Relationships between levels of urinary podocalyxin, number of urinary podocytes, and histologic injury in adult patients with IgA nephropathy

Abstract

Background and objectives: Podocalyxin (PCX) is present on the apical cell membrane of podocytes and is shed in urine from injured podocytes. Urinary podocalyxin (u-PCX) is associated with severity of active glomerular injury in patients with glomerular diseases. This study examined the relationship between number of urinary podocytes, levels of u-PCX, and glomerular injury in adults with IgA nephropathy (IgAN).

Design, setting, participants, & measurements: Urine samples voided in the morning on the day of biopsy were obtained from 51 patients with IgAN (18 men and 33 women; mean age, 31 years). All renal biopsy specimens were analyzed histologically. Pathologic variables of IgAN were analyzed per Shigematsu classification, the Oxford classification of IgAN, and the Clinical Guidelines of IgAN in Japan. Levels of u-PCX were measured by sandwich ELISA.

Results: Histologic analysis based on Shigematsu classification revealed a significant correlation between levels of u-PCX and severity of acute extracapillary abnormalities (r=0.72; P<0.001), but levels of urinary protein excretion did not correlate with acute glomerular abnormalities. Levels of urinary protein excretion in patients with segmental sclerosis (n=19) were higher than in patients without (n=22) (0.49 [interquartile range (IQR), 0.20-0.88] g/g creatinine versus 0.20 [IQR, 0.10-0.33] g/g creatinine; P<0.01). The number of urinary podocytes in patients with segmental sclerosis was higher than in patients without (1.05 [IQR, 0.41-1.67] per mg creatinine versus 0.28 [IQR, 0.10-0.66] per mg creatinine; P<0.01).

Conclusions: Levels of u-PCX and the number of urinary podocytes are associated with histologic abnormalities in adults with IgAN.

Figure 1.

Western blot analysis for urinary podocalyxin (u-PCX) and immunofluorescence for u-PCX and urinary podocytes. (A) Western blot analysis of urine from patients with IgA nephropathy (IgAN) and human glomerular lysate (GL) using monoclonal antibody against PCX. The 160- to 170-kD bands were seen in urine from patients with IgAN. Pt1 and Pt2, urinary sediments of two patients with IgAN. (B) Immunfluorescence of urine from a patient with IgAN. Urinary sediments were stained with anti-PCX monoclonal antibody. PCX staining showed a granular structure on urine from a patient with IgAN. Original magnification, ×400. (C) Immunfluorescence of urinary podocytes of a patient with IgAN showed double-staining for 4′, 6-diamidine-2-phenylindole (DAPI) (blue) and PCX (red). Some nucleated cells in urinary sediments showed positive PCX staining (arrows). Original magnification, ×200.

Figure 2.

Relationship between levels of urinary podocalyxin (u-PCX), levels of urinary protein excretion, and number of urinary podocytes. (A) Relationship between levels of u-PCX and levels of urinary protein excretion. (B) Relationship between levels of u-PCX and number of urinary podocytes. (C) Relationship between number of urinary podocytes and levels of urinary protein excretion. There was low correlation between number of urinary podocytes and levels of urinary protein excretion.

Figure 3.

Representative histologic findings of acute extracapillary abnormalities stained with periodic acid-Schiff and periodic acid methenamine silver-Masson trichrome. (A and E) In grade 0, no acute extracapillary abnormality was observed. (B and F) In grade 1, a small cellular crescent formation was observed (arrow). (C and G) In grade 2, exudates that have escaped into the urinary space and a cellular crescent were observed (arrows). (D and H) In grade 3, three cellular crescents were observed (arrows). Original magnification ×200. (I) Relationship between acute extracapillary abnormalities and levels of urinary podocalyxin. (J) Relationship between chronic extracapillary abnormalities and levels of urinary protein excretion. (K) Relationship between chronic endocapillary abnormalities and levels of urinary protein excretion. There was a positive correlation between the severity of acute extracapillary abnormalities and levels of urinary podocalyxin. There were low correlations between the severity of chronic glomerular abnormalities and levels of urinary protein excretion.

Figure 4.

Relationship of segmental sclerosis to each urinary biomarker. (A) Levels of urinary protein excretion. (B) Number of urinary podocytes. (C) Levels of urinary podocalyxin (PCX). Levels of urinary protein excretion and number of urinary podocytes in patients with IgA nephropathy with segmental sclerosis were higher than those in patients without (P<0.01, P<0.01, respectively). Error bars represent mean ± SEM.

Figure 5.

Relationship between the Clinical Guidelines of IgAN in Japan and urinary biomarkers. Group A, good prognosis and relatively good prognosis groups; group B, relatively poor prognosis and poor prognosis groups. Some patients with relatively poor or poor prognosis had lower levels of urinary protein excretion and higher levels of urinary podocalyxin.