Periostin is induced in glomerular injury and expressed de novo in interstitial renal fibrosis

Abstract

Matricellular proteins participate in the pathogenesis of chronic kidney diseases. We analyzed glomerular gene expression profiles from patients with proteinuric diseases to identify matricellular proteins contributing to the progression of human nephropathies. Several genes encoding matricellular proteins, such as SPARC, THBS1, and CTGF, were induced in progressive nephropathies, but not in nonprogressive minimal-change disease. Periostin showed the highest induction, and its transcript levels correlated negatively with glomerular filtration rate in both glomerular and tubulointerstitial specimen. In well-preserved renal tissue, periostin localized to the glomerular tuft, the vascular pole, and along Bowman's capsule; no signal was detected in the tubulointerstitial compartment. Biopsies from patients with glomerulopathies and renal dysfunction showed enhanced periostin expression in the mesangium, tubular interstitium, and sites of fibrosis. Periostin staining correlated negatively with renal function. α-smooth muscle actin-positive mesangial and interstitial cells localized close to periostin-positive sites, as indicated by co-immunofluorescence. In vitro stimulation of mesangial cells by external addition of TGF-β1 resulted in robust induction of periostin. Addition of periostin to mesangial cells induced cell proliferation and decreased the number of cells expressing activated caspase-3, a marker of apoptosis. These human data indicate for the first time a role of periostin in glomerular and interstitial injury in acquired nephropathies.

Figure 1

Venn diagram of matricellular proteins induced in proteinuric diseases. Transcript levels of 15 matricellular proteins that were found to be induced in glomeruli from patients with proteinuric diseases are indicated. In both progressive glomerulopathies, FSGS and MGN, more matricellular proteins were induced than in the nonprogressive disease MCD. Periostin, although constantly expressed in this renal compartment, showed the highest induction of all matricellular proteins (see Supplemental Table S1 at http://ajp.amjpathol.org). Abbreviations represent the official gene symbols of the matricellular proteins: IBSP, bone sialoprotein; POSTN, periostin; TNC, tenascin-C; WISP1, -3, WNT1 Inducible Signaling Pathway protein-1, -3.

Figure 2

Periostin mRNA is enriched in the glomerular compartment. Expression of periostin mRNA was analyzed by real-time RT-PCR in glomerular and tubulointerstitial samples from pretransplant biopsies. The expression in the glomerular compartment was >30-fold higher compared with the tubulointerstitium. **P < 0.01; data shown are normalized to GAPDH, and tubulointerstitial expression was set as 1.

Figure 3

Localization of periostin in human pretransplant biopsies and transplant nephrectomies. Immunohistochemistry was performed with a polyclonal antiserum against periostin (A–D) or the combination of antiserum and the corresponding peptide (E) on pretransplant biopsies [original magnification, ×250 (A and B)] and allograft nephrectomies [original magnification: ×100 (C); ×250 (D and E)]. A: In well-preserved tissue from pretransplant biopsies, a positivity was rarely found for periostin within the glomerular tuft (arrowhead), but commonly at the vascular pole and along the Bowman‘s capsule (arrow). B: No periostin staining was detected in the well-preserved tubulointerstitium from pretransplant biopsies. C and D: In transplant nephrectomies due to chronic dysfunction, periostin was strongly positive in regions with interstitial fibrosis (arrowheads) and constitutively expressed in the wall of larger arteries (arrow in C). E: Addition of recombinant periostin completely blocked the specific staining of the primary antibody (consecutive section to D).

Figure 4

Periostin mRNA expression in different glomerulopathies. Periostin mRNA is induced in progressive glomerulopathies such as FSGS and LN. A: The expression of periostin mRNA is shown as fold-change compared with living donors (LD). FSGS, focal-segmental glomerulosclerosis; IGAN, IgA nephropathy; MCD, minimal change disease; MGN, membranous glomerulopathy. B: Periostin mRNA showed the highest induction in proliferative lupus nephritis compared with LD. *P < 0.05, ***P < 0.001. Data are shown as fold-change compared to expression in LD (set as 1) and are normalized to GAPDH.

