Question:

The epithelial sodium channel (ENaC) plays a critical role in the regulation of renal sodium excretion. We have recently shown that plasmin is present in urine of PAN-nephrotic rats and can proteolytically activate ENaC (Svenningsen et al., 2009). Thus, in nephrotic syndrome filtered plasminogen converted to plasmin by tubular urokinase may activate ENaC. This may contribute to sodium retention and edema formation in nephrotic syndrome. However, in preliminary studies using urine samples from adult and pediatric patients with proteinuria, we found that although protease activity was present in most urine samples, only a small minority of the samples modestly stimulated the activity of ENaC heterologously expressed in Xenopus laevis oocytes. Moreover, urine samples from doxorubicin treated mice, a model of nephrotic syndrome, also failed to have a consistent stimulatory effect on ENaC. We speculated that proteolytic activation of ENaC by urinary proteases may be prevented by protease inhibitors in the urine, e.g. the known urinary serine protease inhibitor uristatin. Therefore, we investigated whether human and murine urine samples contain protease inhibitors.

Methods:

Dipsticks for the detection of urinary trypsin inhibitors (uTi) were used and analysed with a CLINITEK 50 dipstick reader. A competitive ELISA, sensitive to uristatin was performed to estimate its concentration in the urine samples.

Results:

Urine samples from all adult patients (n=19) and 5 out of 8 pediatric patients contained elevated uTi concentrations (estimated uristatin concentration = 10 µg/ml). In 8 of these samples uristatin concentrations > 40 µg/ml were detected. In contrast, in urine of healthy adults (n=3) uTis were undetectable. An ELISA revealed the presence of uristatin in all urine samples from adult patients and in one sample of the pediatric patients. All murine urine samples tested showed elevated concentrations of uTi (estimated uristatin > 10 µg/ml) at day one after doxorubicin treatment, but a strong increase in uTi was detected on day 5 or 6. Urine samples collected before treatment contained less than 10 µg/ml uTi.

Conclusions:

Our data demonstrate that proteinuria is associated with an increased concentration of urinary protease inhibitors, in particular uristatin. The presence of protease inhibitors may prevent ENaC activation by urinary proteases like plasmin known to occur in nephrotic urine. Thus, the balance between urinary proteases and urinary protease inhibitors may determine the degree of proteolytic ENaC activation.