Renal progenitor cells contribute to hyperplastic lesions of podocytopathies and crescentic glomerulonephritis

Abstract

Glomerular injury can involve excessive proliferation of glomerular epithelial cells, resulting in crescent formation and obliteration of Bowman's space. The origin of these hyperplastic epithelial cells in different glomerular disorders is controversial. Renal progenitors localized to the inner surface of Bowman's capsule can regenerate podocytes, but whether dysregulated proliferation of these progenitors contributes to crescent formation is unknown. In this study, we used confocal microscopy, laser capture microdissection, and real-time quantitative reverse transcriptase-PCR to demonstrate that hypercellular lesions of different podocytopathies and crescentic glomerulonephritis consist of three distinct populations: CD133(+)CD24(+)podocalyxin (PDX)(-)nestin(-) renal progenitors, CD133(+)CD24(+)PDX(+)nestin(+) transitional cells, and CD133(-)CD24(-)PDX(+)nestin(+) differentiated podocytes. In addition, TGF-beta induced CD133(+)CD24(+) progenitors to produce extracellular matrix, and these were the only cells to express the proliferation marker Ki67. Taken together, these results suggest that glomerular hyperplastic lesions derive from the proliferation of renal progenitors at different stages of their differentiation toward mature podocytes, providing an explanation for the pathogenesis of hyperplastic lesions in podocytopathies and crescentic glomerulonephritis.

Figures

Figure 1.

Assessment of CD24 and CD133 expression in normal human kidney is shown. (A) Both CD24 and CD133 are, within the glomerular structure, exclusively expressed by the epithelial cells lining the Bowman's capsule in adult human glomeruli. NHK, normal human kidney. (B) CD24+CD133+ renal progenitors also expressed claudin-1 (blue). (C) Double-label immunofluorescence for CD133 (green) and nestin (red) demonstrating the existence of cells expressing CD133 in absence of the podocyte marker nestin, which localize to the urinary pole (UP) of the Bowman's capsule, and cells expressing CD133 and nestin, which localize between the UP and the vascular pole (VP) of the Bowman's capsule (arrow). Glomerular podocytes also appear as CD133−nestin+. To-pro-3 counterstains nuclei. (D) Double-label immunofluorescence for CD133 (green) and PDX (red) demonstrating the existence of cells expressing CD133, which localize to the UP of the Bowman's capsule, and cells expressing CD133 and PDX (arrows), which localize between the UP and the VP of the Bowman's capsule. Glomerular podocytes also appear as CD133−PDX+. To-pro-3 counterstains nuclei. (D′) High magnification of the UP. Arrow points to cells showing double labeling for CD133 and PDX. To-pro-3 counterstains nuclei. (D″) High magnification of the VP. Arrow points to cells showing double labeling for CD133 and PDX. To-pro-3 counterstains nuclei. Magnifications: ×400 in A through C; ×200 in D; and ×560 in D′ and D″.

Figure 2.

Phenotypic analysis of the cells involved in hyperplastic lesions of podocytopathies is shown. (A) Assessment of CD24 and CD133 expression in kidneys with collapsing glomerulopathy. The involved epithelial cells were positive for both CD24 and CD133 (arrow). (B) In collapsing glomerulopathy, the involved epithelial cells were positive for both CD24 (red) and nestin (green). Merged images are in yellow (arrow). (B′) High magnification of the lesion in collapsing glomerulopathy. (C) Assessment of the co-localization among CD24, CD133, and PDX. In collapsing glomerulopathy lesions, the involved epithelial cells were positive for both CD24 (red) and CD133 (green). Merged images are in yellow. Rare cells within the lesion exhibited a triple labeling for PDX (blue), CD133, and CD24 (arrows). Merged images are in white. (C′) High magnification of collapsing glomerulopathy lesion. (D) Assessment of CD24 and CD133 expression in kidneys with FSGS. The involved epithelial cells were positive for both CD24 (green) and CD133 (red). Merged images are in yellow (arrow). (E) Assessment of the co-localization among CD24, PDX, and nestin. In FSGS lesions, the involved epithelial cells were mostly positive for CD24 (red) but negative for PDX (blue) or nestin (green). Rare cells within the lesion exhibiting a triple labeling for CD24, PDX, and nestin are also visible (white). (E′) High magnification of a FSGS lesion. (F) Assessment of the co-localization between the progenitor markers CD24 (green) and CD133 (red) and the PEC marker claudin-1 (blue) in a biopsy showing FSGS with mild hyperplasia. All markers were expressed by the epithelial cells lining the Bowman's capsule and the cells involved in the lesion (arrow). (F′) High magnification of FSGS lesion. (G and H) CD133, CD24, Oct-4, BmI-1, nestin, podocin, and nephrin mRNA quantification in identical areas of hyperplastic lesions of different podocytopathies and healthy glomerular tuft as obtained with laser capture microdissection. Data are means ± SEM of five independent experiments. Magnifications: ×200 in D; ×400 in A, B, B′, C, C′, E, F, and F′; and ×600 in E′.

Figure 3.

