Proteinuria impairs podocyte regeneration by sequestering retinoic acid

Abstract

In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.

Figure 1.

Transferrin and IgG overload affects human RPC survival, while albumin overload impairs RPC differentiation into podocytes. (A) Effect of HSA, transferrin, or IgG treatment on the proliferative capacity of RPC, as assessed by [³H]-thymidine incorporation (n=4). (B) Flow cytometry analysis of RPC viability after treatment with HSA, transferrin, or IgG, as assessed by annexin-V and propidium iodide costaining. One representative of four experiments is shown (control: 90.82%±0.69% live cells; HSA: 90.35%±1.44%, transferrin: 51.68%±4.83%, IgG: 72.48%±5.28%; P=0.75 control versus HSA, P=0.01 control versus transferrin-treated and control versus IgG-treated cells by Mann-Whitney test, n=4). (C) NPHS1 mRNA levels in RPCs cultured in control medium or VRAD medium alone, or supplemented with HSA, transferrin, or IgG (n=6). (D) Nephrin protein expression assessed by FACS analysis in RPCs cultured in control medium or VRAD medium alone or supplemented with HSA, transferrin, or IgG. Representative histograms obtained in one of three independent experiments are shown. Percentages of nephrin-positive cells are calculated over their respective isotype control. All data are means ± SEM, **P<0.01 by ANOVA with Bonferroni post hoc analysis (A) and by Mann-Whitney test (C).

Figure 2.

Albumin overload impairs RPC differentiation into podocyte by sequestering RA. (A) Effect of HSA (10 mg/ml) on NPHS1 mRNA levels in human cells cultured in control medium, VRAD medium, or RA- or vitamin D3 (vitD3)–containing medium (n=5). (B) Nephrin protein expression assessed by FACS analysis in RPCs cultured in control medium, VRAD medium, or RA- or vitamin D3–containing medium in presence or absence of HSA (10 mg/ml). Representative histograms obtained in one of three independent experiments are shown. Percentages of nephrin-positive cells are calculated over their respective isotype control. (C) Dose-response effect of HSA on NPHS1 mRNA levels in human RPCs cultured in RA-containing medium (n=4); P=0.0006 by ANOVA. (D) Nephrin protein expression assessed by FACS analysis in human RPCs cultured in control medium or in RA-containing medium, in presence of different doses of HSA (0.1, 1, or 10 mg/ml). Representative histograms obtained in one of three independent experiments are shown. Percentages of nephrin-positive cells are calculated over their respective isotype control. (E) Effect of increasing concentrations of HSA on the uptake of radiolabeled-RA by human RPCs (n=6); P<0.0001 by ANOVA for both curves. (F) Effect of HSA (10 mg/ml) on the transcriptional activity of RARE-reporter plasmid in infected human RPCs in control or RA-containing medium (n=6). (G) NPHS1 mRNA levels in human RPCs in response to increasing concentrations of RA (n=6). (H) Flow cytometry analysis of ALDH1a activity of human RPCs in presence (top) or absence (bottom) of the ALDH1a inhibitor DEAB. The flow cytometry gatings used are marked by boxes (ALDH1a^positive fraction+DEAB: 0.67%±0.17% and–DEAB: 40.1%±1.83; n=4; P=0.02). (I) Effect of HSA (10 mg/ml) on NPHS1 mRNA induced by retinol treatment in human RPCs (n=6). (L) Nephrin protein expression assessed by FACS analysis in human RPCs cultured in control medium, retinol-containing medium, or retinol+HSA (10 mg/ml)–containing medium. Representative histograms obtained in one of three independent experiments are shown. Percentages of nephrin-positive cells are calculated over their respective isotype control. (M) Confocal microscopy analysis of FITC-HSA uptake by human RPC (green HSA signal, blue Topro-3 nuclear staining). One representative experiment of four is shown. Bars = 20 μm. All data are means ± SEM. *P<0.05, **P<0.01, ***P<0.001 by ANOVA with Bonferroni post hoc analysis for C, E, and G and by Mann-Whitney test for A, F, H, and I.

Figure 3.

