Origin of parietal podocytes in atubular glomeruli mapped by lineage tracing

Abstract

Parietal podocytes are fully differentiated podocytes lining Bowman's capsule where normally only parietal epithelial cells (PECs) are found. Parietal podocytes form throughout life and are regularly observed in human biopsies, particularly in atubular glomeruli of diseased kidneys; however, the origin of parietal podocytes is unresolved. To assess the capacity of PECs to transdifferentiate into parietal podocytes, we developed and characterized a novel method for creating atubular glomeruli by electrocoagulation of the renal cortex in mice. Electrocoagulation produced multiple atubular glomeruli containing PECs as well as parietal podocytes that projected from the vascular pole and lined Bowman's capsule. Notably, induction of cell death was evident in some PECs. In contrast, Bowman's capsules of control animals and normal glomeruli of electrocoagulated kidneys rarely contained podocytes. PECs and podocytes were traced by inducible and irreversible genetic tagging using triple transgenic mice (PEC- or Pod-rtTA/LC1/R26R). Examination of serial cryosections indicated that visceral podocytes migrated onto Bowman's capsule via the vascular stalk; direct transdifferentiation from PECs to podocytes was not observed. Similar results were obtained in a unilateral ureter obstruction model and in human diseased kidney biopsies, in which overlap of PEC- or podocyte-specific antibody staining indicative of gradual differentiation did not occur. These results suggest that induction of atubular glomeruli leads to ablation of PECs and subsequent migration of visceral podocytes onto Bowman's capsule, rather than transdifferentiation from PECs to parietal podocytes.

Figure 1.

Characterization of the early events in the coagulation model. (A) Schematic of a parietal podocyte on Bowman’s capsule forming foot processes (arrow) and recruiting periglomerular capillaries (asterisk). (B and B’) Schematic of the electrocoagulation model. Along the lateral margin of the kidney, a necrotic area is visible after coagulation (arrowheads, right kidney). (C) Immediately after coagulation, a triangular area of necrosis is observed, reaching from the renal cortex into the medulla. (D) Higher magnification of the interface between the coagulated renal tissue (arrow) and remaining parenchyma shows several necrotic tubules 3 days after coagulation (arrowheads) with inflammatory demarcation. (E) After 1 week, multiple tubules are dilated (arrowhead). (F) After 3 weeks, glomeruli with obliterated urinary poles (arrow) and atrophic tubular remnants (arrowheads) are observed. C–F show periodic acid–Schiff stainings of paraffin sections. (G) Immunohistologic double staining shows that cells obstructing the tubular outlet are SSeCKS-positive parietal cells (arrow) and that Bowman’s capsule is lined by parietal podocytes projecting from the vascular pole (arrowheads). (H–I) TUNEL assays consistently show positive nuclear staining in PECs (approximately 1–2 positive nuclei per 100 glomeruli) 1–3 weeks after electrocoagulation (G, arrow). (H) No nuclear staining is observed in healthy controls.

Figure 2.

Characterization of the coagulation model. (A) Atubular glomerular cysts (arrowheads) and adjacent intact renal parenchyma (interface highlighted with arrows). (B) Higher magnification of a large glomerular cyst with a characteristic hypotropic tuft (arrowhead). The urinary pole is obstructed by cells and extracellular matrix (arrow). (C) Normal tubules are located immediately adjacent to atrophic, protein-filled tubules (arrow). A–C show periodic acid–Schiff stainings of kidneys from 6-month-old Sv129 mice, electrocoagulated at 3 weeks of age. (D) In C57BL/6 mice, significant cystic dilation of Bowman’s capsule occurs with a lower frequency in atubular glomeruli after electrocoagulation (arrows). (E) The urinary poles of small atubular glomeruli are similarly obstructed (white arrows) and surrounded by atrophic tubules and increased extracellular matrix (arrowheads). (F) The papilla contains mostly atrophic tubules (arrows), whereas other segments still appear unaffected (arrowhead). D–F show periodic acid–Schiff stainings of kidneys from 7-month-old C57BL/6 mice electrocoagulated at age 5 months. (G–J) Influence of the genetic background (38 Sv129 mice versus 36 C57BL/6 mice) (G), the postoperative observation time until euthanasia (30 mice at 2 months versus 44 mice at 6 months) (H), sex (39 female mice versus 35 male mile) (I), and age of the mice at coagulation (37 mice at 3 weeks versus 37 mice at 20 weeks) (J) on the development of glomerular cysts, which are shown as the percentage of glomerular cysts of all glomeruli, with 100 glomeruli per mouse analyzed (+, right coagulated kidney; −, left control kidney). (K) Factors facilitating the formation of larger glomerular cysts (>150 μm) shown as percentage of all glomerular cysts. The highest frequency of larger cysts is observed in Sv129 mice coagulated at 3 weeks of age and euthanized after 6 months (right panel). (L) Ki67-positive cells on Bowman’s capsule were counted in 50 glomeruli of each experimental animal (36 Bl6 mice and 38 Sv139 mice). Most proliferating cells are observed in Sv129 animals, which also develop the largest cysts. *P<0.05; **P<0.01; ***P<0.001.

