Urinary Plasminogen Activator Inhibitor-1: A Biomarker of Acute Tubular Injury.

INTRODUCTION: Acute kidney injury (AKI) is a threatening, multiaetiological syndrome encompassing a variety of forms and damage patterns. AKI lacks sufficiently specific diagnostic tools to evaluate the distinct combination of pathophysiological events underlying each case, which limits personalized and optimized handling. Therefore, a pathophysiological diagnosis based on new urinary biomarkers is sought for practical (readiness and noninvasiveness) and conceptual reasons, as the urine is a direct product of the kidneys. However, biomarkers found in the urine may also have extrarenal origin, thus conveying pathophysiological information from other organs or tissues. Urinary plasminogen activator inhibitor-1 (PAI-1) has been associated to AKI, although its origin and traffic to the urine are not known. METHODS: Herein, we studied the blood or renal origin of urinary PAI-1 (uPAI-1) in experimental AKI in Wistar rats, by means of the in situ renal perfusion method. For this purpose, urine was collected while the kidneys of rats with AKI showing increased uPAI-1 excretion, and controls, were in situ perfused with a saline solution. RESULTS: Our results show that during perfusion, PAI-1 remained in the urine of AKI rats, suggesting that renal cells shed this protein directly to the urine. PAI-1 is also significantly increased in the urine of AKI patients. Its low correlation with other urinary markers such as NGAL or NAG suggests that PAI-1 provides complementary and distinct phenotypical information. CONCLUSION: In conclusion, uPAI-1 is a biomarker produced by damaged kidneys following AKI, whose precise pathophysiological meaning in AKI needs to be further investigated.

Clinical value of inflammatory urinary biomarkers in overt diabetic nephropathy: a prospective study.

AIMS: The evolution of diabetic nephropathy is incompletely accounted by current clinical tools. New biomarkers may refine patient assessment and help monitor therapy. We compared the added predictive value of 7 candidate inflammatory urinary biomarkers to known risk factors of progression. METHODS: We prospectively followed 83 patients with overt diabetic nephropathy for a median 2.1 years and obtained repeated measurements of proteinuria, IL-1ß, IL-6, IL-8, MCP-1, TNF-α, TGF-ß1, and PAI-1. RESULTS: Patients had an initial estimated glomerular filtration rate of 25 ± 9 mL/min/1.73 m(2), blood pressure of 142/69 mmHg and used a median of 4 anti-hypertensive medications over the course of the study. The observed rate of renal function decline was 2.9 ± 3.0 mL/min/1.73 m(2)/year. All urinary biomarkers levels were collinear and for each one except IL-1ß, elevated levels predicted a more rapid progression. MCP-1 was the only biomarker increasing during follow-up, which also correlated with a worst outcome. Using multivariate linear regression adjusting for clinical risk factors of progression, urinary MCP-1 and TGF-ß1 predicted progression independently and additively to the degree of proteinuria. We dichotomized these 3 biomarkers and observed a renal function decline with 0, 1, 2 or 3 elevated biomarkers of -0.8 ± 1.4, -2.1 ± 2.1, -4.2 ± 2.8 and -6.0 ± 2.8 mL/min/1.73 m(2)/year, respectively (p<0.001). CONCLUSIONS: Multiple urinary biomarkers predict outcome in overt diabetic nephropathy. However, urinary MCP-1 and TGF-ß1 are also independent and additive to proteinuria in predicting the rate of renal function decline and could serve as useful clinical tools in patient risk stratification.

CCL18 in a multiplex urine-based assay for the detection of bladder cancer.

The early detection of bladder cancer (BCa) is pivotal for successful patient treatment and management. Through genomic and proteomic studies, we have identified a number of bladder cancer-associated biomarkers that have potential clinical utility. In a case-control study, we examined voided urines from 127 subjects: 64 tumor-bearing subjects and 63 controls. The urine concentrations of the following proteins were assessed by enzyme-linked immunosorbent assay (ELISA); C-C motif chemokine 18 (CCL18), Plasminogen Activator Inhibitor 1 (PAI-1) and CD44. Data were compared to a commercial ELISA-based BCa detection assay (BTA-Trak©) and voided urinary cytology. We used analysis of the area under the curve of receiver operating characteristic curves to compare the ability of CCL18, PAI-1, CD44, and BTA to detect BCa in voided urine samples. Urinary concentrations of CCL18, PAI-1, and BTA were significantly elevated in subjects with BCa. CCL18 was the most accurate biomarker (AUC; 0.919; 95% confidence interval [CI], 0.8704-0.9674). Multivariate regression analysis highlighted CCL18 (OR; 18.31; 95% CI, 4.95-67.70, p<0.0001) and BTA (OR; 6.43; 95% CI, 1.86-22.21, p = 0.0033) as independent predictors of BCa in voided urine samples. The combination of CCL18, PAI-1 and CD44 improved the area under the curve to 0.938. Preliminary results indicate that CCL18 was a highly accurate biomarker for BCa detection in this cohort. Monitoring CCL18 in voided urine samples has the potential to improve non-invasive tests for BCa diagnosis. Furthermore using the combination of CCL18, PAI-1 and CD44 may make the model more robust to errors to detect BCa over the individual biomarkers or BTA.

