Gα12 Activation in Podocytes Leads to Cumulative Changes in Glomerular Collagen Expression, Proteinuria and Glomerulosclerosis

Ilene Boucher; Wanfeng Yu; Sarah Beaudry; Hideyuki Negoro; Mei Tran; Martin R Pollak; Joel M Henderson; Bradley M Denker

Disclosures

Lab Invest. 2012;92(5):662-675. 

In This Article

Results

Gα12 is Expressed in the Glomeruli

Although Gα12 is expressed in proximal and distal tubular epithelial cells[26] and glomerular proteomics identified Gα12,[27] its localization in the glomeruli has not been described. Endogenous Gα12 was localized in normal mouse glomeruli by immunohistochemistry. At lower power, Gα12 was detected in the glomeruli (Figure 1a, arrow heads) and throughout the nephron. At higher power (Figure 1a, black/white arrows), several cell types, including podocytes, appear to express Gα12 with only background staining in the controls (antibody preincubated with peptide) (Figure 1a, b and d). To confirm podocyte Gα12 localization, immunogold EM was performed (Figure 1b). Gold particles were visible in several locations, including FPs, major processes (MPs) and at the branch points proximal to the FPs (arrows). The localization of Gα12 in MPs suggests that Gα12 may have functions not directly related to slit diaphragm permeability.

Figure 1.

Endogenous Gα12 is expressed in normal mouse kidney. (a) Immunohistochemistry of normal mouse kidney demonstrates Gα12 expression in glomeruli. Sections were probed with rabbit anti-Gα12 and visualized with Vectastain. Two magnifications ( × 20 and × 40) are shown (panels A and B) Negative controls were performed by pre-incubating the antibody with excess blocking peptide (Gα12+Pep). Kidney sections using the blocked Gα12 antibody showed a significant reduction in staining (panels B and D). (b) Immunoelectron microscopy shows that Gα12 localizes to interdigitating foot processes (FP) and major processes (MP). Immunogold labeling and electron microscopy were performed as described in Materials and Methods. Magnification, × ~100 000. Arrows denote gold particles. Glomerular basement membrane (GBM), FP, fenestrated endothelium (E) and larger major processes (MP) are labeled.

Establishing Transgenic Mice With Podocyte Expression of Activated Gα12 (QLα12)

Mice expressing EE-tagged, human Gα12 (Q229L) were established using a LacZ/floxed transgenic approach[24] (Figure 2a). For conditional expression,[28] QLα12LacZ+/Cre− mice were crossed with Nphs2-Cre mice (Nphs2 is efficiently expressed in podocytes and no other glomerular cells).[29] Cre-mediated excision of the LacZ/stop in podocytes resulted in QLα12 expression (QLα12LacZ+/Cre+) (Figure 2). The CMV promoter is associated with the mosaic expression due to random inactivation,[24] although Cre efficiently excises LacZ in these podocytes. QLα12LacZ+/Cre− (Control) and QLα12LacZ+/Cre+ mice at 2 months of age (littermates) were stained for β-gal using standard techniques. β-Gal staining revealed that transgene expression was low in proximal tubules, but higher in the papilla (as reported,[24] not shown). QLα12LacZ+/Cre+ mice showed a major reduction in glomerular LacZ staining (Figure 2b) compared with Cre− controls. A semiquantitative analysis of β-gal staining suggested that random inactivation of the CMV promoter in podocytes led to LacZ expression in approximately half of the targeted cells (Supplementary Figure 1). Note that all the Cre+ mice showed significantly less or no β-gal staining, while the controls were evenly distributed (Supplementary Figure 1). The small amount of residual LacZ staining in QLα12LacZ+/Cre+ mice likely reflects LacZ expression in mesangial or endothelial cells. To distinguish EE-QLα12 expression from endogenous Gα12, control and QLα12LacZ+/Cre+ kidneys were co-stained with anti-nephrin and anti-EE antibodies (Figure 2c). In control mice, nephrin was seen throughout the glomerulus, with no detectable EE staining (Figure 2c and a). In QLα12LacZ+/Cre+ mice, both proteins were detected (Figure 2c and b), and as expected from immunoEM (Figure 1b), there was little overlap with nephrin in the slit diaphragm. ImmunoEM using the EE epitope antibodies confirmed transgenic QLα12 expression (not shown) in a similar distribution to the endogenous Gα12 (Figure 1b). To confirm that active Gα12 was expressed in these glomeruli, GST-TPR pull downs were performed as described previously;[16] the TPR domain of PP5 binds the active conformation of Gα12/13.[30] Figure 2d shows GST-TPR pull downs of thrombin-stimulated MDCK cells compared with cortical kidney lysates from QLα12LacZ+/Cre+ and QLα12LacZ+/Cre− mice.

