New-onset diabetes after kidney transplantation: Assessing urinary Wilm's tumor-1 protein to predict renal allograft dysfunction.

PURPOSE: New-onset diabetes after transplantation (NODAT) is a frequent metabolic complication associated with podocyte damage and renal allograft dysfunction. Thus, Wilm's tumor-1 (WT-1) protein, as a podocyte marker, holds promise as an option to evaluate renal allograft dysfunction in NODAT. Therefore, the study aimed to investigate urinary WT-1 levels in NODAT patients during the first year after kidney transplantation (KTx). MATERIALS AND METHODS: KTx patients were categorized into non-NODAT and NODAT groups. Fasting blood glucose, glycated hemoglobin (HbA1c), urinary albumin/creatinine ratio (ACR), serum creatinine, estimated glomerular filtration rate (eGFR), and urinary WT-1 were measured at 3, 6, 9, and 12-months post-KTx. RESULTS: The NODAT group manifested elevated levels of blood glucose and HbA1c during the first year post-KTx. Also, exhibited elevations in ACR and serum creatinine levels at 6, 9, and 12-months post-KTx when compared to non-NODAT group. Conversely, eGFR values in the NODAT group demonstrated significant declines at 3, 6, and 9-months post-KTx relative to non-NODAT. Furthermore, NODAT group exhibited a median annual eGFR of 47 â€‹mL/min/1.73 â€‹m2. Urinary WT-1 levels at 3, 6, 9, and 12-months post-KTx were significantly higher in the NODAT group compared to non-NODAT. Additionally, noteworthy positive correlations were identified between urinary WT-1 and HbA1c levels, along with significant negative correlations between urinary WT-1 and eGFR at the 3, 6, 9, and 12-months post-KTx. CONCLUSION: The increased urinary WT-1 levels from 3-months post-KTx in NODAT patients may indicate the first sign of podocyte injury, predicting a renal allograft dysfunction in these patients.

Wilm's tumor-1 protein levels in urinary exosomes from diabetic patients with or without proteinuria.

BACKGROUND: Podocyte injury is an early feature of diabetic nephropathy (DN). Recently, urinary exosomal Wilm's tumor-1 protein (WT1), shed by renal epithelial cells, has been proposed as a novel biomarker for podocyte injury. However, its usefulness as biomarker for early diabetic nephropathy has not been verified yet. We investigated urinary exosomal WT1 in type-1 diabetic patients to confirm its role as a non-invasive biomarker for predicting early renal function decline. METHODS: The expression of WT1 protein in urinary exosomes from spot urine samples of type-1 diabetes mellitus patients (n = 48) and healthy controls (n = 25) were analyzed. Patients were divided based on their urinary albumin excretion, ACR (mg/g creatinine) into non- proteinuria group (ACR<30 mg/g, n = 30) and proteinuria group (ACR>30 mg/g, n = 18). Regression analysis was used to assess the association between urinary exosomal levels of WT1 with parameters for renal function. Receiver Operating Characteristic (ROC) curve analysis was used to determine the diagnostic performance of exosomal WT-1. RESULTS: WT1 protein was detected in 33 out of 48 diabetic patients and in only 1 healthy control. The levels of urinary exosomal WT1 protein is significantly higher (p = 0.001) in patients with proteinuria than in those without proteinuria. In addition, all the patients with proteinuria but only half of the patients without proteinuria were positive for exosomal WT1. We found that the level of exosomal WT1 were associated with a significant increase in urine protein-to-creatinine ratio, albumin-to-creatinine ratio, and serum creatinine as well as a decline in eGFR. Furthermore, patients exhibiting WT1-positive urinary exosomes had decreased renal function compared to WT1-negative patients. ROC analysis shows that WT-1 effectively predict GFR<60 ml. min-1/1.73 m(2). CONCLUSION: The predominant presence of WT1 protein in urinary exosomes of diabetic patients and increase in its expression level with decline in renal function suggest that it could be useful as early non-invasive marker for diabetic nephropathy.

Dynamics of absolute amount of nephrin in a single podocyte in puromycin aminonucleoside nephrosis rats calculated by quantitative glomerular proteomics approach with selected reaction monitoring mode.