[Determination of urinary markers of proteolysis/fibrinolysis and fibroangiogenesis in the kidney in hypertensive patients].

AIM: To determine clinical significance of urinary biomarkers of proteolysis/fibrinolysis and fibroangiogenesis in essential hypertension (EH). MATERIAL AND METHODS: Examination of the kidneys was made in 71 patients with EH degree 1-3. Renal function was assessed by 24-h albuminuria, calculated glomerular filtration rate (GFR) by Cockroft-Golt. Early signs of renal damage were microalbuminuria--MAU (diurnal albuminuria 30-300 mg/day), reduction of GFR (< 90 ml/min/1.73 m2). EH patients with hypercreatininemia and GFR under 60 ml/min/1.73m2 corresponding to stage III of chronic kidney disease were not included in the study. An additional nephropathy marker was an elevated index of resistance of interlobular renal arteries (RI > 0.65) as shown by dopplerometry. ELISA examined urinary biomarkers of intercellular and cell-matrix interactions in the kidney in EHpatients and healthy controls (n = 12). RESULTS: MAU was detected in 54 (76%) of 71 EH patients, elevated RI > 0.65--in 37 (52%) patients. Urinary biomarkers of proteolysis/fibrinolysis and fibroangiogenesis were higher in EH patients then in the controls. Urinary excretion of PAI-1, TGF-beta1, VEGF and collagen of type IV in EH patients with MAU was significantly higher than in patients with normoalbuminuria. A strong direct correlation between MAU and the rest above urinary biomarkers was found as well as between urinary excretion of collagen IV and RI. An inverse negative relationship was seen between RI and GFR. CONCLUSION: Renal impairment in EHpatients is a progressive disorder. Each stage of this process has its own clinicodiagnostic markers. Urinary biomarkers ofproteolysis/fibrinolysis and fibroangiogenesis in the kidney are informative for monitoring of early HNP.

Prognostic impact of plasminogen activator inhibitor type 1 expression in bladder cancer.

BACKGROUND: Recent studies have demonstrated a poor prognosis for patients who have altered expression of plasminogen activator inhibitor type 1 (PAI-1) in several cancer types. The objective of the current study was to investigate the prognostic impact of PAI-1 on patients with transitional cell carcinoma (TCC) of the urinary bladder. METHODS: PAI-1 expression was quantified using real-time polymerase chain reaction in 91 TCCs and in 6 normal tissue specimens. PAI-1 concentrations were analyzed by enzyme-linked immunoadsorbent assay in plasma from 104 patients and 10 controls and in urine from 244 patients and 74 controls. PAI-1 expression was evaluated immunohistochemically in paraffin-embedded tissues (94 tumor samples and 10 adjacent normal tissue samples). The results were analyzed in relation to clinical features and follow-up. RESULTS: Significantly higher PAI-1 levels were detected in tissue and plasma samples, but not in urine, from patients with bladder cancer compared with controls (P=.001 and P=.008, respectively). Elevated gene expression and plasma protein concentrations were independent of tumor stage and grade. Immunostaining revealed a subgroup of patients with single tumor cells that strongly expressed PAI-1. These patients' survival was significantly shorter, and their clinical presentation was correlated significantly with lymph node-positive disease. CONCLUSIONS: PAI-1 gene expression in tissues and plasma protein levels were elevated in patients with TCC compared with controls. PAI-1 gene or protein expression was not associated with the clinical characteristics of bladder TCC. Although the assessment of PAI-1 expression in plasma or urine may not serve as an independent predictor of prognosis in patients with TCC, the immunohistochemical detection of single PAI-1-positive cells may serve as a predictor of survival and a possible indicator of metastasis.

[Significance of the markers of endothelial dysfunction and hemorheological disorders for assessing the activity and prognosis of chronic glomerulonephritis].