Figure 2.

Development of transgenic mice with podocyte-specific expression of QLα12. (a) Schematic of targeting epitope tagged (EE) human QLα12 to podocytes. The floxed LacZ/stop is driven by cytomegalovirus (CMV) promoter and Nphs-2 podocin-Cre was used for podocyte expression. (b) Transgenic mice show mosaic expression of LacZ. Control and QLα12LacZ+/Cre+ mice at 2 months of age (littermates) were stained for β-gal as describe in Materials and Methods. Insets (C and D) show an individual glomerulus. (c) QLα12LacZ+/Cre+ mice express EE-tagged QLα12 in podocytes. Immunofluorescent staining was performed on control (A) and QLα12LacZ+/Cre+ (B) mice using fluorescein isothiocyanate (FITC)-conjugated goat anti-EE (shown in green) and guinea-pig anti-nephrin (Progen) and Cy3 secondary antibody (shown in red) (d) Activated Gα12 was pulled down from kidney lysates of QLα12 LacZ+/Cre+ or thrombin-stimulated Madin–Darby canine kidney (MDCK) cells using GST-TPR or GST alone.

QLα12 Expressed in Podocytes Leads to Age-dependent Proteinuria

Development of microalbuminuria is a sensitive marker for podocyte injury.[31] Urine was analyzed for albumin/creatinine ratio (Figure 3a) from QLα12LacZ+/Cre+ and control mice every 2 months. Microalbuminuria (albumin/creatinine ratios ≥34) appeared in a few control mice at 4–6 months, but was seen in ~40% of QLα12LacZ+/Cre+ mice (some with ratio >200; Figure 3 and Table 1 ). Most of the QLα12LacZ+/Cre+ mice developed proteinuria as they aged, whereas only a few controls had mildly increased levels. Coomassie blue staining of urine confirmed the expression of albumin (Figure 3b). The magnitude of proteinuria continued to increase until mice were sacrificed at 22–26 months. On the basis of this phenotype, we divided the mice into young (<6 months; occasional mild proteinuria) and older (>12 months; frequent and often severe proteinuria) for further analysis.

Figure 3.

QLα12LacZ+/Cre+ mice develop of proteinuria with age. (a) Urine microalbumin/creatinine ratio in QLα12 mice is higher than in controls. Urine from control and QLα12 mice was monitored every 2 months using a BCA analyzer. Albumin/creatinine from individual control (○) and QLα12 (▪) mice are shown. Lines indicate median value (dashed, control; solid, QLα12). (b) Urine from QLα12 mice contains high levels of albumin. Urine from 12- and 16-month QLα12 (QL) and littermate control mice (c) was collected and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. The arrow denotes ~66 kDa, the size of excreted albumin. Note that the 12-month old mice had more concentrated urine (based on the nonspecific low-molecular-weight bands). (c) QLα12 mice are more susceptible to lipopolysaccharide (LPS)-induced injury. Control (n=17) and QLα12 (n=21) mice were injected with 10 μg/g body weight of LPS. Urine was collected 18 h post-injection and analyzed for urine microalbumin/creatinine ratio using a BCA analyzer. Statistical analysis was performed using two-way analysis of variance (ANOVA), followed by Bonferroni's post-hoc test (# P<0.001; * P<0.0001).

The lack of proteinuria in most younger mice suggests that podocytes compensate for the expression of activated Gα12. To test whether QLα12 expression predisposed younger mice (<6 months) to injury, proteinuria was examined after LPS stimulation (a model of transient podocyte injury) (Figure 3c).[32] Baseline proteinuria was similar, and 18 h after LPS stimulation, control mice increased proteinuria 1.9-fold, whereas QLα12LacZ+/Cre+ mice showed a significantly larger increase (3.6-fold). However, these short-term experiments did not detect any differences in ultrastructural or light microscopy findings (not shown). These findings are consistent with LPS-stimulated podocyte actin cytoskeleton changes leading to proteinuria and reveal that podocyte expression of QLα12 in young mice enhances the injury response.

