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

Discussion

Understanding the mechanisms of CKD progression is important for finding new therapeutic targets. Podocytes are exposed to approximately 180 l of ultrafiltrate per day that contains biologically active molecules, including hormones, cytokines and filtered proteins. Herein, we demonstrate that activation of Gα12 in podocytes leads to age-dependent proteinuria and focal GS through a distinct mechanism involving dysregulated collagen α(IV) expression without podocyte depletion. This supports the hypothesis that filtered agonists activate podocyte signaling pathways and can contribute to progressive glomerular injury. Although Gα12 regulates numerous processes that could lead to podocyte damage, including apoptosis, proliferation, cell attachment, actin cytoskeletal changes and junctional regulation, these do not explain the time-dependent phenotype in these mice. Rather, a novel mechanism of collagen α(IV) abnormalities appears to be responsible.

Testing the notion that podocytes respond to filtered molecules is difficult to examine in vivo. Expressing a constitutively active G protein allows for the identification of downstream effector pathways, and this approach led to the identification of h-Ras as an oncogene.[36] This strategy could identify novel therapeutic targets to prevent GS without the need to identify the specific GPCR or its ligand. Analogous to our studies, activated (QL) Gαq was targeted to podocytes,[37] and these mice developed proteinuria through a different mechanism. QLαq mice had developmental defects, including smaller kidneys and reduced nephron number. There was downregulation of nephrin and other podocyte genes, although Col4a was not examined. QLαq mice were more susceptible to puromycin aminonucleoside injury, and a subset of mice developed GS at 6 months of age, a finding that could occur from reduced nephron number. In contrast, QLα12 in podocytes did not affect renal development, nephron or podocyte number, and revealed altered regulation of collagen and GBM abnormalities.

Proteinuria developed in some QLα12LacZ+/Cre+ mice by 4–6 months before morphological changes (Figures 3, 4 and 5). By 12–14 months, there was some FP fusion that could account for the increase in proteinuria; however, the CMV promoter results in mosaic expression, and we estimate that only about half of podocytes express QLα12 (Figure 2). This focal expression is likely to account for the mild phenotype, but also recapitulates local differences in glomerular response to stress and the focal nature of GS. In fact, the majority of sclerotic glomeruli was located at the cortico-medullary junction, a region where the glomeruli are more susceptible to hemodynamic stress.[38,39] Why some younger mice manifest proteinuria before morphological changes are detected is not clear. They do not show enhanced baseline Rho or Src activity, but they are more susceptible to LPS-induced proteinuria (without changes in Col4 gene expression (not shown)), suggesting that QLα12 can promote functional effects on permeability. The absence of ultrastructural changes in young mice or after LPS suggests that perhaps through Gα12 localization in the major FPs, it regulates cortical actin and permeability. Although only about 5% of the glomeruli develop GS, proteinuria is likely to arise from a more global effect on significantly more glomeruli. This is based on the observation that QLα12 is expressed in approximately 50% of glomeruli (Supplementary Figure 1) and QLα12 expression increased proteinuria in LPS-treated mice without morphological or ultrastructural changes.

Mesangial expansion was prominent in the QLα12LacZ+/Cre+ mice, and mesangial expansion is particularly characteristic of FSGS, membranous and diabetic nephropathy, Alport's syndrome and Denys–Drash syndrome.[40] Mesangial expansion can contribute to proteinuria through a mechanism that does not require podocyte depletion (as seen in this study).[41] In addition, both nephropathy and Alport's syndrome are both characterized by changes in collagen expression.[42–44] Transcription of Col4a1 and A2 is co-regulated by the same promoter owing to head-to-head orientation in chromosome 13q34.[45] As a result, diseases affecting collagen IV expression will be associated with changes in both α1(IV) and α2(IV)) as seen in both the above disease and QLα12 mice (Figure 8). However, Alport's syndrome is caused by mutations in α3, α4 or α5(IV) collagen, leading to sustained α1 and α2(IV) expression, whereas membranous nephropathy results from autoantibodies[46] that promote podocyte injury and subepithelial deposits.

On the basis of the time course of proteinuria and morphological changes, we speculate that Gα12 modifies COL4 gene expression and the resulting basement membrane and endothelial abnormalities accrue with time. However, whether these changes reflect age-dependent effects or are the result of cumulative Gα12 activation will require additional study. Since C57/B6 mice develop mild ultrastructural changes, cytokine activation and proteinuria with age (as seen in some control mice), the phenotypic differences seen in the older mice with Gα12 activation could represent indirect effects on cumulative age-related changes in this mouse strain, or a cumulative time effect of Gα12 activation. Although the control mice do develop mild changes with age, the dramatic differences seen in the age- and sex-matched QLα12 mice strongly implicates the persistent Gα12 activation as the etiology. Furthermore, the finding of upregulated COL4A1/2 seen with QLα12 expression in MDCK cells (Supplementary Figure 3) supports an important link between Gα12 activation and Col4A1/2 gene expression.

Although Gα12 stimulates TGFβ[20,21] and could promote GS, we were unable to detect increased TGFβ in either young or old mice (not shown). However, an alternative mechanism is suggested by the increased number of subepithelial GBM membrane projections (Figure 5) similar to findings seen in DDR1 and integrin α2 knockout mice.[47,48] DDR1 is a tyrosine kinase receptor for collagen IV, and we previously showed that Gα12 regulates α2β1 integrin signaling and attachment,[17] thus suggesting that persistent Gα12 activation might lead to podocyte changes in integrin signaling. The onset of proteinuria is during adulthood and virtually all mice have proteinuria by mid-life (12–16 months, normal C57/B6 mouse lifespan ~3 years[49]). With aging, podocytes may accumulate damage from a variety of sources, including GPCR-linked inflammatory and vasoactive mediators. ROS, inflammation and vascular changes occur in hypertensive and diabetic kidney disease, and ROS directly activates Gα subunits.[50] On the basis of these observations, we suggest that this model of slowly progressive kidney disease will be valuable for understanding CKD progression in humans. Consistent with GPCR pathways regulating age-related kidney damage, AT1 receptor knockout mice exhibit decreased oxidative damage and outlive their wild-type littermates.[51] The glomerular filtration rate declines with age, but it is unclear whether this is part of normal aging or represents injury from cumulative exposures. If decreased renal function and GS occur through repetitive activation of podocyte signaling pathways, it is tempting to speculate that inhibiting Gα12 pathways in podocytes may provide new treatments to protect renal function and delay CKD progression.

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