Urine Angiotensin II Signature Proteins as Markers of Fibrosis in Kidney Transplant Recipients.

BACKGROUND: Interstitial fibrosis/tubular atrophy (IFTA) is an important cause of kidney allograft loss; however, noninvasive markers to identify IFTA or guide antifibrotic therapy are lacking. Using angiotensin II (AngII) as the prototypical inducer of IFTA, we previously identified 83 AngII-regulated proteins in vitro. We developed mass spectrometry-based assays for quantification of 6 AngII signature proteins (bone marrow stromal cell antigen 1, glutamine synthetase [GLNA], laminin subunit beta-2, lysophospholipase I, ras homolog family member B, and thrombospondin-I [TSP1]) and hypothesized that their urine excretion will correlate with IFTA in kidney transplant patients. METHODS: Urine excretion of 6 AngII-regulated proteins was quantified using selected reaction monitoring and normalized by urine creatinine. Immunohistochemistry was used to assess protein expression of TSP1 and GLNA in kidney biopsies. RESULTS: The urine excretion rates of AngII-regulated proteins were found to be increased in 15 kidney transplant recipients with IFTA compared with 20 matched controls with no IFTA (mean log2[fmol/µmol of creatinine], bone marrow stromal cell antigen 1: 3.8 versus 3.0, P = 0.03; GLNA: 1.2 versus -0.4, P = 0.03; laminin subunit beta-2: 6.1 versus 5.4, P = 0.06; lysophospholipase I: 2.1 versus 0.6, P = 0.002; ras homolog family member B: 1.2 versus -0.1, P = 0.006; TSP1_GGV: 2.5 versus 1.9; P = 0.15; and TSP1_TIV: 2.0 versus 0.6, P = 0.0006). Receiver operating characteristic curve analysis demonstrated an area under the curve = 0.86 for the ability of urine AngII signature proteins to discriminate IFTA from controls. Urine excretion of AngII signature proteins correlated strongly with chronic IFTA and total inflammation. In a separate cohort of 19 kidney transplant recipients, the urine excretion of these 6 proteins was significantly lower following therapy with AngII inhibitors (P < 0.05). CONCLUSIONS: AngII-regulated proteins may represent markers of IFTA and guide antifibrotic therapies.

Proteomics analysis of altered proteins in kidney of mice with aristolochic acid nephropathy using the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method.

Aristolochic acid (AA) causes interstitial renal fibrosis, called aristolochic acid nephropathy (AAN). There is no specific indicator for diagnosing AAN, so this study aimed to investigate the biomarkers for AAN using a proteomics method. The C3H/He female mice were given ad libitum AA-distilled water (0.5 mg/kg/day) and distilled water for 56 days in the AA and normal groups, respectively. The AA-induced proteins in the kidney were investigated using a proteomics study, including fluorogenic derivatization with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide, followed by high-performance liquid chromatography analysis and liquid chromatography tandem mass spectrometry with a MASCOT database searching system. There were two altered proteins, thrombospondin type 1 (TSP1) and G protein-coupled receptor 87 (GPR87), in the kidney of AA-group mice on day 56. GPR87, a tumorigenesis-related protein, is reported for the first time in the current study. The renal interstitial fibrosis was certainly induced in the AA-group mice under histological examination. Based on the results of histological examination and the proteomics study, this model might be applied to AAN studies in the future. TSP1 might be a novel biomarker for AAN, and the further role of GPR87 leading to AA-induced tumorigenesis should be researched in future studies.

[Urinary excretion of angiogenesis regulatory factors and renal injury markers in chronic glomerulonephritis: Significance in the assessment of progression].

AIM: To study the urinary excretion of the molecular factors regulating angiogenesis, such as vascular endothelial growth factor type A (VEGF-A), thrombospondin 1 (THBS1), and angiopoietin 2 (ANGPT2), versus that of the urinary markers of renal injury and fibrogenesis, such as neutrophil gelatinase-associated lipocalin (NGAL), type IV collagen (COL4), and known clinical risk factors for accelerated disease progression to estimate the prognostic value of urinary excretion in patients with chronic glomerulonephritis (CGN). SUBJECTS AND METHODS: Eighty-two patients (45% men, 55% women; mean age, 36.5 years) with a clinical diagnosis of CGN were examined. 31.7% of the examinees presented with nephrotic syndrome; 31.7% had a glomerular filtration rate (GFR) of less than 60 ml/min/1.73 m2. Morning urine samples were analyzed by Elisa to determine the urinary excretion of biomarkers (VEGF-A, THBS1, ANGPT2, NGAL, and COL4). The results were adjusted to urinary creatinine concentrations. RESULTS: The urinary excretion of the angiogenesis regulators VEGF-A, THBS1, and ANGPT2 correlated between them, with that of the renal injury markers NGAL and COL4, with the level of proteinuria. That was found to be unassociated with blood pressure and GFR. In the presence and absence of nephrotic syndrome, high (> 75th percentile) urinary excretion rates were 46.2 and 14.8% for VEGF-A (p < 0.01); 50 and 13% for THBS1 (p < 0.001); and 46.2 and 14.8% for ANGPT2 (p < 0.01), respectively. That for ANGPT2 was also high in the presence of anemia (63.2 versus 11.7%; p < 0.001). CONCLUSION: The finding of the high urinary excretion of the angiogenesis regulators VEGF-A, THBS1, and ANGPT2 and its association with that of kidney injury markers in the patients with the proteinuric forms of CGN suggest that this excretion may be considered as an integral index that displays glomerular injury and indicates tubulointerstitial proteinuric/hypoxic remodeling.

Evaluation of gene panel mRNAs in urine samples of kidney transplant recipients as a non-invasive tool of graft function.

Non-invasive monitoring may be useful after kidney transplantation (KT), particularly for predicting acute rejection (AR). It is less clear whether chronic allograft nephropathy (CAN) is also associated with changes in urine cells. To identify non-invasive markers of allograft function in kidney transplant patients (KTP), mRNA levels of AGT, TGF-beta1, EGFR, IFN-gamma, TSP-1, and IL-10 in urine (Ur) samples were studied using QRT-PCR. Ninety-five KTP and 111 Ur samples were evaluated. Patients (Pts) were divided as, within six months (N = 31), and with more than six months post-KT (N = 64). KTP with more than six months post-KT were classified as KTP with stable kidney function (SKF) (N = 32), KTP with SKF (creatinine < 2 mg/dL) and proteinuria > 500 mg/24 h (N = 18), and KTP with biopsy proven CAN (N = 14). F-test was used to test for equality of variances between groups. IL-10 mRNA was decreased in Ur samples from KTP with less than six months post-KT (P = 0.005). For KTR groups with more than six months post-KT, AGT and EGFR mRNA were statistically different among KTP with SKF, KTP with SKF and proteinuria, and CAN Pts (P = 0.003, and P = 0.01), with KTP with SKF having higher mean expression. TSP-1 mRNA levels also were significantly different among these three groups (P = 0.04), with higher expression observed in CAN Pts. Using the random forest algorithm, AGT, EGFR, and TGF-beta1 were identified as predictors of CAN, SKF, SKF with proteinuria. A characteristic pattern of mRNA levels in the different KTP groups was observed indicating that the mRNA levels in Ur cells might reflect allograft function.