Extensive podocyte loss triggers a rapid parietal epithelial cell response

Abstract

Damage to podocytes is a central pathomechanism of proteinuric kidney disease. However, it is not fully understood how podocyte injury evolves to progressive glomerulopathies such as FSGS or collapsing glomerulopathy. In particular, the role of parietal epithelial cells remains controversial. Here, we show that adriamycin induces DNA damage and podocyte lysis in mice without evidence of autophagy, endoplasmic reticulum stress, or necroptosis. After extensive podocyte loss, activated parietal cells mediated tuft re-epithelialization by two distinct mechanisms. In the majority of glomeruli, vacuolized parietal epithelial cells attached to denuded glomerular basement membrane and, occasionally, disengaged from the parietal basement membrane. Less frequently, parietal epithelial cells covered the denuded visceral basement membrane via formation of proliferative pseudocrescents. Notably, "visceralized" parietal epithelial cells did not express vascular endothelial growth factor but upregulated hypoxia-inducible factor 1 expression. The presence of visceralized parietal epithelial cells in sclerosing and collapsing lesions in a kidney biopsy from a patient with diabetes underscores the human relevance of our findings. In conclusion, repopulation of the glomerular tuft by parietal cells may represent a compensatory response to extensive podocyte loss. Our results suggest, however, that visceralized parietal epithelial cells cannot induce revascularization of the hyalinized tuft, resulting in hypoxic cell death and irreversible destruction of the glomerulus.

Figure 1.

ADR causes proteinuric kidney disease and mortality in mice. (A) Twenty-four–hour albuminuria at baseline (0) and after ADR injection (1, 2, 3, 4 weeks). Albuminuria in control mice remains at baseline. Mice treated with 11 µg ADR develop mild proteinuria within 1 week, which peaks at 2 weeks and subsequently recovers to low levels. Mice injected with 17 or 22 µg ADR both develop high albuminuria with a peak at 1 week. (B) Differences in total body weight from baseline to 1, 2, 3, and 4 weeks. Mice treated with 11 µg ADR show significant but mild weight loss compared with controls by week 2. Mice treated with 17 or 22 µg ADR exhibit a significant increase in weight loss compared with controls as early as 2 or 1 weeks, respectively. (C) Survival rates of control and ADR-injected mice. Results show that 100% of the control animals survive to 4 weeks, whereas ADR-treated animals show diminished survival rates. Survival appears to be inversely correlated with ADR dosage, with 11 µg ADR–injected mice showing the best and 22 µg ADR–injected mice showing the worst survival. *P≤0.05; **P≤0.01; ****P≤0.0001. (Note: due to death or euthanasia of mice due to severe ADR toxicity, no data on albuminuria, body weight, or survival were obtained on 22 µg ADR–injected mice past 1 week and on 17 µg ADR–injected mice past 2 weeks.)

Figure 2.

Overview of histologic changes in the renal cortex in ADR nephropathy. (A) Control mouse kidney 4 weeks after injection of saline. The renal cortex is preserved with normal glomerular and tubulointerstitial architecture. (B) Mouse kidney 4 weeks after injection of 11 µg ADR. The renal cortex shows mild chronic changes with proteinaceous casts and focal atrophy of the renal parenchyma. Only isolated glomerular changes are seen. (C) Mouse kidney 2 weeks after injection of 17 µg ADR. The renal cortex shows moderate chronic changes of the tubulointerstitium with tubular atrophy and frequent proteinaceous casts. Many glomeruli demonstrate pathologic changes. (D) Mouse kidney 1 week after injection of 22 µg ADR. The renal cortex shows only few glomerular lesions; the majority of tubules demonstrate severe ATN, characterized by low epithelium and epithelial cell necrosis. Scale bar, 100 µm.

Figure 3.

The spectrum of glomerular lesions in ADR nephropathy. (A) Light-microscopically normal glomerulus. Exclusively normal glomeruli are found in control mice, and represent the majority of glomeruli in all ADR-injected animals at 1 week. In contrast, few normal glomeruli are observed in 17 µg ADR–injected animals at 2 weeks. (B) Glomerulus with parietal epithelial cell vacuolization (PEC Vac). Asterisks mark large vacuoles in PECs, spanning from the parietal basement membrane to the glomerular tuft. (C) Glomeruli with sclerosed segments are a frequent finding in ADR-injected animals and are absent in controls. (D) Glomeruli with GS are frequent in 17 µg ADR–injected animals at 2 weeks, whereas they are rare in other ADR-injected groups and absent in controls. (E and F) Glomeruli with single-layered PCF (E) and multi-layered PCF (F). Epithelial cell proliferations covering a collapsed tuft, reminiscent of collapsing glomerulopathy, are encountered in 17 and 22 µg ADR–injected animals. The arrow points to an epithelial bridge (E), and the asterisk marks a multi-layered pseudocrescent (F). Scale bar, 20 µm.

