Kidney function during the course of PAN.

<p>A: Development of massive proteinuria 5 days after induction of PAN as indicated by an increase in protein/creatinine ratio. B: Decrease in urine output 5 days after the induction of PAN. C: Urea and creatinine on days 0, 5 and 10 after induction of PAN. Values represent mean ± SD. Statistical significant differences: *** p<0.001.</p

Immunohistochemistry staining of cystatin C in renal cortex of rats with PAN.

<p>A, C and E: Magnification 40-fold; B, D and F: magnification 400-fold; sections counterstained with methylen green. A+B: Cortex day 0, staining of the prox. tubules and minimal staining of the glomerular podocytes. C+D: PAN day 5, staining of additional segments of the proximal tubule as well as increased podocyte staining. E+F: PAN day 10, tubular staining similar to day 5, persistent cystatin C staining in podocytes.</p

Cystatin C mRNA expression in the renal cortex.

<p>Cystatin C mRNA expression in lysates of the renal cortex of rats with PAN during the course of the disease (day 0, day 5 and day 10 after injection of puromycin). Results are expressed as rel. expression of cystatin c mRNA normalized to cyclophillin A relative to the means of day 0 animals. Bars represent mean ± SD. Statistical significant differences: * p<0.05; *** p<0.001.</p

Exosomal gene expression of cystatin C mRNA.

<p>mRNA expression analysis in urinary exosomes isolated from the urine of rats during the course of PAN (day 0 before disease induction, day 5 after injection of puromycin and day 10 after disease induction). Cyclophilin A was used as reference gene. A: Cystatin C mRNA expression in gene array analysis from urinary exosomes relative to exosomes isolated from day 0, n = 3 animals. Bars represent mean ± SD. Significance level was assessed by Affymetrix Power Tools, using constitutive probe sets with detection above background p-values <0.01 and a raw expression threshold of 50. Threefold changes with a p-value of ≤0.01 were used as cut-off for up/down regulation. B: cystatin C mRNA from urinary exosomes relative to exosomes isolated from day 0 measured by RT-qPCR in separate animals, n = 3 animals. Bars represent mean ± SD. Statistical significant differences: *p<0.05.</p

Results of gene array analysis in urinary exosomes of rats during the course of PAN.

<p>Top 10 differentially regulated genes in urinary exosomes of rats during the course of PAN. Gene expression is presented in log intervals of relative gene expression on the different days of PAN (d0, d5 and d10). In addition, the table shows changes in gene expression from day 0 to day 5 and from day 5 to day 10 also expressed in log intervals. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109631#pone-0109631-t002" target="_blank">Table 2</a> (a) shows the genes with the highest increase in expression during the course of PAN while (b) depicts the top 10 genes with reduced expression in urinary exosomes.</p><p>Results of gene array analysis in urinary exosomes of rats during the course of PAN.</p

Sequences of primers used for real-time reverse transcriptase PCR.

<p>Sequences of primers used for real-time reverse transcriptase PCR.</p

Urinary cystatin C expression.

<p>Cystatin C excretion in the urine (mg/l) of rats with PAN on the different days after induction of PAN. Bars represent mean ± SD. Statistical significant differences: ** p<0.01.</p