R (P)RR in kidney sections was performed using the EnVisionTM + Dual Link System-HRP (Dakocytomation,

R (P)RR in kidney sections was performed using the EnVisionTM + Dual Link System-HRP (Dakocytomation, Glostrup, Denmark), as previously described [11?4]. The primary antibody was rabbit anti-(P)RR antibody, which detected within residues 300 to the C-terminus (Abcam, Tokyo, Japan). Sections incubated without the primary antibody were used as controls. To examine the expression levels of (P)RR, 10 small vessels and collecting ducts or connecting tubular cells and infiltrated cells in 10 microscopic fields at ?00 magnification were evaluated for representative samples according to the stained levels for (P)RR: unstained, 0; weak, 1; moderate, 2; and severe, 3, and mean values were calculated.Immunohistochemical analysis of infiltrated cells using cell surface markers in serial sectionsInfiltrated cells were stained by using cell surface markers to determine what PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21108687 kinds of cells were infiltrated. CD3-, CD19-, or CD68-positive cells indicate T cell, B cell, and monocyte/PLOS ONE | DOI:10.1371/journal.pone.0156165 May 26,3 /Plasma S(P)RR for Renal Damagemacrophage lines, respectively. The primary antibody for CD3 was mouse monoclonal from Novocastra/Leica Biosystems, Heiderberg, Germany, and those for CD19 and CD68 were mouse monoclonal from Dakocytomation. The patient with eGFR of 27 mL/min/1.73m2 was selected for immunohistochemical analyses because remarkable infiltrated cells were present in the tissue section.Double staining by immunofluorescenceDouble staining of paraffin sections was performed using anti-(P)RR antibody with MedChemExpress COH29 either antiCD3, anti-CD19, or anti-CD68 antibody. The sections were incubated with Alexa Fluor 546and 488-labeled secondary antibodies (Molecular Probes, Eugene, OR, USA) for the anti-(P) RR and cell surface marker antibodies, respectively. The patient whose eGFR was 27 mL/min/ 1.73m2 was selected for the immunofluorescence analyses. Localization of (P)RR and cell surface markers was investigated with an immunofluorescence microscope (BX50, Olympus, Tokyo, Japan).Statistical analysisResults were expressed as the mean ?standard deviation. Dry weight was used as body weight for analyses in patients on HD. All parameters, except for PRA, showed a normal distribution. On the other hand, because PRA did not show a normal distribution, logarithmic transformation was applied. The correlations between plasma s(P)RR and other parameters were evaluated by using Pearson’s product-moment correlation coefficient. Multiple linear regression analyses for plasma s(P)RR levels were adjusted for age, sex, body weight, systolic BP, plasma AngII, and the extent of interstitial fibrosis. Age, sex, and body weight were selected as variables because these parameters are common in performing multiple linear regression analyses. In addition, systolic BP and plasma AngII, component of circulating RAS, were also adjusted as they are associated with renal damage. We considered p < 0.05 statistically significant. Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software version 20 (SPSS Inc., Chicago, Illinois, USA).Results Characteristics of all the patientsThe patient characteristics are listed in Table 1. Dialysis was performed for all the patients with an eGFR < 15 mL/min/1.73m2; 8 patients required HD and 1 required PD. The extent of tubulointerstitial damage was proportional to the extent of renal dysfunction (Fig 1).Relationships between plasma s(P)RR levels and other parameters in all the pati.