Urinary podocyte-associated molecules and albuminuria in hypertension.

OBJECTIVE: Hypertension-induced podocyte damage and the relationship with UAE is analyzed in diabetic and nondiabetic participants. PATIENTS AND METHODS: Sixty-four hypertensive patients, 30 diabetics, with glomerular filtration rate (eGFR) greater than 60 ml/min per 1.73 m were included. Urinary albumin excretion was measured in morning urine using a nephelometric immunoassay and expressed as albumin/creatinine ratio. Urinary pellets were obtained from fresh urine and mRNA was assessed by real-time quantitative PCR. Likewise, protein podocyte-specific molecules were measured by western blot using specific antibodies. RESULTS: Fourteen nondiabetics and 20 diabetics had increased UAE greater than 30 mg/g. In individuals with increased EUA, the mRNA expression of nephrin and CD2AP was low in diabetics, whereas only nephrin mRNA in nondiabetics. No differences were observed in podocalyxin and aquaporin-1 mRNA levels. Concerning the protein values, in both nondiabetic and diabetic patients, nephrin, CD2AP and podocalyxin were increased in patients with increased UAE, with no differences in aquaporin-1. A significant positive relationship was observed between log UAE and nephrin protein values, and an inverse association observed with mRNA. CONCLUSION: Hypertensive patients who had elevated UAE showed increased urinary excretion of podocyte-specific proteins coupled with a phenotype of decreased mRNA expression. The phenotype of podocyte-specific mRNA and the increment of nephrin can be used as a valuable marker of early glomerular injury.

Influence of hematuria and infection on diagnostic accuracy of urinary LASP1: a new biomarker for bladder carcinoma.

AIM: To further promote the clinical use of urinary LASP1 as biomarker for urothelial carcinoma of the bladder regarding limitations and alternative testing systems. PATIENTS & METHODS: Urine stabilization, alternative measurement systems and limitations by erythrocyte contamination and infection were investigated in 246 patients. RESULTS: Thimerosal allowed sufficient stabilization. Fluorescence-activated cell sorting analysis was not influenced by presence of erythrocytes or leukocytes and reliably urothelial carcinoma of the bladder but cell counts in specimen were low. Cut-off values of <25 leukocytes and <200 erythrocytes/µl resulted in sensitivity, specificity, positive and negative predictive values of 0.59, 0.80, 0.80 and 0.59, respectively. CONCLUSION: Hematuria up to 200 erythrocytes/µl but not presence of leukocytes may be tolerated for this promising marker.

CD2AP mRNA in urinary exosome as biomarker of kidney disease.

AIMS: Podocyte injury plays an important role in the pathogenesis of kidney disease. Urinary exosomes are microvesicles released by tubular epithelial cells and podocytes containing information of their originated cells. This study investigated for the first time whether podocyte related mRNA in urinary exosome could serve as novel biomarkers for kidney disease. METHODS: Urine samples were collected from 32 patients of kidney disease who underwent kidney biopsy and 7 controls. CD2AP, NPHS2 and synaptopodin were detected by real-time RT-PCR on RNA isolated from urinary exosome. RESULTS: The pellet microvesicles were positively stained with exosome and podocyte marker, AQP2, CD9 and nephrin. CD2AP mRNA was lower (p=0.008) in kidney disease patients compared with controls and decreased with the increasing severity of proteinuria (p=0.06). CD2AP correlated with serum creatinine (r=-0.373, p=0.035), BUN (r=-0.445, p=0.009) and eGFR (r=0.351, p=0.046). Neither NPHS2 nor synaptopodin correlated with parameters of renal function. CD2AP mRNA correlated negatively with 24 hour urine protein (r=-0.403, p=0.022), severity of tubulointerstitial fibrosis (r=-0.394, p=0.026) and glomerulosclerosis (r=-0.389, p=0.031) and could discriminate kidney disease from controls with AUC of 0.821 (p=0.008). CONCLUSIONS: Urinary exosome mRNA of CD2AP might be a non-invasive tool for detecting both renal function and fibrosis of kidney disease.

LASP-1, a novel urinary marker for detection of bladder cancer.

OBJECTIVES: The LIM and SH3 (LASP)-1 protein is a focal adhesion protein that has been linked to oncogenesis. The aim of this study was to evaluate the diagnostic use of the detection of LASP-1 in tumor specimens and in urine for noninvasive detection of transitional cell carcinoma (TCC). MATERIALS AND METHODS: Immunohistochemical staining for LASP-1 was performed on 72 archived bladder tumor specimens, and LASP-1 content was measured in 132 spontaneous urine sample sediments by Western blot analysis. RESULTS: In the histologic specimens, immunohistochemical staining for LASP-1 showed abundant expression throughout the urothelium of the bladder and ureter. However, modest overexpression of LASP-1 was observed in the TCC specimens. Measurement of the LASP-1 protein concentrations in urinary cell pellets from healthy donors and bladder cancer patients was highly sensitive for the presence of TCC. The cut-off value was determined by receiver operating characteristic (ROC) analysis. When a cut-off value of 1 ng LASP-1/500 µl of urine was used, the sensitivity, specificity, and positive and negative predictive values of the assay were 83.1%, 85.3%, 83.1%, and 80.6%, respectively.The increased urinary LASP-1 content in TCC patients was attributable in part to a specific increase in cell shedding presumably caused by changes in cell adhesion, as confirmed by LASP-1 knockdown. Contamination with erythrocytes above 250 cells/µl and urinary infection gave false positive results and are therefore sample exclusion criteria. CONCLUSIONS: In the absence of urinary infection or gross hematuria, urinary LASP-1 level is a promising marker for transitional cell carcinoma.

Pioglitazone reduces urinary podocyte excretion in type 2 diabetes patients with microalbuminuria.

In various renal diseases, including diabetic nephropathy, detection of podocytes in the urine indicates severe injury to podocytes in the glomeruli. Pioglitazone is a newly developed antidiabetic agent that attenuates insulin resistance. The aim of the present study was to determine whether pioglitazone affects urinary albumin excretion (UAE) or the number of urinary podocytes or both in type 2 diabetes patients with microalbuminuria. Twenty-eight patients with normotensive type 2 diabetes and microalbuminuria (18 men and 10 women; mean age, 52.5 years) and 30 age-matched normotensive controls (20 men and 10 women; mean age, 51.5 years) were included in the study. Urinary podocytes were detected by immunofluorescence with a monoclonal antibody against podocalyxin. Patients were randomly assigned to 2 groups: a pioglitazone-treatment group (30 mg/day, n = 14) and a placebo group (n = 14). Treatment was continued for 6 months. Podocytes were absent in the urine of healthy controls, but detected in 17 of 28 diabetic patients (60.7%). UAE was reduced from 96.7 +/- 50.5 microg/min to 39.7 +/- 22.9 microg/min (P <.01) in the pioglitazone-treatment group, and the number of urinary podocytes was reduced from 0.9 +/- 1.0 cells/mL to 0.1 +/- 0.2 cells/mL (P <.001). Neither UAE nor the number of urinary podocytes was affected in the placebo group. These data indicate that pioglitazone is effective for reducing UAE and podocyte injury in early-stage diabetic nephropathy.