Phenotypic analysis of the cells involved in the generation of the tip lesion is shown. (A) Assessment of CD24 and CD133 expression in kidneys with tip lesion. The involved epithelial cells were positive for both CD24 and CD133 (white arrows). Black arrows indicate the tip lesion. (B) Assessment of the co-localization among CD24, CD133, and nestin in a tip lesion. The involved epithelial cells were positive for both CD24 (green) and CD133 (red). Merged images are in yellow (arrow). (B′) High magnification of the lesion. (C) Assessment of the co-localization among CD24 (red), CD133 (green), and PDX (blue) in a tip lesion. Rare cells exhibited a triple labeling for CD133, CD24, and PDX (arrows). Merged images are in white. (C′) High magnification of the lesion. The image was rotated to show the triple-labeled cells better. (D and E) CD133, CD24, Oct-4, BmI-1, nestin, podocin, and nephrin mRNA quantification in identical areas of tip lesions or healthy glomerular tuft as obtained with laser capture microdissection. Data are means ± SEM of five independent experiments. Magnifications: ×400 in A, B, B′, and C; and ×600 in C′.

Figure 4.

Phenotypic analysis of the cells involved in hyperplastic lesions of crescentic glomerulonephritis is shown. (A) Assessment of the co-localization among CD24, CD133, and nestin. In crescents, the involved epithelial cells were positive for both CD24 (red) and CD133 (green). Merged images are in yellow. Rare hyperplastic cells exhibited a triple labeling for CD133, CD24, and nestin (blue). Merged images are in white. (A′) High magnification of the hyperplastic lesion. (B) Assessment of the co-localization among CD24, CD133, and PDX. In crescents, the involved epithelial cells were positive for both CD24 (red) and CD133 (green). Merged images are in yellow. Rare hyperplastic cells exhibited a triple labeling for CD133, CD24, and PDX (blue). Merged images are in white. (B′) High magnification of the hyperplastic lesion. (C) Assessment of the co-localization among CD24 (red), PDX (blue), and nestin (green). In crescents, the involved epithelial cells were mostly positive for CD24 but negative for PDX or nestin. Rare hyperplastic cells exhibited a triple labeling for CD24, PDX, and nestin. Merged images are in white. (C′) High magnification of the hyperplastic lesion. (D and E) Assessment of the co-localization between the progenitor cell markers CD24 (green) and CD133 (red) and the PEC markers claudin-1 (D, blue) and caveolin-1 (E, blue). All four markers were expressed by the epithelial cells lining the Bowman's capsule and the proliferative cells involved in the lesions. A great overlap was observed in the staining pattern of the three markers (white). (D′ and E′) High magnification of the hyperplastic lesion. (F) Absence of co-localization between CD24 (red) and CD68 (green) in hyperplastic lesions. Arrow points to CD68+ cells within the lesion. (G and H) CD133, CD24, Oct-4, BmI-1, nestin, podocin, and nephrin mRNA quantification in identical areas of hyperplastic lesions of crescentic glomerulonephritis or healthy glomerular tuft as obtained with laser capture microdissection. Data are means ± SEM of four independent experiments. Magnifications: ×400 in A, A′, B, C, and F; ×200 in C′, D, D′, E, and E′; and ×600 in B′.

Figure 5.

Proliferating cells in hyperplastic glomerular lesions derive from CD133+CD24+nestin− and CD133+CD24+nestin+ cells. (A) Quantification of the percentages of CD133+CD24+nestin− (range 50 to 81%), CD133+CD24+nestin+ (range 5 to 46%) and CD133−CD24−nestin+ cells (range 0 to 3%) over the total of Ki67+ cells in hyperplastic lesions of podocytopathies. (B) A representative image showing triple-label immunofluorescence for CD24 (red), PDX (blue), and Ki67 (green), demonstrating that proliferating epithelial cells in a glomerulus of a patient with collapsing glomerulopathy express CD24 in absence of PDX or coexpress CD24 and PDX. No PDX+ but CD24− cells express Ki67. (B′) High magnification of the lesion. (C) Quantification of the percentages of CD133+CD24+nestin− (range 48 to 90%), CD133+CD24+nestin+ (range 3 to 42%), and CD133−CD24−nestin+ cells (range 0 to 3%) over the total of Ki67+ cells in a glomerulus of a patient with crescentic glomerulonephritis (GN). (D) Representative image showing triple-label immunofluorescence for CD24 (red), PDX (blue), and Ki67 (green), demonstrating that proliferating epithelial cells in crescentic glomerulonephritis express CD24 in the absence of PDX or coexpress CD24 and PDX. No PDX+ but CD24− cells express Ki67. (D′) High magnification of the lesion. Magnification: ×400 in B, D, and D′; and ×600 in B′.

Figure 6.

Differential upregulation of ECM components after treatment with TGF-β in CD133+CD24+ renal progenitors and podocytes. (A and B) Double-label immunofluorescence for CD133 (red) and WT-1 (green) on podocytes (A) and on CD133+CD24+ renal progenitors (B). To-pro-3 counterstains nuclei (blue). (C, E, G, and I) Assessment of mRNA levels for collagen α3(IV) and collagen α1(IV) (C), collagen α1(I) (E), fibronectin (G), and laminin (I) by real-time quantitative RT-PCR in podocytes treated with 5 ng/ml TGF-β. (D, F, H, and J) Assessment of mRNA levels for collagen α3(IV) and collagen α1(IV) (D), collagen α1(I) (F), fibronectin (H), and laminin (J) by real-time quantitative RT-PCR in CD133+CD24+ renal progenitors treated with 5 ng/ml TGF-β. Data are means ± SEM of triplicate assessment from three different experiments. *P < 0.05. Magnification, ×200 in A and B.