Focal GS Develops With Age in QLα12LacZ+/Cre+ Mice

In mice aged <6 months, no significant renal histopathological changes were observed in control or QLα12LacZ+/Cre+ mice (Figure 4a and e). However, by 12–18 months, QLα12LacZ+/Cre+ mice showed numerous globally and segmentally sclerosed glomeruli, whereas controls exhibited only rare sclerosed glomeruli. Quantifying affected glomeruli showed a >6-fold increase in sclerosed glomeruli from kidneys of QLα12LacZ+/Cre+ mice (n=7) compared with controls (n=7) (Figure 4b and f). Segmentally sclerosed glomeruli were characterized by hyalinosis, GBM reduplication, isolated epithelial cells containing PAS+ protein reabsorption granules and adhesion of the tuft to Bowman's capsule. Mesangial areas of QLα12LacZ+/Cre+ mice exhibited mild to focally moderate expansion by matrix and cells, whereas the mesangial areas of control mice exhibited minimal expansion (Figure 4b and f, arrow). The kidneys of 18-month-old mice exhibited changes similar to the 12- to 18-month group, but more prominent. On average, 4.79% of glomeruli in QLα12LacZ+/Cre+ mice were globally or segmentally sclerosed, whereas only 0.16% of glomeruli in control mice were sclerosed (Figure 4c–e and h). Over 73% of these sclerosed glomeruli were juxtamedullary. Focal segmental double contours (areas of GBM redundancy) were more commonly seen in QLα12LacZ+/Cre+ mice than in controls. Occasional PAS+ hyaline casts were observed in the medulla to varying degrees in older animals as well as focal interstitial mononuclear inflammation, usually in association with focal GS (Figure 4g and h, arrows). Features of focal tubular injury, including tubular luminal distension, epithelial flattening and microcyst formation, were seen in a subset of the oldest QLα12LacZ+/Cre+ mice, and these findings were not seen in the age-matched controls. The non-lesional glomeruli (Figure 4h, arrowhead) exhibit moderate mesangial expansion by matrix and cells (*). Other features of active or chronic tubulointerstitial or vascular pathology were not observed in any animals. There were no detectable differences in serum creatinine between the 14- and 19-month-old QLα12LacZ+/Cre+ mice and controls as all values were ≤0.2 mg/dl.

Figure 4.

Light micrographs show focal and segmental glomerulosclerosis in the juxtamedullary region of older QLα12LacZ+/Cre+ mice. Representative light micrographs of murine juxtamedullary kidney cortex in control (top row) and QLα12LacZ+/Cre+ (bottom row) mice aged 4.5, 13 and 24 months are shown. (a, e) Kidneys of mice aged <6 months, regardless of genotype, show no significant pathological changes in glomeruli, tubulointerstitium or vasculature. (b, f) Kidney of QLα12LacZ+/Cre+ mice (f) aged 12–18 months exhibit focal glomerulosclerosis involving juxtamedullary glomerulus (arrow). The parenchyma is otherwise well preserved. Age-matched controls (b) show no significant pathological changes. (c, d, g, h) Kidneys of QLα12LacZ+/Cre+ mice aged >18 months show focal global (g) and segmental (h) glomerulosclerosis involving multiple juxtamedullary glomeruli (arrows), accompanied by focal interstitial mononuclear inflammation. The non-lesional glomeruli (h; arrowhead) exhibit moderate mesangial expansion by matrix and cells (*). Age-matched controls (c, d) show mild mesangial expansion, but no other cortical parenchymal lesions are apparent. PAS; bar=100 μm (left 3 columns) and 50 μm (right column).