Evaluation of urinary protein levels and podocyte markers in mice with genetically tagged podocytes validate AN as a model to study podocyte loss and regeneration. (A) Evaluation of albumin, transferrin, and IgG levels in the urine of three different mice strains (n=15 each) after induction of AN (day 11). (B) Three-dimensional reconstruction of glomerulus of healthy and Adriamycin-treated NPHS2.Cre;mT/mG transgenic mouse (green = GFP). (C) Representative electron micrographs of glomerular podocytes in AN show podocyte loss with extensive denudation of the capillary loops (arrows). Bars = 2 μm. (D) WT1 staining (blue) in renal sections of healthy (left) and of Adriamycin-treated (right) NPHS2.Cre;mT/mG transgenic mice (green = GFP; red = Tomato). Bars = 20 μm. (E) Nephrin staining (blue) in renal sections of healthy (left) and of Adriamycin-treated (right) NPHS2.Cre;mT/mG transgenic mice (green = GFP; red = Tomato). Bars = 20 μm. (F) Number of GFP, WT1, or nephrin-positive cells/glomerulus area in healthy (n=6) and in Adriamycin-treated (n=10) NPHS2.Cre;mT/mG transgenic mice. (G) Percentage of GFP cells coexpressing WT1 (left) or nephrin (right) in healthy (n=6) and in Adriamycin-treated (n=10) NPHS2.Cre;mT/mG transgenic mice. Data are means ± SEM; P=NS by Mann-Whitney test.

Figure 4.

Blocking endogenous retinoic acid synthesis decreases podocyte number, increases proteinuria and enhances mortality in SCID mice with FSGS. (A) Survival curve of Adriamycin-treated SCID mice exposed to DSF 100 mg/kg (purple line) (n=15) or 200 mg/kg (green line) (n=24). Survival curve of healthy mice (n=6), mice treated with DSF 100 mg/kg (n=6) and 200 mg/kg (n=6), and mice with Adriamycin alone (n=14) was 100%. (B) Time course assessment of albumin-to-creatinine ratio in mice with AN (blue triangle) (n=14) in comparison with mice with AN treated with DSF 100 mg/kg (pink inverted triangle) (n=11) or 200 mg/kg (green diamond) (n=11). Albumin-to-creatinine ratio in healthy mice and healthy mice treated with DSF 100 and 200 mg/kg (n=6 for each group) was all around 0. P=0.0001 by ANOVA test. (C) Representative periodic acid-Schiff staining of renal sections of healthy mice and of mice with AN, untreated or treated with DSF 100 and 200 mg/kg. (D) Representative WT1 (green) and claudin1 (red) costaining of renal sections of healthy mice and of mice with AN, untreated or treated with DSF 100 and 200 mg/kg. (E) Left: index of glomerular injury assessed as described in the Concise Methods section. Right: quantitation of number of podocytes (WT1+claudin1− cells)/glomerulus is shown (healthy, n=6; Adriamycin, n=14; Adriamycin plus DSF 100 mg/kg, n=11; Adriamycin plus DSF 200 mg/kg, n=11). Topro-3 counterstains nuclei (blue). Bars = 20 µm. All data are means ± SEM. *P<0.05, **P<0.01 by Kaplan-Meier survival analysis and log-rank test for A, by ANOVA with Bonferroni post hoc analysis for B, and by Mann-Whitney test for E.

Figure 5.

