Protease nexin-1, tPA, and PAI-1 are upregulated in cryoglobulinemic membranoproliferative glomerulonephritis

Abstract

Thymic stromal lymphopoietin (TSLP) transgenic mice develop cryoglobulin-associated membranoproliferative glomerulonephritis, characterized by renal monocyte/macrophage infiltration, marked expansion of extracellular matrix, and variable intraluminal and mesangial deposits of cryoglobulins. A microarray approach was used to study global gene expression in glomerular RNA obtained from these mice, as well as from combined TSLP transgenic and Fcγ receptor IIb null mice (TSLP/FcIIb^−/−), which develop aggravated membranoproliferative glomerulonephritis. Protease nexin-1 (PN-1) and tissue plasminogen activator (tPA), two potential regulators of fibrosis that are involved in the fibrinolytic and coagulation pathways, were dramatically upregulated in TSLP mice compared with wild-type controls. In situ hybridization revealed minimal expression of PN-1 mRNA in the glomeruli of wild-type mice, increased expression in TSLP mice, and the greatest expression in the mesangial cells of TSLP/FcIIb^−/− mice. Immunohistochemistry demonstrated greater expression of PN-1, tPA, and PAI-1 in the mesangial cells of TSLP mice compared with wild-type and the greatest in TSLP/FcIIb^−/− mice. In cultured mesangial cells, incubation with cryoglobulins induced an upregulation of PN-1 mRNA; increased expression of PN-1, tPA, and PAI-1 proteins; and stimulated secretion of TGF-β1. It is concluded that PN-1, tPA, PAI-1, and TGF-β1 are likely important mediators of murine cryoglobulinemic glomerulonephritis and that the cryoglobulins may directly upregulate their expression.

Figure 1.

Histology of TSLP transgenic mice and human cryoglobulinemic MPGN. Glomeruli from human cryoglobulinemic MPGN (A and C) and from TSLP transgenic mice (B and D) show a range of morphologic features, including intraluminal as well as mesangial cryoglobulin deposits (arrows, A and B), mesangial expansion and increased cellularity (A through D), and splitting of basement membranes (* in A, B, and C). In both humans and mice, the deposition of cryoglobulins can be histologically inapparent, and in these cases, the association of the MPGN with cryoglobulinemia is determined only by serologic evidence of cryoglobulins. Influx of monocytes, common but not invariably so in both humans and mice, is prominent in the capillaries and probably also in mesangial regions in these examples of human and murine MPGN (C and D, respectively) and confirmed by monocyte-specific immunohistochemical labeling.

Figure 2.

Upregulation of PN-1 mRNA in mesangial cells of TSLP transgenic and TSLP/FcγIIb−/− mice. WT mice demonstrate little expression of PN-1 mRNA in glomeruli (A), with increased expression in TSLP transgenic mice (B), and greatest expression in glomeruli of TSLP/FcIIb−/− mice (C). Negative control hybridized with sense probe (D). In both TSLP mice (data not shown) and TSLP/FcIIb−/− mice (E through G), most of the cells immunolabeled with α-SMA also expressed PN-1 mRNA (E), whereas Mac-2+ monocyte/macrophages expressing PN-1 were a small subset of overall PN-1–expressing cells (F and G, arrow). (G) Negative control hybridized with the sense probe. Magnification, ×400.

Figure 3.

Mesangial expression of PN-1, tPA, and PAI-1 proteins were increased in TSLP transgenic and TSLP/FcγIIb−/− mice. PN-1 is localized to podocytes in WT mice (A), and de novo mesangial expression is present in TSLP transgenic mice (B) and further upregulated in TSLP/FcIIb −/− mice (C). Mesangial expression of tPA protein was increased in TSLP transgenic mice (E) compared with WT mice (D) and was even more pronounced in TSLP/FcIIb−/− mice (F). Focal PAI-1 protein expression in WT mice was present in mesangial areas (G), which was upregulated in TSLP transgenic mice (H) and more so in TSLP/FcIIb−/− mice (I). Magnification, ×400.

Figure 4.

Upregulation of PN-1 RNA in mesangial cells exposed to cryoglobulins. (A) Northern blot analysis. Mouse mesangial cells were incubated with cryoglobulin or mouse IgG (control Ig) for the indicated time. RNA extracted from each was analyzed by Northern blot (10 μg/lane) with a cDNA probe specific for PN-1. (B) PN-1 RNA expression. Mouse mesangial cells incubated with cryoglobulins demonstrate upregulated PN-1 RNA expression, increasing of 2.6-fold over control at 24 h. Upregulation became apparent at 6 h and peaked at 24 h. Mesangial cells incubated with control mouse IgG showed no significant change of PN-1 RNA expression. Values are normalized for differences in RNA loading.

Figure 5.

Upregulation of PN-1, tPA, and PAI-1 protein by cryoglobulin in WT mesangial cells. (A) Western blot analysis. Mouse mesangial cells were incubated with cryoglobulins or HA IgG/IgM or mouse IgG (control Ig) for the indicated time. Cryoglobulin-induced upregulation of PN-1, tPA, and PAI-1 proteins became apparent at 24 h and maximal at 48 h. No significant increase in PN-1 or tPA was observed in mesangial cells incubated with HA-IgG/IgM (shown) or mouse IgG. Similarly, no increase was seen in PAI-1 expression after incubation with mouse IgG. γ-Tubulin was used as an internal control to ensure equal protein loading. (B) PN-1, tPA, and PAI-1 expression. Mesangial cells incubated with cryoglobulins demonstrated upregulated expression levels for PN-1 (2.8-fold), tPA (3.1-fold), and PAI-1 (1.5-fold) compared with control. Values were normalized for differences in protein loading.

Figure 6.

Expression of TGF-β1 mRNA and protein by mesangial cells incubated with cryoglobulin or mouse IgG. (A) Northern blot analysis. Mouse mesangial cells were incubated with cryoglobulin or control mouse IgG (control Ig) for the indicated time. Both cryoglobulin and mouse IgG induced a strong increase in TGF-β1 mRNA. Cryoglobulin induced a strong increase in TGF-β1 mRNA at 45 min, which gradually decreased up to 48 h. In contrast, mesangial cells incubated with mouse IgG showed upregulation of TGF-β1 mRNA at 45 min, which peaked at 2 h, and then decreased. (B) TGF-β1 RNA expression. There are no significant differences seen in expression levels of TGF-β1 resulting from exposure to cryoglobulins or IgG; values are normalized for equal RNA loading. (C) Analysis of conditioned medium derived from mouse mesangial cells incubated with cryoglobulin or HA IgG/IgM or mouse IgG by an ELISA specific for TGF-β1. Incubation with cryoglobulin induced increased release of TGF-β1 into culture medium as compared with control Ig. Values are the average of two experiments.