Urinary Thrombin - Semantic Scholar

RESEARCH ARTICLE

Urinary Thrombin: A Novel Marker of Glomerular Inflammation for the Diagnosis of Crescentic Glomerulonephritis (Prospective Observational Study) Yasunori Kitamoto1*, Kenji Arizono2, Hiroyoshi Fukui2, Kimio Tomita3, Hiroshi Kitamura4, Yoshio Taguma4, Takahisa Imamura5

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1 Department of Laboratory Medicine, JCHO Sendai Hospital, Sendai, Japan, 2 Department of Nephrology, Kumamoto Chuo Hospital, Kumamoto, Japan, 3 Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan, 4 Department of Nephrology, JCHO Sendai Hospital, Sendai, Japan, 5 Department of Molecular Pathology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan * [email protected]

Abstract OPEN ACCESS Citation: Kitamoto Y, Arizono K, Fukui H, Tomita K, Kitamura H, Taguma Y, et al. (2015) Urinary Thrombin: A Novel Marker of Glomerular Inflammation for the Diagnosis of Crescentic Glomerulonephritis (Prospective Observational Study). PLoS ONE 10(3): e0118704. doi:10.1371/ journal.pone.0118704 Academic Editor: Utpal Sen, University of Louisville, UNITED STATES Received: October 24, 2014 Accepted: January 14, 2015 Published: March 5, 2015 Copyright: © 2015 Kitamoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Miyagi Kidney Foundation; Grant in Aid #H16; YK, http://www.miyajin.or.jp/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Background Crescentic glomerulonephritis (CresGN), an uncommon rapidly progressive disease, is characterized by severe glomerular inflammation with fibrin deposition. The lack of specific CresGN biomarkers delays diagnosis and threatens life. Because fibrin deposits in CresGN glomeruli indicate thrombin generation, we hypothesized that thrombin is excreted in urine and is a specific CresGN biomarker.

Methods We measured urinary thrombin activity in 200 untreated patients (17 with CresGN, 183 with primary glomerulonephritis) and controls (8 patients with healed CresGN, 11 with nephrosclerosis, and 10 with tubulointerstitial nephritis, and 66 healthy volunteers). CresGN types included 15 pauci-immune and 2 immune complex. We assessed the diagnostic accuracy of thrombinuria in 169 patients with hematuria and proteinuria. Renal biopsy tissues were immunostained for tissue factor and fibrin. We analyzed the relationship of thrombinuria to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, glomerular fibrin deposition, antineutrophil cytoplasmic antibodies (ANCAs), and C-reactive protein (CRP). We studied changes in thrombin activities after glucocorticoid treatment in 12 patients with thrombinuria.

Results The highest thrombinuria occurrence was in CresGN (70.6%), followed by membranoproliferative glomerulonephritis (41.7%), IgA nephropathy (9.2%), and acute glomerulonephritis (0%). More than 75% of patients with nonproliferative glomerulonephritis manifested no

PLOS ONE | DOI:10.1371/journal.pone.0118704 March 5, 2015

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Urinary Thrombin in Patients with Crescentic Glomerulonephritis

thrombinuria. No controls had thrombinuria. Thrombinuria showed high CresGN specificity (90.1%) and moderate sensitivity (70.6%) and was detected in 4 of 7 patients with ANCAnegative CresGN. In CresGN, thrombinuria was associated with fibrin deposition in glomerular extracapillary tissue, where monocytes/macrophages expressed tissue factor. Thrombinuria in CresGN was unrelated to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, and CRP. After glucocorticoid treatment, thrombinuria in patients with CresGN rapidly disappeared but proteinuria and hematuria persisted.

Conclusions Thrombinuria was specific for glomerular inflammation, was unaffected by systemic inflammation or coagulation, and demonstrated good diagnostic accuracy for CresGN including ANCA-negative cases. Thrombinuria measurement may provide risk-free diagnosis and screening for CresGN.