Figure 3.

Cysts induced by electrocoagulation are glomerular cysts. (A) In normal glomeruli, synaptopodin (A, arrow) and SSeCKS (A’, arrow) are specifically detected in podocytes and PECs, respectively. The boundary from synaptopodin-positive podocytes and SSeCKS-positive PECs is at the VP (arrow). (B) Overview kidney section from a 6-month-old Sv129 mouse coagulated at 3 weeks of age containing a normal segment and a segment containing atubular and cystic glomeruli costained for synaptopodin and SSeCKS. Both cysts with (arrows) and without (asterisks) a visible glomerular tuft show expression of the PEC marker SSeCKS, indicating that all represent glomerular cysts. (C) Synaptopodin expression is observed on Bowman’s capsule in multiple atubular glomerular cysts (arrows). These parietal podocytes are negative for SSeCKS (C’, arrows). In these glomeruli, the boundary between podocytes and PECs shifts from the vascular stalk onto Bowman’s capsule (3C”, arrowheads). VP, vascular pole; UP, urinary pole.

Figure 4.

Parietal podocytes in atubular glomeruli. (A and B) Bowman's capsules of atubular glomeruli are lined by synaptopodin-positive parietal podocytes regardless of the cyst diameter. (C–F) Influence of genetic background (38 Sv129 mice versus 36 C57BL/6 mice) (C), postoperative observation time until euthanasia (30 mice at 2 months versus 44 mice at 6 months) (D), sex of the mice (39 female mice versus 35 male mice) (E), or age at coagulation (37 mice at 3 weeks versus 37 mice at 20 weeks) (F) on the frequency of atubular glomeruli with parietal podocytes. The y-axis shows the percentage of glomeruli with synaptopodin-positive cells on Bowman’s capsule versus all glomeruli on a renal cross-section, with 50 glomeruli per mouse analyzed. *P<0.05; **P<0.01; ***P<0.001.

Figure 5.

Ultrastructure of atubular glomeruli. (A) Transmission electron microscopic images of an atubular glomerulus forming a glomerular cyst. Parietal podocyte differentiation varies along Bowman’s capsule, with areas of partial foot process effacement (arrow) between areas with interdigitating foot processes (arrowheads). (B) At higher magnification, a parietal podocyte forms interdigitating foot processes (arrowhead), resides on a thin GBM-like basement membrane, and recruits a periglomerular capillary indicating vascular endothelial growth factor expression (arrow). (C) A podocyte adheres to the glomerular tuft as well as to Bowman’s capsule forming foot processes on both sides (arrowheads).

Figure 6.

Lineage tracing of podocytes or PECs in atubular glomeruli. (A1–A4) Serial cryosections of a triple transgenic Pod-rtTA/LC1/R26R mouse kidney (coagulation at 6 weeks of age and euthanasia at 6 months later). (A1 and A3) Genetically tagged podocytes are visualized using β-gal staining. (A2 and A4) Differentiated podocytes are stained in brown (antinephrin). In these serial sections, the same areas of Bowman’s capsule costain for β-gal and nephrin, indicating the presence of differentiated visceral podocytes on Bowman’s capsule (same arrows in consecutive sections indicate colocalization). (B1 and B2) Serial cryosections of a transgenic Pec-rtTA/LC1/R26R mouse kidney (coagulation at 6 weeks of age, euthanized 6 months later) stained for PEC marker β-gal or nephrin, respectively. In the vicinity of the vascular pole, genetically tagged PECs can no longer be found on Bowman’s capsule (B1, arrows). Instead, differentiated podocytes are detected, which are not derived from PECs (B2, arrows). (C) A parietal podocyte with multiple differentiated foot processes on Bowman’s capsule (arrowheads) is marked by the genetic marker for podocytes (black deposits of Bluo-Gal in a Pod-rtTA mouse). Similar Bluo-Gal deposits can be observed on the visceral podocyte on the glomerular tuft (arrows with tail).