AIM: to define the clinical value of changes in blood rheological properties and renal endothelial function in patients with hematuric and nephritic forms of chronic glomerulonephritis (CGN) and to ascertain whether the indices under study can be applied to assess the activity (progression) of nephritis and used as a prognostic criteria. SUBJECTS AND METHODS: Sixty-one patients, including 30 with hematuric nephritis (Group 1) and 31 with nephrotic nephritis (Group 2), were examined. A control group consisted of 12 healthy individuals. The rheological properties of blood, such as its viscosity; kinetics of spontaneous aggregation and disaggregation of red blood cells in shear flow; their deformability; urinary excretion of functionally active von Willebrandt factor (WF), a plasminogen activator inhibitor type 1 (PAl-1); urine total fibrinolytic activity (UTFA), activity of urinary urokinase-type plasminogen activator (UPA) were studied. RESULTS: The patients with CGN were found to have signs of impaired blood rheological properties (increased viscosity, an accelerated rapid phase of erythrocyte aggregation, increased strength of erythrocyte aggregates) and vascular endothelial dysfunction in the microcirculatory bed, among other factors, increased urinary excretion of functionally active WF, PA-1, which correlated with the activity of CGN. Data were obtained on the negative impact of the level of urinary PAl-1 excretion, red blood cell aggregation on the prognosis of CGN regardless of its form, the markers of endothelial damage/activation. Low urokinase activity and decreased red blood cell deformability in parallel with higher diurnal proteinuria are of poor prognostic value for hematuric nephritis. CONCLUSION: The findings illustrate two ways of the involvement of the endothelium in the mechanisms contributing to the development of tubular interstitial fibrosis, namely: endothelial dysfunction and as a substrate that links the processes of immune inflammation, hemorheology, and fibrinolysis/proteolysis in the kidney. The regularities revealed by clinical and laboratory comparison suggest that the indices under study may be used to determine the prognosis of the disease and may serve as a basis for the application of treatments aimed at correcting the detected disorders.

[Clinical implications of urine matrix metalloproteinases and their inhibitors in patients with chronic glomerulonephritis].

AIM: Estimation of urinary excretion of matrix metalloproteinases (MMP) and their inhibitors in patients with chronic glomerulonephritis (CGN), specification of the role of MMP and inhibitors as criteria of CGN activity and prognosis. MATERIAL AND METHODS: ELISA was used for measurement of urinary levels of basic proteolysis system components (MMP-2 and MMP-9), tissue inhibitor TIMP-2 and plasminogen activator inhibitor PAI-1 in four groups of patients. Patients of group 1 (n = 23) had CGN with manifest urinary syndrome (US), of group 2 (n = 26)--CGN with nephritic syndrome (NS), of group 3 (n = 22)--CGN with marked proteinuria (PU) and transient renal failure (RF), group 4 (n = 15)--CGN with high PU and persistent RF. RESULTS: Patients with enhancing CGN activity (marked US, developing NS, acute nephritic syndrome) had balanced elevation of urinary levels of MMP, TIMP and PAI-1. Development of persistent RF in CGN occurred with imbalance between components of proteolysis system--low urine excretion of MMP and elevation of PAI-1. Urine excretion of MMP and TIMP in patients with progressive CGN directly correlated with 24-h PU and negatively correlated with blood serum creatinine. PAI-1 correlated with severity of RF and fibrosis in renal tissue. CONCLUSION: Correlation of changes in urinary excretion of MMP, TIMP and PAI-1 with CGN activity, RF and fibrosis in the kidney confirm the importance of the above urinary tests for estimation of local renal proteolysis and validity of their use for monitoring of extracellulary matrix accumulation (fibrosis) in the kidney and for CGN prognosis.

Effect of renin-angiotensin-aldosterone system triple blockade on non-diabetic renal disease: addition of an aldosterone blocker, spironolactone, to combination treatment with an angiotensin-converting enzyme inhibitor and angiotensin II receptor blocker.