Older Qlα12LacZ+/Cre+ Mice Develop GBM Irregularities, Mesangial Expansion and FP Fusion

EM of QLα12LacZ+/Cre+ and control mice <6, 12–18 and >18 months were scored for ultrastructural abnormalities. The youngest QLα12LacZ+/Cre+ mice (Figure 5a) appeared normal. At 12–18 months, there was little difference in the GBM or the podocytes, but most QLα12LacZ+/Cre+ mice had focal features of endothelial injury, including double contours, subendothelial granular electron-dense deposits and cell swelling (Figure 5e, arrowhead; not seen in the controls). In addition, QLα12LacZ+/Cre+ scored higher for mesangial expansion by matrix and cells (Figure 5e (*); Table 2 ). Older QLα12LacZ+/Cre+ mice (>18 months) (Figure 5f) mice exhibited a greater degree of FP effacement and irregularity, presumably associated with aging (Figure 5f, arrows) than was seen in the controls (although controls did reveal some age-related changes). There were few differences in the GBM, but there were an increased number of subepithelial GBM membrane projections in the oldest transgenics (Figure 5f, ♦). All mice exhibited mild mesangial expansion at >18 months; however, QLα12LacZ+/Cre+ mice showed more severe signs of mesangial abnormalities (Figure 5f, *), and overall scores ( Table 2 ) for glomerular injury were twice as high in the QLα12LacZ+/Cre+ mice (Figure 5c).

Figure 5.

QLα12LacZ+/Cre+ develop foot process fusion and ultrastructural changes that worsen with age. Transmission electron microscopy (EM) was performed on kidneys from control and QLα12LacZ+/Cre+ mice at <6 (a, d), 12–18 (b, e) and >18 months (c, f) were analyzed in a blinded manner and scored for severity of injury. Representative micrographs in control (top row) and QLα12LacZ+/Cre+ (bottom row) mice aged 4, 14 and 23 months are shown. At <6 months, both control (a) and QLα12LacZ+/Cre+ (d) show normal glomerular structure. By 12–18 months, the QLα12LacZ+/Cre+ mice (e) show more signs of endothelial injury (arrowhead) and mesangial expansion (*) than controls (b). All of the oldest mice examined show significant GBM thickening, but the QLα12LacZ+/Cre+ (f) show increased development of subepithelial basement membrane projections (♦) along the GBM. The podocytes have more foot process effacement and irregularity (arrow) in addition to the mesangial expansion (*) and endothelial injury (arrowhead) seen in the 12–18 months mice compared with controls (c).

QLα12LacZ+/Cre+ Mice Have Normal Numbers of Podocytes

On the basis of the morphological changes, we examined podocyte number by WT-1 staining.[33] Surprisingly, podocyte number/glomerulus showed no difference between QLα12LacZ+/Cre+ and controls >12 months (Figure 6a and b). As Gα12 can stimulate both proliferation and apoptosis,[16] WT-1-positive cells were quantified for apoptosis (TUNEL) and proliferation (Ki67) to exclude the possibility that podocyte number was preserved through QLα12 co-stimulation of proliferation and apoptosis. No apoptotic podocytes were seen in QLα12LacZ+/Cre+ or controls (Figure 6a, middle panels), nor any difference observed in proliferation (not shown). In addition, there were no differences in the number of glomeruli in kidneys obtained from QLα12LacZ+/Cre+ mice as compared with controls (data not shown). This indicates that the development of proteinuria and GS does not result from developmental effects on glomeruli number or podocyte depletion, and other mechanisms must be responsible.

Figure 6.

QLα12LacZ+/Cre+ mice have normal numbers of podocytes. (a) Wilms's tumor-1 (WT-1) staining shows little podocyte apoptosis in both control and QLα12 mice. In all, 24 fields and total >200 podocytes were counted for control and QLα12LacZ+/Cre+ mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed on kidney sections from QLα12 and control mice 2–6 and 12–16 months of age. In addition, sections were probed for WT-1 and stained for 4′,6-diamidino-2-phenylindole (DAPI). (b) WT-1 quantification shows similar number of podocytes in both control and QLα12 mice. The number of cells per glomeruli stained for both WT-1 and DAPI quantified for 100 glomeruli and averaged.

QLα12 Expression in Podocytes Does Not Lead to RHO or SRC Activation

Next, Rho activity was examined in QLα12Lac+/Cre+ and control mice. Rho activity was determined by ELISA on glomerular isolates from young and old mice. There were no significant differences in Rho activity from QLα12Lac+/Cre+ mice compared with controls, nor was there any difference in Rho protein expression (Figure 7a). Gα12 also activates Src,[34] and western blot using pY419 antibodies failed to demonstrate any differences in Src activation (not shown). This finding suggests that podocytes compensate for persistent QLα12 expression and employs other mechanisms to prevent sustained Rho or Src activation.