Introduction Crescentic glomerulonephritis (CresGN), an uncommon [1] but devastating disease, rapidly progresses to renal failure [2]; however, patients receiving proper treatment at early stages may heal without impaired renal function. Therefore, early diagnosis and treatment are crucial for improving the poor prognosis of this disease. Since Bright’s report in 1827 [3,4], proteinuria, caused by an impaired barrier against plasma protein leakage at the glomerular capillary wall [5], has been a marker for glomerular diseases caused by inflammation, hypertension, and metabolic or hereditary disorders, but proteinuria is not specific for CresGN. Serum antineutrophil cytoplasmic antibodies (ANCAs) are used to diagnose pauci-immune CresGN, but a significant proportion (10–30%) of patients with pauci-immune CresGN [6] and most patients with immune-complex CresGN are negative for ANCAs [7]. Thus, a substantial number of patients with CresGN remain undiagnosed if only the ANCA test is used. Definitive diagnosis of CresGN requires histopathological examination of renal biopsy tissues. However, biopsy is invasive, causes patients pain, and risks serious bleeding, and routine sections from biopsy tissues do not always include the characteristic crescentic lesions, which are focally or segmentally distributed. Thus, noninvasive and specific diagnostic methods have been sought for early CresGN diagnosis. Inflammation triggers the tissue factor pathway of blood coagulation [8]. Tissue factor is expressed in extravascular inflamed tissues, where plasma leaks because of increased vascular permeability [9], and activates blood coagulation factors (inactive precursors) in the leaked plasma. The ultimate product, thrombin, converts fibrinogen to fibrin, which deposits in lesions. CresGN features severe glomerular inflammation and glomerular crescent formation, often with fibrin deposition in glomerular extracapillary tissue [10] that is caused by thrombin probably generated via the monocyte/macrophage tissue factor-dependent coagulation pathway [11–15]. The pathogenesis of experimental CresGN [16] involved thrombin and stimulation of its receptor, protease-activated receptor-1 [17]. Thrombin may also contribute to glomerular inflammation by modulating monocyte/macrophage chemotaxis [18]. These close associations of thrombin with CresGN suggest that this protease may be a CresGN biomarker. However, thrombin has not been investigated in patients with CresGN, probably because no assay method exists for lesional thrombin activity.


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Urine reflects the glomerular milieu and is obtained without pain or risk, so urine may be an appropriate material for estimating thrombin generation in inflamed glomeruli. Therefore, to investigate the feasibility of using urinary thrombin as a diagnostic indicator of CresGN, we utilized a urinary thrombin assay [19,20] to evaluate patients with various glomerulonephritides, which led us to propose the application of urinary thrombin activity to CresGN diagnosis and screening.

