Gα12 activation in podocytes leads to cumulative changes in glomerular collagen expression, proteinuria and glomerulosclerosis

Abstract

Glomerulosclerosis is a common pathological finding that often progresses to renal failure. The mechanisms of chronic kidney disease progression are not well defined, but may include activation of numerous vasoactive and inflammatory pathways. We hypothesized that podocytes are susceptible to filtered plasma components, including hormones and growth factors that stimulate signaling pathways leading to glomerulosclerosis. Gα12 couples to numerous G-protein-coupled receptors (GPCRs) and regulates multiple epithelial responses, including proliferation, apoptosis, permeability and the actin cytoskeleton. Herein, we report that genetic activation of Gα12 in podocytes leads to time-dependent increases in proteinuria and glomerulosclerosis. To mimic activation of Gα12 pathways, constitutively active Gα12 (QL) was conditionally expressed in podocytes using Nphs2-Cre and LacZ/floxed QLα12 transgenic mice. Some QLα12(LacZ+/Cre+) mice developed proteinuria at 4-6 months, and most were proteinuric by 12 months. Proteinuria increased with age, and by 12-14 months, many demonstrated glomerulosclerosis with ultrastructural changes, including foot process fusion and both mesangial and subendothelial deposits. QLα12(LacZ+/Cre+) mice showed no changes in podocyte number, apoptosis, proliferation or Rho/Src activation. Real-time PCR revealed no significant changes in Nphs1, Nphs2, Cd2ap or Trpc6 expression, but Col4a2 message was increased in younger and older mice, while Col4a5 was decreased in older mice. Confocal microscopy revealed disordered collagen IVα1/2 staining in older mice and loss of α5 without changes in other collagen IV subunits. Taken together, these studies suggest that Gα12 activation promotes glomerular injury without podocyte depletion through a novel mechanism regulating collagen (α)IV expression, and supports the notion that glomerular damage may accrue through persistent GPCR activation in podocytes.

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 (20x and 40x) are shown (panel a, 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 (panel b, d). (B) Immunoelectron microscopy shows 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 ~100,000x. Arrows denote gold particles. Glomerular basement membrane (GBM), foot processes (FP), fenestrated endothelium (E), and larger major processes (MP) are labeled.

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 CMV promoter and Nphs-2 Podocin-Cre was used for podocyte expression. (B) Transgenic mice show mosaic expression of LacZ. Control and QLα12^LacZ+/Cre+ mice at 2m of age (littermates) were stained for β-gal as describe in materials and methods. Insets (c, d) show an individual glomerulus. (C) QLα12^LacZ+/Cre+ mice express EE-tagged QLα12 in podocytes. Immunofluorescent staining was performed on control (a) and QLα12^LacZ+/Cre+ (b) mice using 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 MDCK cells using GST-TPR or GST alone.

Figure 3. QLα12^LacZ+/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 were monitored every two 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 SDS-PAGE and Coommassie Blue staining. The arrow denotes ~66kDa, the size of excreted albumin. Note that the 12m mice had more concentrated urine (based on the non-specific low molecular weight bands). (C) QLα12 mice are more susceptible to LPS induced injury. Control (n=17) and QLα12 (n=21) mice were injected with 10μg/g body weight LPS. Urine was collected 18h post injection and analyzed for urine microalbumin/creatinine ratio a BCA Analyzer. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s post hoc test (# p<0.001, * p<0.0001)

Figure 4. Light micrographs show focal and segmental glomerulosclerosis in the juxtamedullary region of older QLα12^LacZ+/Cre+ mice

Representative light micrographs of murine juxtamedullary kidney cortex in control (top row) and QLα12^LacZ+/Cre+ (bottom row) mice aged 4.5, 13, and 24 m are shown (A,E) Kidneys of mice aged <6 m, regardless of genotype, show no significant pathologic changes in glomeruli, tubulointerstitium, or vasculature. (B, F) Kidney of QLα12^LacZ+/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 pathologic changes. (C,D,G,H) Kidneys of QLα12^LacZ+/Cre+ mice aged >18m 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 microns (left 3 columns), 50 microns (right column).

Figure 5. QLα12^LacZ+/Cre+ develop foot process fusion and ultrastructural changes that worsen with age

Transmission electron microscopy (EM) was performed on kidneys from control and QLα12^LacZ+/Cre+ mice at <6m(A, D), 12-18m (B, E), and >18 m (C, F) were analyzed in a blindly and scored for severity of injury. Representative micrographs in control (top row) and QLα12^LacZ+/Cre+ (bottom row) mice aged 4, 14, and 23m are shown. At <6m, both control (A) and QLα12^LacZ+/Cre+ (D) show normal glomerular structure. By 12-18m, the QLα12^LacZ+/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 QLα12^LacZ+/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 the 12-18m mice compared with controls (C).

Figure 6. QLα12^LacZ+/Cre+ mice have normal numbers of podocytes

(A) WT-1 staining shows little podocyte apoptosis in both control and QLα12 mice. 24 fields and total >200 podocytes were counted for control and QLα12^LacZ+/Cre+ mice. TUNEL assays were performed on kidney sections from QLα12 and control mice 2-6 and 12-16m of age. Additionally, sections were probed for WT-1 and stained for 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.

Figure 7. RhoA activity is not altered in QLα12^LacZ+/Cre+ mice

(A) ELISA for activated RhoA was performed on young (2-6mo) and old (>12mo) QLα12^LacZ+/Cre+ and control mice. (B) Total RhoA did not change in QLα12^LacZ+/Cre+ mice as they age. Western blot analysis was performed to examine total RhoA. Blots were stripped and re-probed for GAPDH and ImageJ was used to determine RhoA/GAPDH expression.

Figure 8. Collagen (α)IV is misregulated QLα12^LacZ+/Cre+ mice

(A-B) Col4a1, Col4a2, and Col4a5 transcript expression are altered in QLα12^LacZ+/Cre+ mice >12m of age. Various α chains of COL4 were examined in A (2-6 month) or B (>12 month) mice. Results were normalized to the 18S ribosomal subunit and graphed as relative expression compared to non-targeting control (normalized to 1). n≥ 6 mice for each experiment. (C) Immunofluorescent staining was performed on frozen kidney sections using collagen antibodies [α1/2, Rockland; α3NC1 (mAb 8D1), α3α4α5NC1 (mAb 26-20), α5 (polyclonal)] (52) and Alexa488 anti-mouse or anti-rabbit secondary antibody. Representative images are shown from QLα12^LacZ+/Cre- and QLα12^LacZ+/Cre+ mice 2-6 month or 12-16 month mice. Scale bar=100 microns