Figure 4.

Immunohistochemical analysis of early glomerular changes. (A) Immunoperoxidase staining for H2AX in controls, and in 17 µg ADR–treated mice at 1 week. The controls show minimal glomerular staining, whereas strong specific staining is present in occasional tubule cell nuclei. In glomeruli of ADR-injected mice, intense nuclear staining is present in podocytes and weak staining is seen in endothelial cells. (B) Quantification of H2AX-positive tuft cells per glomerulus. The increase in H2AX-positive tuft cells compared with controls is statistically significant in animals treated with 17 and 22 µg ADR at 1 week (****P≤0.001); this increase is not statistically significant at 2 weeks and does not differ from controls in animals injected with 11 µg ADR. (C) Representative images of immunoperoxidase staining for BiP in controls, and in 17 µg ADR–treated mice at 1 week. No statistically significant difference in glomerular staining is present (data quantification not shown). (D) Representative images of immunoperoxidase staining for LC-3 in controls and in 17 µg ADR–treated mice at 1 week. Although autophagosome staining was observed in distal tubules, glomeruli are negative for LC-3 in controls as well as in ADR-injected mice. n=50 glomeruli per animal. Scale bars, 20 µm.

Figure 5.

Ultrastructural changes of the glomerulus in ADR nephropathy. (A) Representative electron micrograph of a control glomerulus. The architecture of the kidney filter is normal, with preservation of podocyte FPs with intact slit diaphragms, regular GBM, and fenestrated glomerular endothelium (lower panel). Podocyte cell bodies and primary processes show normal-appearing nuclei, organelle composition, and cytoskeleton, and are devoid of vacuoles. (B) Representative electron micrograph of a glomerulus at 1 week after injection of 17 µg ADR. Podocytes show extensive FPE and irregular thickening of the GBM. Notably, the typical cytoplasmic actin condensation is seen along the GBM (lower panel, arrow). (C) Representative electron micrograph of a glomerulus at 1 week after injection of 17 µg ADR with focal podocyte vacuolization. Effaced podocytes show vacuolization of their cytoplasm (asterisk), whereas the parietal epithelium is still intact. Note the presence of the dense actin band, typical for podocyte FPE, along an irregularly thickened GBM. Endothelial fenestrations are absent. (D) Left panel: representative electron micrograph of an injured podocyte at 1 week after injection of 17 µg ADR. Note the focal rupture of the podocyte plasma membrane, leaking cytoplasmic content into Bowman’s space, indicative of beginning cell lysis. Right panel: representative electron micrograph of a lysed podocyte at 2 weeks after injection of 17 µg ADR. Occasional podocytes show complete cell lysis. Scale bar, 2 µm.

Figure 6.

PECs form giant vacuoles spanning across Bowman’s space and attach to the denuded glomerular tuft. (A) Representative electron micrograph of a glomerulus with tuft adhesion 2 weeks after injection of 17 µg ADR. In a subset of glomeruli, enlarged PECs (green, yellow, blue) bearing large nucleoli and giant vacuoles (asterisks) are spanning from the parietal basement membrane (PBM) (orange) across Bowman’s space to the opposite denuded GBM. (B) Representative electron micrograph of a glomerulus with tuft adhesion 2 weeks after injection of 17 µg ADR. In a subset of glomeruli, activated PECs (green) already adherent to the denuded GBM (pink) are partially detaching from the PBM (orange; arrow). (C) Representative electron micrograph of a glomerulus with segmental hyalinosis at 2 weeks after injection of 17 µg ADR. Hyalinized capillaries (double asterisks) with denuded GBMs (pink) are focally covered with epithelial cells (green, blue) with morphologic features of PECs. Note that the PBM (orange) is devoid of an epithelial layer. Scale bar, 10 µm in A; 2 µm in B and C.