Methods Patients and sample collection We studied 200 patients with untreated glomerulonephritis (17 patients with CresGN and 183 patients with other types of primary glomerulonephritis) who were admitted to Sendai Shakaihoken Hospital or Kumamoto Chuo Hospital from 2003 to 2011 (Table 1). In our 17 patients with CresGN (11 from Kumamoto Chuo Hospital, 6 from Sendai Shakaihoken Hospital), more than 50% of glomeruli had crescentic lesions in renal biopsy specimens (diffuse CresGN); 15 patients had the pauci-immune type of the disease and 2 had the immune-complex type (1 IgA nephropathy [IgAN], 1 rheumatoid arthritis). Patients with healed CresGN (N = 8) were age- and sex-matched with patients who had untreated CresGN. We judged CresGN healed when the abnormal urinary findings (proteinuria and hematuria) disappeared. For ANCA-associated CresGN, we added the disappearance of serum ANCAs to judge healing. Nephrosclerosis (N = 11) and tubulointerstitial nephritis (TIN; N = 10) were studied as a representative non-inflammatory glomerular disease and as a representative tubulointerstitial inflammatory disease, respectively. Our study also included healthy volunteers (N = 66) who visited our hospital for a routine health check and had no abnormal urinary findings or impaired renal function and were receiving no anticoagulant, antiplatelet, or anti-inflammatory drugs. Patients with primary glomerulonephritis included 87 patients with IgAN, 9 with nonIgA mesangial proliferative glomerulonephritis (non-IgA mesPGN), 12 with membranoproliferative glomerulonephritis (MPGN), 9 with acute glomerulonephritis (AGN), 13 with minimal change glomerulopathy (MCNS), 17 with focal segmental glomerulosclerosis (FSGS), and 36 with membranous glomerulopathy (MN). Ethics Committees of Sendai Shakaihoken Hospital (approval number: 2004003) and Kumamoto Chuo Hospital (approval number: H15003) approved this study, and all patients and volunteers gave written informed consent for participation. We used clinical examination findings, including pathological studies of renal biopsy specimens, to diagnose all consecutive patients, after which they received standard treatment. We diagnosed AGN by evaluating a combination of clinical features and abnormal serological findings including hypocomplementemia and an elevated titer of antistreptolysin O (7 patients) or human parvovirus B19-specific IgM antibodies [21] (2 patients). We collected blood and urine samples within 24 hours before renal biopsy except for patients with AGN, whose samples we collected when the patients manifested hypocomplementemia. Urine samples obtained at the second morning void within 2 hours after the first void were centrifuged at 370 × g for 10 minutes at 4°C; supernatants were promptly stored at -80°C until use.

Thrombin measurement We measured urinary thrombin activity by using the cleaving activity of t-butyloxycarbonylL-valyl-L-prolyl-L-arginine 4-methyl-coumaryl-7-amide (Peptide Research Institute, Minoh, Japan), which was inhibited by hirudin, a thrombin-specific inhibitor, as reported previously [19]. Briefly, we measured the hydrolytic activity of the substrate in urine with a fluorescence spectrophotometer (excitation at 380 nm, emission at 440 nm), which was connected to a


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Table 1. Characteristics of the patients. Disease

No. of patients (%)

Sex (male/ female)

Age (yr) (mean±SD)*

Proteinuria (g/gCr) (mean±SD)†

eGFR (ml/min/1.73 m2) (mean±SD)‡

Crescentic glomerulonephritis (CresGN)

17 (8.5)

IgA nephropathy (IgAN)

87 (43.5)

9/8

64.1±12.7

2.79±2.58

14.7±9.5

35/52

32.6±14.4

1.09±2.22

81.1±30.7

Non-IgA mesangial proliferative glomerulonephritis (non-IgA mesPGN)

9 (4.5)

3/6

46.7±17.3

0.75±1.37

72.6±24.1

Membranoproliferative glomerulonephritis (MPGN)

12 (6.0)

7/5

39.9±15.5

2.21±2.08

64.7±21.5

Acute glomerulonephritis (AGN)

9 (4.5)

6/3

41.9±20.7

1.37±1.87

68.7±24.2

Minimal change glomerulopathy (MCNS)

13 (6.5)

8/5

41.6±18.9

6.04±2.43

83.4±20.0

Focal segmental glomerulosclerosis (FSGS)

17 (8.5)

6/11

46.1±14.8

4.05±3.80

67.9±34.7

Membranous glomerulopathy (MN)

36 (18.0)

21/15

62.9±10.8

4.99±6.76

74.0±24.2

Subtotal

200 (100)

Healed crescentic glomerulonephritis

8

4/4

66.9 ± 10.7

Negative§

40.8 ± 22.8

Nephrosclerosis

11

4/7

61.0 ± 9.7

0.63±0.90

54.2 ± 29.9

Tubulointerstitial nephritis (TIN)

10

5/5

50.8. ± 19.6

0.54±0.56

27,9 ± 17.2

Healthy volunteers

66

42/24

41.4±10.3

Negative§

82.9±15.7

*The ages of patients with healed CresGN, CresGN, MN, or nephrosclerosis were signiﬁcantly higher than those of the other groups (P