Kirac et al. 2014) and often rises by systemic inflammation complicated with type 2 diabetes (van Eeden et al. 1988).
Renal injury as shown by decreased eGFR and increased proteinuria was associated with glomerular inflammation indicated by the presence of thrombinuria (Fig. 4A, B), which suggests that glomerular inflammation may participate in glomerular injuries, deteriorating renal function in the patients with type 2 diabetic nephropathy.
Recently, anti-inflammatory effects of hypoglycemic drugs were reported in patients with type 2 diabetic nephropathy (Kothari et al. 2016). No significant difference in serum HbA1c level (Table 1) or no bias in the drug use among the nephropathy stages suggested a less possibility for the drug treatment to have caused the difference in thrombinuria among the stages.
In conclusion, we demonstrated the presence of glomerular inflammation in the patients with type 2 diabetic nephropathy at advanced stages by measuring urinary thrombin and suggested a possible contribution of glomerular inflammation to the progression of the nephropathy. Urinary thrombin may be applied to monitoring an effect of anti-diabetic treatments on glomerular inflammation in the patients with type 2 diabetic nephropathy.
Acknowledgments
We thank Dr. Shin Nakamura for providing anti-TF antibody; Ms. Hisako Sugai, Mr. Hiroshi Kitamura, and Ms. Tatsuko Kubo for technical assistance; Ms. Judith B. Gandy for excellent editing; and Drs. Tomohiro Nakagaki, Kensuke Joh, J. Evan Sadler, Qingyu Wu, Joji Sakurai, Hiroshi Maeda, Tohru Morioka, Sadaaki Iwanaga, Osamu Hotta, Toshinobu Sato, Tsuyoshi Kobayashi, and Shiroaki Shirato for encouragement. This work was supported by the Miyagi Kidney Foundation (Grant-in-Aid #H16 to Y.K.).
Conflict of Interest
The authors declare no conflict of interest.
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