Although dual blockade of the renin-angiotensin-aldosterone system (RAAS) with the combination of an angiotensin-converting enzyme inhibitor (ACE-I) and angiotensin II receptor blocker (ARB) is generally well-established as a treatment for nephropathy, this treatment is not fully effective in some patients. Based on the recent evidence implicating aldosterone in renal disease progression, this study was conducted to examine the efficacy of blockade with three different mechanisms by adding an aldosterone blocker in patients who do not respond adequately to the dual blockade. A 1-year randomized, open-label, multicenter, prospective controlled study was conducted, in which 32 non-diabetic nephropathy patients with proteinuria exceeding 0.5 g/day were enrolled after more than 12 weeks of ACE-I (5 mg enalapril) and ARB (50 mg losartan) combination treatment. These patients were allocated into two groups of 16 patients each: a triple blockade group in which 25 mg of spironolactone daily was added to the ACE-I and ARB combination treatment, and a control group in which 1 mg of trichlormethiazide or 20 mg of furosemide was added to the combination treatment instead of spironolactone depending upon the creatinine level. After 1 year of treatment, the urinary protein level decreased by 58% (p<0.05) with the triple blockade but was unchanged in the controls. Furthermore, urinary type IV collagen level decreased by 40% (p<0.05) with the triple blockade but was unchanged in the controls. The decreases in urinary protein and urinary type IV collagen were not accompanied by a decrease in blood pressure. Mean serum creatinine, potassium and blood pressure did not change significantly by either treatment. In conclusion, triple blockade of the RAAS was effective for the treatment of proteinuria in patients with non-diabetic nephropathy whose increased urinary protein had not responded sufficiently to a dual blockade.

[Clinical implication of urine test for markers of endothelial dysfunction and angiogenesis factors in assessment of tubulointerstitial fibrosis in chronic glomerulonephritis].

AIM: To evaluate contribution of endothelial dysfunction and impairment of endothelial proliferation/ regeneration to mechanisms of development of tubulointerstitial fibrosis (TIF) in chronic glomerulonephritis (CGN) basing on urinary levels of markers of endothelial activation/impairment and angiogenesis factors. MATERIAL AND METHODS: A total of 67 CGN patients entered the study: 19 patients with moderate urinary syndrome (group 1), 37 patients with nephrotic syndrome (group 2), 11 patients with nephrotic syndrome and persistent renal failure (RF). A control group consisted of 12 healthy subjects. The examination covered excretion with urine of Willebrand factor (WF), plasminogen activator inhibitor I (PAL-I), fibrin degradation products (FDP), vascular endothelial growth factor (VEGF). These values were compared with severity of fibrous changes in renal interstitium estimated by biopsy morphometry. RESULTS: CGN patients had signs of affection of parietal effects of vascular endothelium. In particular, increased excretion of functionally active WF, PAI-I and FDP correlating with activity/severity of CGN. The changes were especially noticeable in patients with progressive forms of CGN (with NS and RF). Patients with morphologically verified TIF (interstitial area more than 20%) excretion of endothelial dysfunction markers was higher than in CGN patients free of TIF In a progressive course of nephritis endothelial dysfunction deteriorates by endothelial proliferation/regeneration impairment as shown by reduced urinary excretion of angiogenic factor VEGF and parallel elevation of functionally active WF in urine of patients with severe forms of CGN. Combined contribution of endothelial dysfunction and angiogenesis impairment to mechanisms of TIF development is seen from these values relations with severity of creatinemia and fibrous alterations in tubulointerstitial tissues of the kidney. CONCLUSION: The results point to participation of endothelium in mechanisms promoting development of TIF and RF in CGN both in terms of endothelial dysfunction and impairment of endothelial repair capacity. Clinicomorphological comparisons confirm the significance of WF, PAI-I and VEGF in assessment of local-renal endothelial changes and severity of fibrosis in renal tissue in CGN. Due to availability of the study material, perspectives of fibrogenesis monitoring in the kidneys with the tests appear which is essential for making prognosis and treatment policy in CGN patients.

Effects of losartan on urinary secretion of extracellular matrix and their modulators in type 2 diabetes mellitus patients with microalbuminuria.