Figure 7.

vPECs express PAX2 and are negative for synaptopodin. (A) Representative images of glomeruli stained for PAX2. PAX2 staining in control glomeruli is limited to the PECs; no specific staining is present in the glomerular tuft. In glomeruli with PEC vacuolization (PEC Vac), vacuolated cells, some of them attached to the tuft, show nuclear PAX2 staining (arrowheads). Globally sclerosed glomeruli exhibit scattered PAX2-positive cells within the sclerosed tuft (arrowhead). (B) Representative images of glomeruli stained for synpo. Controls show strong staining for synpo in podocytes. Only remnant podocyte synpo staining or nonspecific staining of large intracapillary hyalin deposits is present in glomeruli with PEC vacuolization or global sclerosis. (C) Representative images of glomeruli stained for synpo (brown) and PAX2 (red). The tuft in control glomeruli stains exclusive for synpo. PAX2 is only seen within the flat parietal epithelium. In glomeruli with PEC vacuolization, vacuolated cells are exclusively PAX2 positive, whereas synpo stains remnant podocytes in the tuft that are negative for PAX2. In global sclerosis, scattered cells in the glomerular tuft are positive for PAX2 (arrow), whereas nonspecific synpo staining is limited to hyalin deposits. Scale bar, 20 µm.

Figure 8.

vPECs form the proliferative pseudocrescents of collapsing lesions. (A) Representative electron micrograph of a glomerulus with a collapsed tuft segment and epithelial cell proliferation. The cells composing a single layer (orange), which is loosely covering a partially denuded tuft with only residual underlying podocyte coverage (red), show features of PECs. Note the open capillaries still covered by podocyte remnants. (B) Representative image of a glomerulus with a segmental epithelial cell proliferation stained for PAX2. The staining is limited to the parietal epithelium and palisading cells cover the glomerular tuft (arrows). (C) Representative image of a glomerulus with a segmental epithelial cell proliferation stained for synpo. The staining is negative in the palisading cells covering the tuft (arrows), whereas residual podocytes within the tuft are positive. (D) Representative image of a glomerulus with parietal cell vacuolization stained for Ki-67. Activated vacuolated cells show strong nuclear staining for Ki-67 (arrows), indicating cell cycle entry. Scale bar, 10 µm in A; 20 µm in B–D.

Figure 9.

vPECs are negative for VEGF but express high levels of HIF-1. (A) Control glomerulus stained for VEGF and HIF-1. Whereas VEGF expression is strong in podocytes (arrows), glomerular expression of HIF-1 is weak. (B) Glomerulus with GS stained for VEGF and HIF-1. VEGF expression in the sclerosed glomerular tuft is virtually negative. In contrast, scattered epithelial cells within the tuft are positive for HIF-1 (arrows). (C) Glomerulus with PCF stained for VEGF and HIF-1. VEGF expression in the cells forming the proliferative lesion is virtually negative, whereas they are strongly positive for HIF-1 (arrows). Scale bar, 20 µm.

Figure 10.

Renal biopsy from a patient with diabetic nephropathy (DN), and superimposed segmental sclerosis or glomerular collapse in the same sample, stained with periodic acid–Schiff, and for synpo and PAX2. (A) Glomerulus with mesangial expansion and SGS. The sclerosed segment (asterisk) shows loss of synpo staining and PAX2-positive, synpo-negative vPECs lining the tuft (arrows). (B) Glomerulus with a globally collapsed tuft. The collapsed tuft is repopulated by synpo-negative, PAX2-positive vPECs (arrows). (C) Glomerulus with mesangial expansion only. Note the intact podocyte staining for synaptopodin and absence of PAX2-positive vPECs on the tuft. Scale bar, 50 μm.

Figure 11.

Pathways of re-epithelialization of the tuft by vPECs after podocyte dropout in sclerosing and proliferative glomerular lesions. (A) Schematic chain of events leading to tuft sclerosis or proliferative glomerulopathy: (1) normal epithelial coverage by podocyte FPs (blue) in a healthy glomerulus, (2) stress leads to podocyte FPE, and (3) localized podocyte lysis leaves behind areas of denuded GBM. These events can trigger one of two different pathways. Pathway I is as follows: (4) PECs (green) vacuolize to span Bowman’s space and attach to the denuded GBM, (5) PECs detach from the parietal basement membrane, and (6) a fully detached vPEC is covering aspects of a hyalinized capillary wall. Alternatively, in pathway II, the GBM denudation triggers proliferation of PECs across a small epithelial bridge (7) onto the denuded tuft, forming a pseudocrescent (8). (B) Model of pathologic events in ADR nephropathy. Adriamycin causes DNA damage in podocytes. Low doses lead to mild podocyte injury with FPE, followed by recovery of the visceral epithelium. High doses of ADR lead to podocyte lysis, whereas remaining podocytes attempt to compensate by hypertrophy. However, if too many podocytes drop out, denuded GBM is left behind, enabling extensive protein leakage. This in turn activates PECs, which either directly migrate onto the hyalinized tuft, eventually leading to glomerulosclerosis, or continuously proliferate along the denuded tuft to cover a collapsed glomerular segment.