Figure 5

Periostin staining in membranous nephropathy and proliferative lupus nephritis. Immunohistochemistry was performed with a polyclonal antiserum against periostin (A–D) on renal biopsies with membranous nephropathy (A and B), lupus nephritis (C and D), [original magnification, ×200 (A–D)]. A: The typical constitutive staining of the vascular pole is illustrated in combination with a prominent staining of the glomerular tuft with a predominant mesangial pattern in a biopsy with membranous nephropathy. B: Three glomeruli with different grades of glomerular staining are illustrated ranging from focal staining (left glomerulus), to global staining in a completely sclerosed glomerulus (in the middle). C and D: In two patients with LN ISN/RPS IV and comparably high proteinuria (7 to 8 g/day), the glomerular periostin staining was similar. However, the tubulointerstitial periostin staining was mild in the patient with preserved eGFR (>60 mL/min; C, arrow: glomerular periphery) and prominent in the patient with impaired renal function and interstitial fibrosis (eGFR <30 mL/min; D, arrowhead).

Figure 6

Periostin correlates negatively with renal function. A and B: Digital image analysis was performed to quantify the periostin-positive area. A: Periostin staining was most prominent in the interstitium of biopsies from patients with an eGFR <30 mL/min (**P < 0.01 vs. eGFR 30–60 mL/min and ***P < 0.001 vs. eGFR >60 mL/min, respectively). B: The same enhanced periostin positivity can be seen in the glomerular compartment of biopsies with reduced GFR (*P < 0.05 eGFR <30 mL/min vs eGFR >60 mL/min). C and D: In a comprehensive transcriptomic screen on biopsies from patient with different nephropathies (n = 221), periostin mRNA expression was correlated with eGFR of the patients. Intrarenal periostin mRNA levels showed a significant, negative correlation with eGFR in both tubulointerstitium (C) and glomeruli (D) (tubulointerstitium: r = −0.47, P = 6.9⁻¹⁴, glomeruli: r = −0.18, P = 8.1⁻⁰³).

Figure 7

Periostin mRNA expression in tubulointerstitium. Periostin mRNA levels in the tubulointerstitium were quantified by real-time RT-PCR in biopsies from patients with FSGS (n = 25) and MGN (n = 28), with a wide range of eGFR. Highest periostin mRNA levels were found in biopsies from patients with eGFR <30 mL/min (**P < 0.01, data are shown as fold-change compared to the eGFR >60 mL/min group).

Figure 8

Co-immunofluorescence and consecutive sections for periostin, α-SMA, PECAM1, and WT1. A transplant nephrectomy was stained for periostin (A) and α-smooth muscle actin (α-SMA) (B). The merge (C) indicates no overlap of both signals but clear and consistent proximity of both molecules in both glomeruli (arrows) and in the tubulointerstitium. Focal staining in the periphery of glomerular tuft is illustrated by the arrowhead. No such proximity or any overlap was seen by co-immunofluorescence for periostin and PECAM1 (D–F). Consecutive sections of human transplant nephrectomy tissue were also stained for the cell-type markers WT1 (G), PECAM1 (H), α-SMA (I), and for periostin (J). Again, a clear proximity of α-SMA and periostin were observed, illustrated by arrow and arrowhead, and no overlap or obvious proximity with WT1 or PECAM1.

Figure 9

Periostin is produced by mesangial cells in vitro and induces cell proliferation. A: Western blot analysis for periostin showed a single band of the expected size in human and murine mesangial cell supernatants (SN1, SN2, respectively); a truncated recombinant periostin protein served as positive control (RCP; 75 kDa; Biovendor). Water served as negative control (NC). B: Murine mesangial cells were incubated with TGF-β1 at the indicated concentrations for 4 hours. This stimulation led to a dose-dependent induction of periostin mRNA (*P < 0.05 vs vehicle control; n = 4 each). C: Cell proliferation was tested on mesangial cells incubated in the absence and presence of varying concentrations of periostin. Highest proliferative effect was found for 10 ng/mL periostin. The results are shown from human mesangial cells and are summarized from four independent experiments with three to eight stimulations each. **P < 0.01, ***P < 0.001 versus vehicle control. Murine mesangial cells gave comparable results with highest induction at 10 ng/mL periostin.