Figure 7.

Lineage tracing of parietal podocytes in the UUO model. (A) Typical morphologic changes are observed 3 weeks after UUO on sections stained with periodic acid–Schiff. These include degeneration of renal medulla (black arrows), dilated proximal tubules with protein casts (white arrows), and atubular glomeruli (arrowheads). (B) Degeneration of the glomerulotubular junction. A thin basement membrane with a layer of cubic cells (arrowheads) has disconnected the proximal tubule (arrow). (C–D’) Three weeks after UUO, the Bowman’s capsule of the atubular glomeruli is lined by synaptopodin- and nephrin-positive parietal podocytes (arrows). (E and E’) Serial cryosections of a Pod-rtTA/LC1/R26R mouse show colocalization of β-gal and nephrin staining in parietal podocytes 3 weeks after UUO (arrows). (F) Statistical analysis of the percentage of glomeruli with synaptopodin-positive cells on Bowman’s capsule 1–3 weeks after UUO (12 mice total, 50 glomeruli per time point). Using ANOVA and Bonferroni analysis, no statistically significant differences are observed between experimental animals over time. (G) Negative control for nephrin immunostaining using irrelevant guinea pig antiserum as a primary antibody shows no reactivity. (H) Seven days after UUO, nuclear TUNEL staining is observed in PECs (arrows; SSeCKS, TUNEL, Hoechst triple staining). Nonspecific green fluorescence arises from erythrocytes in glomerular and tubulointerstitial capillaries. ***P<0.001.

Figure 8.

Podocyte and PEC marker expression in humans. (A–A’’’) Immunofluorescence double stainings of an apparently normal kidney of an 18-year-old old healthy man show no colocalization of the podocyte marker synaptopodin and the PEC marker claudin-1 on Bowman’s capsule. (B–E’’) Sporadic atubular glomeruli in human kidneys. Synaptopodin-positive parietal podocytes (arrowhead in B) does not show costaining for the PEC marker claudin-1 in atubular glomeruli (arrowheads in B’ and B’’). A sharp border between synaptopodin and claudin-1–stained cells is always observed in atubular glomeruli (B’’, arrowhead). Atubular glomeruli in which the entire Bowman’s capsule stained for synaptopodin are negative for claudin-1 (C–C’’, arrows). (D–E’’) Similarly, no costaining for synaptopodin (podocytes) and CD133 (PECs) is observed in glomeruli in which Bowman’s capsule is totally (D) or partially (E) lined by synaptopodin-positive parietal podocytes.

Figure 9.

Statistical analysis of the proportion of Bowman’s capsules covered by synaptopodin-positive cells (presumptive parietal podocytes). More than half of the glomeruli do not show parietal podocytes. In a significant portion of glomeruli (approximately 30%–40%), few parietal podocytes are detected (mostly close to the vascular pole, indicating that minor shifts of the podocyte to parietal cell interface occur in human kidneys over time). Bowman’s capsules populated more than one third by parietal podocytes occur predominantly in diseased kidneys and represent atubular glomeruli. Four and eight total kidney specimens with or without renal pathologies, including all 1404 random cross-sections of individual glomeruli, are analyzed.

Figure 10.

Schematic for the origin of parietal podocytes. In atubular glomeruli, the urinary outlet is disconnected from the tubule (orange) by a layer of epithelial cells (blue, PECs and/or proximal tubular cells) forming their own basement membrane (arrowhead) and becomes obstructed. PECs (blue) are progressively lost and visceral podocytes (green) migrate from the capillary tuft onto Bowman’s capsule via the vascular stalk to become parietal podocytes (arrow).