Expression of Ser729 phosphorylated PKCepsilon in experimental crescentic glomerulonephritis: an immunohistochemical study

Abstract

PKCε, a DAG-dependent, Ca2+- independent kinase attenuates extent of fibrosis following tissue injury, suppresses apoptosis and promotes cell quiescence. In crescentic glomerulonephritis (CGN), glomerular epithelial cells (GEC) contribute to fibro-cellular crescent formation while they also transdifferentiate to a mesenchymal phenotype. The aim of this study was to assess PKCε expression in CGN. Using an antibody against PKC-ε phosphorylated at Ser729, we assessed its localization in rat model of immune-mediated rapidly progressive CGN. In glomeruli of control animals, pPKCε was undetectable. In animals with CGN, pPKCε was expressed exclusively in glomerular epithelial cells (GEC) and in GEC comprising fibrocellular crescents that had acquired a myofibroblast-type phenotype. In non-immune GEC injury induced by puromycin aminonucleoside and resulting in proteinuria of similar magnitude as in CGN, pPKCε expression was absent. There was constitutive pPKCε expression in distal convoluted tubules, collecting ducts and thick segments of Henley's loops in both control and experimental animals. We propose that pPKCε expression occurring in GEC and in fibrocellular crescentic lesions in CGN may facilitate PKCε dependent pathologic processes.

Figure 1.

Trichrome stains of control (A) and nephritic glomerulus (B) with fibrocellular crescent. Scarring (collagen) stains as light green. Scale bar: 50 µm.

Figure 2.

Immunolocalization of pPKCε in glomerulus of control (A), and nephritic animal (B); pPKCε is barely detectable or absent in intrinsic cells of the control glomerulus. pPKCε is intensely expressed in glomerular epithelial cells and within cellular crescent of the nephritic glomerulus (B); immunolocalization of the visceral epithelial cell marker, WT-1 in the nephritic glomerulus (C); lack of pPKCε staining in GEC of a PAN-treated rat (D). Scale bar: 50 μm.

Figure 3.

E-cadherin immunohistochemical expression in glomerulus and non-proximal tubules of a control kidney (A) and in glomerulus of a kidney with CGN (B); there is loss of E-cadherin expression in the nephritic glomerulus. (C), crescentic glomerulus with evidence for epithelial-to-mesenchymal (E-to-M) transformation of GEC identified by strong expression of α-smooth muscle actin in cells populating the crescent. Black arrows point to visceral and parietal GEC with E-to-M transformation, and to the vascular pole of the glomerulus. Scale bar: A and B, 100 µm; C, 50 µm.

Figure 4.

Differential expression of pPKCε (A) and macrophage marker ED1 (B) in nephritic glomerulus: pPKCε expression is mainly restricted in GEC while that of ED1 is observed throughout the glomerulus. Scale bar: 50 µm.

Figure 5.

Non-proximal tubules (black stars) that are positive for pPKCε and are surrounded by inflammatory cell infiltrates and scarring. Scale bar: 50 µm

Figure 6.

Serial sections of nephritic kidney stained for pPKCε and AH lectin. pPKCε positive tubules (A, black arrows) are also positive for the AH lectin (B, black arrows), which is specific for distal tubules and collecting ducts. In glomeruli, pPKCε localized in GEC (A, black arrow heads). Scale bar: 50 µm.

Figure 7.

Serial sections of tubules strongly positive for pPKCε (demarcated by the arrow heads in A) but negative for the TP lectin (demarcated by the arrow heads in B). Also shown, are tubules weakly positive for pPKCε (A, black arrows). The same tubules are strongly positive for the TP lectin (B, black arrows). Scale bar: 50 µm.

Figure 8.

Strongly pPKCε positive tubules in which there is heterogeneity of pPKCε staining amongst epithelial cells (positively alternating with negatively stained cells pointed by black arrows in A and B). These tubules were AH lectin positive and TP lectin negative. Scale bar: 50 µm.