Circulating TNF Receptors 1 and 2 Are Associated with the Severity of Renal Interstitial Fibrosis in IgA Nephropathy

<div><p>The current study aimed to examine whether the levels of TNF receptors 1 and 2 (TNFR1 and TNFR2) in serum and urine were associated with other markers of kidney injury and renal histological findings, including TNFR expression, in IgA nephropathy (IgAN). The levels of the parameters of interest were measured by immunoassay in 106 biopsy-proven IgAN patients using samples obtained immediately before renal biopsy and in 34 healthy subjects. Renal histological findings were evaluated using immunohistochemistry. The levels of serum TNFRs were higher in IgAN patients than in healthy subjects. The levels of both TNFRs in serum or urine were strongly correlated with each other (<i>r</i> > 0.9). Serum TNFR levels were positively correlated with the urinary protein to creatinine ratio (UPCR) and four markers of tubular damage of interest (N-acetyl-β-D-glucosaminidase [NAG], β2 microglobulin [β2m], liver-type fatty acid-binding protein [L-FABP], and kidney injury molecule-1 [KIM-1]) and negatively correlated with estimated glomerular filtration rate (eGFR). Patients in the highest tertile of serum TNFR levels showed more severe renal interstitial fibrosis than did those in the lowest or second tertiles. The tubulointerstitial TNFR2-, but not TNFR1-, positive area was significantly correlated with the serum levels of TNFRs and eGFR. Stepwise multiple regression analysis revealed that elevated serum TNFR1 or TNFR2 levels were a significant determinant of renal interstitial fibrosis after adjusting for eGFR, UPCR, and other markers of tubular damage. In conclusion, elevated serum TNFR levels were significantly associated with the severity of renal interstitial fibrosis in IgAN patients. However, the source of TNFRs in serum and urine remains unclear.</p></div

Simple and stepwise multiple regression analysis of variables that were associated with renal interstitial fibrosis in IgAN patients.

<p>*P<0.05</p><p>**P<0.01</p><p>^†P<0.001</p><p>^††P<0.0001</p><p>Abbreviations used in this table are the same as in Table <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122212#pone.0122212.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122212#pone.0122212.t002" target="_blank">2</a>.</p><p>Simple and stepwise multiple regression analysis of variables that were associated with renal interstitial fibrosis in IgAN patients.</p

Clinical characteristics and levels of inflammatory and tubular damage markers according to tertile of serum TNFR2 levels in IgAN patients.

<p>Data are mean ± SD, median (quartiles), or %. Abbreviations used in this table are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122212#pone.0122212.t001" target="_blank">Table 1</a>. NAG, N-acetyl-β-D-glucosaminidase; β2m, β2-microglobulin; L-FABP, liver-type fatty acid binding protein; KIM-1, kidney injury molecule-1.</p><p>Clinical characteristics and levels of inflammatory and tubular damage markers according to tertile of serum TNFR2 levels in IgAN patients.</p

Histological findings according to serum TNFR2 levels.

<p>Patients were grouped according to distribution tertiles for each histological finding and serum TNFR2 levels. The severity of interstitial fibrosis, tubular atrophy, and glomerulosclerosis was significantly associated with serum TNFR2 levels.</p

Clinical characteristics and levels of inflammatory markers in IgAN patients and healthy subjects.

<p>Data are mean ± SD, median (quartiles), or %.</p><p>BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; Rx, treatment; GFR, glomerular filtration ratio; UPCR, the ratio of urinary protein to creatinine; HPF, high power field; TNFR, TNF receptor; NA, not applicable</p><p>Clinical characteristics and levels of inflammatory markers in IgAN patients and healthy subjects.</p

Spearman correlation coefficients between markers of tubular damage or inflammation and impaired renal function in IgAN patients.

<p>*P<0.05</p><p>**P<0.01</p><p>†P<0.001</p><p>††P<0.0001</p><p>Abbreviations used in this table are the same as in Table <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122212#pone.0122212.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122212#pone.0122212.t002" target="_blank">2</a>. sTNFR, serum TNF receptor; uTNFR, urinary TNF receptor.</p><p>Spearman correlation coefficients between markers of tubular damage or inflammation and impaired renal function in IgAN patients.</p

Representative immunohistochemical staining for TNFR1 and TNFR2 in the kidneys.

<p>(A) Images were captured at 200× (a, b, c, d, i, j, k, l) and 100× (e, f, g, h, m, n, o, p) magnification. Images show the negative controls in the glomeruli (a, i) and tubulointerstitium (e, m) and TNFR1 and TNFR2 immunostaining in the glomeruli (b, j) and tubulointerstitium (f, n) of the normal kidneys, respectively. TNFR1 and TNFR2 immunostaining is shown in the glomeruli (c, k) and tubulointerstitium (g, o) of the kidneys from selected IgAN patients who had levels of serum TNFR2 that ranked in the lowest 10 [low group (LG)]. TNFR1 and TNFR2 immunostaining in the glomeruli (d, l) and tubulointerstitium (h, p) of the kidneys from selected IgAN patients who had levels of serum TNFR2 that ranked in the highest 10 [high group (HG)], respectively, are also shown. (B) Percentage of the TNFR1 and TNFR2-positive area in the kidneys were evaluated. Glomerular and tubulointerstitial TNFR expression was elevated significantly in IgAN patients compared with those in control (Ctrl) subjects. The tubulointerstitial TNFR2-positive area was significantly larger in HG than in LG. However, there was no significant difference in the tubulointerstitial TNFR1 and glomerular TNFR areas between LG and HG. * <i>P</i> < 0.01, **<i>P</i> < 0.001, ^†<i>P</i> < 0.0001.</p

Incidence rate of ESRD in subjects with T2D stratified by quartiles of FGF-23 and TNFR1.

<p>Quartile cut-off values were 1049, 1302, and 1812 pg/mL for TNFR1 and 42, 60, and 96 RU/mL for FGF-23, respectively.</p

Baseline characteristics of subjects with T2D according to their outcome during 8–12 years of follow-up.

<p>Data are mean ± SD, median (25th, 75th percentiles), or percentage. AER and plasma markers were transformed to base 10 logarithms for the statistical analyses. Bonferroni correction for a number of groups was applied.</p

Univariate and multivariate Cox proportional hazard models assessing risk of all-cause mortality adjusting for relevant baseline clinical characteristics and plasma markers in subjects with T2D followed for 8–12 years.

*<p>Effect measures are expressed as the HR for a one-quartile increase in the distribution of each covariate except for eGFR, for which it is a one-quartile decrease.</p><p>Model #1 included relevant clinical characteristics and plasma TNFR1 and FGF-23 independently.</p><p>Model #2 included relevant clinical characteristics and plasma TNFR1 and FGF-23 together.</p