Following podocyte injury, RA is neutralized by albuminuria. (A) mRNA levels of RDH1, RDH9, RDH10, Aldh1a1, Aldh1a2, Aldh1a3, and Stra6 in glomeruli from healthy or Adriamycin-treated mice (n=4). (B) Flow cytometry analysis of ALDH1a activity of murine glomerular cells isolated from healthy or Adriamycin-treated mice, and stained with Aldefluor in presence (left) or absence (right) of the ALDH1a inhibitor DEAB. The flow cytometry gatings used are marked by boxes (in healthy animals: ALDH1a^positive fraction+DEAB 1.025%±0.4%, and –DEAB 8.875%±1.07%, n=4, P=0.02; in Adriamycin-treated animals: ALDH1a^positive fraction+DEAB 1.325%±0.24%, and –DEAB 7.975%±1.18%, n=4, P=0.02 by Mann-Whitney test). (C) Representative immunofluorescence staining of glomeruli from healthy or Adriamycin-treated mice showing Aldh1a1/2 (green) and PanCK (red). (D) Graph representing retinol (black) or RA (red) concentration (nM) in urine of mice with various levels of proteinuria. n=3–6, P=0.003 for retinol measurement by ANOVA test. (E) Percentage of RA (100 nM) assessed by LC-MS/MS in a urine standard solution in presence of increasing doses of albumin. (F) Hypothetic scheme summarizing the blocking effect of albumin on RARE activation induced by retinoids in renal progenitors. *P<0.05, **P<0.01 by Mann-Whitney test for D. Topro-3 counterstains nuclei (blue). Bars = 20 µm.

Figure 6.

RA treatment increases podocyte number by reversing proteinuria-impaired RARE activation in RPC. (A) Albumin-to-creatinine ratio in RARE-lacZ healthy transgenic mice, in healthy mice treated with RA, and in mice with AN with or without RA treatment. n=32 at days 0 and 4, n=11 for each group at the following time points. P=0.0001 by ANOVA. (B) Quantitation of podocyte number (WT1+claudin1− cells)/glomerulus in healthy (n=10), Adriamycin before treatment (n=10), Adriamycin plus vehicle (n=11), Adriamycin plus RA (n=11). (C) Left: quantitation of podocyte number (WT1+claudin1− cells)/glomerulus in renal biopsy samples (n=12) before (bt) and after (at) RA treatment (3.98±0.6 versus 5.16±0.62). Center: quantitation of number of RPCs that are differentiating toward podocytes (WT1+claudin1+ cells)/glomerulus in renal biopsy specimens (n=12) before (bt) and after (at) RA treatment (2.80±0.31 versus 3.33±0.31). Each symbol represents one single mouse. Right: representative staining of WT1 (green) and claudin1 (red) in renal biopsy specimens of mice with AN before (top) and after (bottom) RA treatment. (D) Left: RARE activity (blue) in healthy mouse kidney. Right: higher magnification of colocalization of β-gal staining (blue) and claudin1 (red) assessed by immunohistochemistry (top) and immunofluorescence (bottom, β-gal green and claudin1 red). (E) Low and high magnification images showing RARE activity in the kidney of mice with AN at day 4 as seen by colocalization of X-gal staining (blue) and claudin1 (red) by immunohistochemistry (left) and by immunofluorescence (right) for β-gal (green) and claudin1 (red). Arrows point to positive glomeruli. (F) Low and high magnification images demonstrating RARE activity in the kidney of mice with AN treated with RA (day 11) as seen by colocalization of X-gal staining (blue) and nestin (red) by immunohistochemistry (left) and by immunofluorescence (center and right) for β-gal (green) and claudin1 (red). White arrows pointed to β-gal+claudin1+ cells entering in the glomerular tuft. (G) Immunofluorescence for β-gal (green), claudin1 (red) and nestin (blue) in the kidney of mice with AN treated with RA (day 11). White arrow points to β-gal+claudin1+nestin+ cells demonstrating the acquisition of podocyte markers by β-gal+ cells. (H) Absence of RARE activity in the glomeruli of mice with AN treated with vehicle (day 11) as seen by colocalization of β-gal staining (blue) and nestin (red) by immunohistochemistry (left) and by immunofluorescence (right) for β-gal (green) and claudin1 (red). (I) Quantitation of number of claudin1+β-gal+ cells/section in healthy (n=10), Adriamycin before treatment (n=10), Adriamycin plus vehicle (n=11), and Adriamycin plus RA mice (n=11). (J) Percentage of claudin+β-gal+ cells outside and inside the lesions in Adriamycin plus RA treated mice (n=11). Topro-3 counterstains nuclei (blue). Bars = 20 µm. All data are means ± SEM. *P<0.05, **P<0.01, ***P<0.001 by ANOVA with Bonferroni post hoc analysis for A; by Mann-Whitney test for B, I, and J; and by Wilcoxon test for C.