IgA production by various spleen cells.

<p>(A) IgA production by B cells increased when cocultured with T cells and/or dendritic cells (DCs). (B) Under low zinc (Zn) conditions, IgA production was significantly increased in the presence of DCs. Under high Zn conditions, IgA production by all cell combinations was suppressed. (C) Coculture of untreated B cells with low Zn-treated T cells and/or DCs. IgA production increased in the presence of DCs. (D) Coculture of B cells and DCs with or without lipopolysaccharide (LPS). LPS significantly enhanced IgA production despite the absence of T cells. (E) Coculture of B cells and DCs from gddY or BALB/c mice. A combination of both B cells and DCs from gddY mice yielded the highest production of IgA. **p<0.01; ***p<0.001.</p

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

The Kinetics of Glomerular Deposition of Nephritogenic IgA

<div><p>Whether IgA nephropathy is attributable to mesangial IgA is unclear as there is no correlation between intensity of deposits and extent of glomerular injury and no clear mechanism explaining how these mesangial deposits induce hematuria and subsequent proteinuria. This hinders the development of a specific therapy. Thus, precise events during deposition still remain clinical challenge to clarify. Since no study assessed induction of IgA nephropathy by nephritogenic IgA, we analyzed sequential events involving nephritogenic IgA from IgA nephropathy-prone mice by real-time imaging systems. Immunofluorescence and electron microscopy showed that serum IgA from susceptible mice had strong affinity to mesangial, subepithelial, and subendothelial lesions, with effacement/actin aggregation in podocytes and arcade formation in endothelial cells. The deposits disappeared 24-h after single IgA injection. The data were supported by a fluorescence molecular tomography system and real-time and 3D in vivo imaging. In vivo imaging showed that IgA from the susceptible mice began depositing along the glomerular capillary from 1 min and accumulated until 2-h on the first stick in a focal and segmental manner. The findings indicate that glomerular IgA depositions in IgAN may be expressed under the balance between deposition and clearance. Since nephritogenic IgA showed mesangial as well as focal and segmental deposition along the capillary with acute cellular activation, all glomerular cellular elements are a plausible target for injury such as hematuria.</p></div

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

Serum concentration of immunoglobulins for each dietary zinc condition.

<p>Dietary zinc intervention altered the serum concentrations of IgA and IgA–IgG immune complexes but not of IgG and IgM. *p<0.05.</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

IgA from gddY mice is deposited along the glomerular capillary wall in a focal and segmental manner.

<p>Detailed kinetics of IgA deposition analyzed from 1 min to 2 h postinjection using confocal laser microscopy. Alexa Fluor 633-labeled IgA from gddY and Balb/c mice (red) and 500-kDa fluorescein-labeled dextran (green) were injected for analyzing kinetics of IgA deposition and visualizing blood vessel wall integrity, respectively. (a) IgA signals were detectable even after 1 min and accumulated up to 2 h in a focal and segmental manner in mice with IgA from gddY mice. In contrast, mice who received Balb/c IgA did not show a signal even after 2 h. (b)(c) Serial images of a glomerulus in mice with IgA from gddY mice showed that these IgA molecules accumulated on top of the initial aggregates along the glomerular capillaries. These aggregates were found in a focal and segmental manner but not in a diffuse and global manner.</p

Expression of Toll-like receptor 4, MyD88, TRIF, and interferon-β in each zinc condition.

<p>(A) Expression of Toll-like receptor 4 (TLR4) for each zinc (Zn) condition with/without lipopolysaccharide (LPS). Expression of TLR4 was significantly increased when stimulated by LPS under low and normal Zn conditions. However, TLR4 expression under high Zn conditions was low despite LPS stimulation. (B) Expression of MyD88 at each Zn condition. Expression of MyD88 remained constant at each Zn condition. (C) Expression of TRIF for each Zn condition. Expression of TRIF under low Zn conditions was significantly higher than that in other groups. (D) Expression of interferon-β in each Zn condition. Expression of interferon-β was higher in low Zn condition and lower in high Zn condition. *p<0.05; **p<0.01.</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

Reactivity against lipopolysaccharide.

<p>Lipopolysaccharide (LPS) was nasally administered to some mice in each dietary group, while others received saline as a vehicle control; samples were collected during the sixth week. We calculated the ratio (sixth:fourth week) of each parameter. Although ACR in all groups increased after LPS stimulation, the elevation in the low zinc (Zn) diet group was very high. Serum concentrations of IgA and IgA–IgG immune complexes (ICs) in the low and normal Zn diet groups tended to be higher after LPS stimulation. However, the high Zn diet group maintained low concentrations of serum IgA and IgA–IgG ICs despite LPS stimulation. *p<0.05.</p