BACKGROUND: The slit diaphragm (SD) is a complex of podocyte-specific proteins and plays a significant role in glomerular filtration. To understand podocyte biology, it is important to determine the expression amount of the SD complex proteins. This study aimed to quantify the absolute amount of nephrin, which is believed to be a major component of SD, in podocytes and to apply that method to normal and puromycin aminonucleoside (PAN) nephrosis rats. METHODS: The counting method for podocyte number in a glomerulus was developed by three-dimensional reconstruction imaging of Wilms tumor (WT-1) immunofluorescence on isolated glomeruli. Absolute amount of nephrin was quantified by mass spectrometry using the selected reaction monitoring (SRM) mode with a stable isotope-labeled peptide. RESULTS: The number of podocytes per glomerulus was 95.5±17.6 in the control rats, 90.7±19.2 on Day 4 and 90.7±26.2 on Day 7 in PAN nephrosis rats. The amount of nephrin per glomerulus in control rats was 1.02±0.11 fmol and those in PAN nephrosis rats were reduced to 0.46±0.06 fmol and 0.35±0.04 fmol on Day 4 and Day 7. The nephrin amount per podocyte was significantly decreased association with the development of proteinuria in PAN nephrosis rats. CONCLUSIONS: This study established the absolute quantification of nephrin and determined the amount of nephrin in a podocyte of normal and PAN nephrosis rat kidneys. This highly sensitive and selective quantification method for protein is a useful tool for the analysis of SD protein in a podocyte.

Urinary exosomal WT1 in childhood nephrotic syndrome.

BACKGROUND: Recently, urinary exosomal WT1 has been proposed as a novel biomarker for simple podocyte injury. We investigated urinary exosomal WT1 to confirm its role as a non-invasive biomarker for predicting steroid responsiveness or renal pathological conditions in patients with idiopathic nephrotic syndrome (NS). CASE DIAGNOSIS: Forty children with active NS were recruited. Twenty-eight (70%) were steroid-sensitive, including 3 with minimal change NS (MCNS) and 1 with focal segmental glomerulosclerosis (FSGS). The remaining 12 (30%) were steroid-resistant, including 8 with FSGS and 4 with MCNS. Urinary exosomes were isolated by a differential centrifugation method, and WT1 was measured by Western blot analysis. RESULTS: WT1 was detected in 25 patients (62.5%). There was no significant difference in the proportion of the patients with a detectable amount of WT1 according to steroid responsiveness or renal pathological condition, the amount of WT1 showed no significant difference according to steroid responsiveness or renal pathological condition, and there was no significant difference in the amount of proteinuria between patients with or without detectable WT1. CONCLUSIONS: Urinary exosomal WT1 was detected in some patients with NS. However, its role as an appropriate biomarker in childhood NS was not verified in this study.

Urinary exosomal transcription factors, a new class of biomarkers for renal disease.

Urinary exosomes are excreted from all nephron segments and constitute a rich source of intracellular kidney injury biomarkers. To study whether they contain transcription factors, we collected urine from two acute kidney injury models (cisplatin or ischemia-reperfusion), two podocyte injury models (puromycin-treated rats or podocin-Vpr transgenic mice) and from patients with focal segmental glomerulosclerosis, acute kidney injury and matched controls. Exosomes were isolated by differential centrifugation and found to contain activating transcription factor 3 (ATF3) and Wilms Tumor 1 (WT-1) proteins detected by Western blot. These factors were found in the concentrated exosomal fraction, but not in whole urine. ATF3 was continuously present in urine exosomes of the rat models following acute injury at times earlier than the increase in serum creatinine. ATF3 was found in exosomes isolated from patients with acute kidney injury but not from patients with chronic kidney disease or controls. Urinary WT-1 was present in animal models before significant glomerular sclerosis and in 9/10 patients with focal segmental glomerulosclerosis but not in 8 controls. Our findings suggest that transcription factor ATF3 may provide a novel renal tubular cell biomarker for acute kidney injury while WT-1 may detect early podocyte injury. Measurement of urinary exosomal transcription factors may offer insight into cellular regulatory pathways.