Figure 7.

RhoA activity is not altered in QLα12LacZ+/Cre+ mice. (a) Enzyme-linked immunosorbent assay (ELISA) for activated RhoA was performed on young (2–6 months) and old (>12 months) QLα12LacZ+/Cre+ and control mice. (b) Total RhoA did not change in QLα12LacZ+/Cre+ mice as they age. Western blot analysis was performed to examine total RhoA. Blots were stripped and re-probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ImageJ was used to determine RhoA/GAPDH expression.

Real-time PCR Showed no Changes in Podocyte-specific Genes, but QLα12LacZ+/Cre+ Mice Exhibit Dysregulated Glomerular Collagen Expression

Real-time PCR of glomerular isolates failed to detect changes in expression levels of several major podocyte genes implicated in FSGS (Nphs1, Nphs2, CD2AP and TRPC6) (Supplementary Figure 2). The normal adult GBM is composed predominantly of α3, α4 and α5(IV) collagen and laminin-11 (α5β2γ1).[35] During development, the normal GBM is composed of α1 and α2(IV) collagen that is followed by a switch to α3, α4 and α5(IV) expression in mature glomeruli. Real-time PCR of Col4a showed increased Col4a2 transcript expression levels in younger mice (Figure 8a), although the variability in phenotype led to wide confidence intervals. These age-dependent changes are likely to contribute to the observed variability, and as this analysis was performed on isolated glomeruli, it is possible that the most severely sclerotic glomeruli were not included and would thus tend to underestimate the differences. In addition, the real-time results were reanalyzed in male and female mice, and no differences were observed to account for the differences in phenotype seen between transgenic and control mice in any age group (not shown). Nevertheless, when considered together, these results suggest a change in the relative balance of specific collagen α(IV) chains, and immunofluorescence microscopy confirmed mildly increased collagen α1/2(IV) expression (Figure 8c). In older mice, Col4a2 expression abnormalities persist, and by 12–14 months, there is also a twofold increase in Col4a1 and decreased Col4a5 (Figure 8c). Consistent with the real-time results, collagen α1/2(IV) staining was increased in older mice, and the localization was disorganized without clear capillary loop staining (Figure 8d). Decreased expression of α5 was confirmed, although the pattern of staining appeared similar to the control (Figure 8d). No other α(IV) chains showed differences in staining. The α3/4/5 antibody showed decreased intensity compared with the control, and based on the α3 staining and real-time results, this difference is most likely explained by the decreased α5 collagen expression. To determine if QLα12 directly regulates Col4 gene expression, a previously characterized inducible (tet off) QLα12-MDCK cell line[10,11,17] was analyzed by microarray. Supplementary Figure 3 shows 7- and 10-fold increase in Col4a1 and Col4a2, respectively, within 72 h of QLα12 expression. Taken together, this analysis indicates that QLα12 expression leads to dysregulated Col4a gene expression before the onset of proteinuria and suggests a mechanism where activated Gα12 alters Col4 gene expression.

Figure 8.

Collagen (α)IV is misregulated QLα12LacZ+/Cre+ mice. (a, b) Col4a1, Col4a2 and Col4a5 transcript expression are altered in QLα12LacZ+/Cre+ mice >12 months of age. Various α chains of COL4 were examined in a (2–6 month) or b (>12 months) mice. Results were normalized to the 18S ribosomal subunit and graphed as relative expression compared with non-targeting control (normalized to 1); n≥6 mice for each experiment. (c, d) Immunofluorescent staining was performed on frozen kidney sections using collagen antibodies (α1/2, Rockland; α3NC1 (monoclonal antibody (mAb) 8D1), α3α4α5NC1 (mAb 26–20), α5 (polyclonal))[52] and Alexa488 anti-mouse or anti-rabbit secondary antibody. Representative images are shown from QLα12LacZ+/Cre− and QLα12LacZ+/Cre+ mice in c (2–6 month) or d (12–16 month). Scale bar=100 μm.

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