PURPOSE: Angiotensin II receptor Type 1 antagonists postpone the development of nephropathy in type 2 diabetes mellitus (DM). We hypothesize that Losartan may ameliorate renal function in diabetic patients through the regulation on the generation of transforming growth factor (TGF)-beta and fibrinolytic regulators. METHODS: Twenty-two type 2 DM patients with microalbuminuria were treated with 50-100 mg/day of Losartan for 6 months. Urinary secretion of TGF-, plasminogen activator inhibitor-1 (PAI-1), tissue and urokinase plasminogen activators (tPA and uPA) fibronectin, collagen IV and plasma levels of TGF-beta, PAI-1, tPA and uPA of the patients before and after the treatment were analyzed using enzyme-linked immunoabosorbance assay. RESULTS: Losartan effectively reduced arterial blood pressure and urinary albumin excretion. The levels of TGF-beta in urine, but not in plasma, were reduced after 2, 4 and 6 months of the treatment (-32% to -48%, P < 0.05 or 0.01). Urinary or plasma levels of PAI-1, tPA or uPA, and urinary secretion of fibronectin or collagen IV were not significantly altered by Losartan treatment. Urinary levels of collagen IV positively correlated with uPA, and that of fibronectin negatively correlated with PAI-1 in the patients (P < 0.01). Urinary TGF-beta negatively correlated uPA in urine of the patients (P < 0.01). CONCLUSION: Losartan reduced urinary excretion of TGF-beta and albumin in type 2 DM patients with microalbuminuria. Fibrinolytic regulators and TGF-beta are implicated in the regulation of ECM turnover in kidneys of the patients with diabetic nephropathy.

[Diabetic nephropathy and plasminogen activator inhibitor 1 in urine samples].

Plasminogen activator inhibitor-1 (PAI-1) may contribute to renal fibrosis because of its involvement in matrix (ECM) accumulation through inhibition of plasmin-dependent ECM degradation. The aim of this study is to determine urinary PAI-1 concentrations and its intrarenal localization in patients with various renal diseases and to identify inducers for PAI-1 expression in human cultured proximal renal tubular cells (HRCs). Urinary PAI-1 concentrations were significantly higher in patients with overt diabetic nephropathy (DN, n=36) than in proliferative glomerulonephritis (PGN, n=8), nephrotic syndrome (NS, n=10) and healthy controls (n=12). Urinary PAI-1 concentrations (ng/gCr) were directly correlated with urinary N-acetyl glucosaminidase (NAG) levels (r=0.58, p<0.05). As for intrarenal localization of PAI-1 antigen, strong stainings for PAI-1 were observed in proximal tubular cells of renal biopsy samples from patients with DN, while no stainings for PAI-1 were found in renal tissues of PGN or NS. Immunoblot analysis revealed the presence of PAI-1 protein in whole cell lyzates from HRCs grown to semiconfluency. Exposure of growth-arrested HRCs with hypoxia (1% O2) or TNF-alpha (10 ng/ml) for 24 hours increased the secretion rate of PAI-1 protein by about 2.0-fold, while 24-hour treatment with high glucose (450 mg/dl) did not increase PAI-1 secretion at all, compared with that of the control cells under normal glucose (100 mg/dl) and normoxia (18% O2). These findings suggest that PAI-1 expression is upregulated especially in the proximal renal tubular cells of DN, which may be explained partially by hypoxia and inflammatory cytokines but not high glucose.

Enhanced expression of plasminogen activator inhibitor 1 in patients with nephrotic syndrome.

Hypercoagulability is present in patients with nephrotic syndrome. However, alterations in coagulation and fibrinolysis reflected in the glomeruli and urine are not fully understood. We examined plasma and urine concentrations of tissue-type plasminogen activator (tPA) and type 1 plasminogen activator inhibitor (PAI-1) in 33 patients with nephrotic syndrome (nephrotic group). We compared these concentrations with the concentrations in 30 nonnephrotic patients with chronic glomerulonephritis (nonnephrotic group) and with the concentrations in 30 healthy volunteers (control group). We also examined fibrin/fibrinogen degradation products in serum and urine and plasma D-dimers. The expression of tPA and PAI-1 was examined in isolated glomeruli using RT-PCR methods. Deposition of fibrinogen/fibrin-related antigen was observed by direct immunofluorescence. The incidence of fibrinogen/fibrin-related antigen deposition in the nephrotic group was significantly higher than that in the nonnephrotic group. The concentrations of fibrin/fibrinogen degradation products in serum and urine and of plasma D-dimers were significantly elevated in the nephrotic group as compared with the nonnephrotic and control groups. The plasma concentrations of tPA in the nephrotic group were significantly higher than those in the control group. The urinary excretion of tPA in the nephrotic group was also significantly higher than in the nonnephrotic and control groups. The urinary excretion of PAI-1 in the nephrotic group was higher than that in the control group. The ratio of PAI-1 mRNA to tPA mRNA in glomeruli was increased in the nephrotic group as compared with the nonnephrotic group. These results indicate that the fibrinolytic activity is increased in patients with nephrotic syndrome despite urinary losses of tPA. However, a relatively enhanced expression of PAI-1 may be involved in the intraglomerular fibrinogen/fibrin-related antigen deposition seen in nephrotic syndrome.