TGF-beta receptor deletion in the renal collecting system exacerbates fibrosis

Abstract

TGF-beta plays a key role in upregulating matrix production in injury-induced renal fibrosis, but how TGF-beta signaling in distinct compartments of the kidney, such as specific segments of the nephron, affects the response to injury is unknown. In this study, we determined the role of TGF-beta signaling both in development of the renal collecting system and in response to injury by selectively deleting the TGF-beta type II receptor in mice at the initiation of ureteric bud development. These mice developed normally but demonstrated a paradoxic increase in fibrosis associated with enhanced levels of active TGF-beta after unilateral ureteral obstruction. Consistent with this observation, TGF-beta type II receptor deletion in cultured collecting duct cells resulted in excessive integrin alphavbeta6-dependent TGF-beta activation that increased collagen synthesis in co-cultured renal interstitial fibroblasts. These results suggest that inhibiting TGF-beta receptor-mediated function in collecting ducts may exacerbate renal fibrosis by enhancing paracrine TGF-beta signaling between epithelial and interstitial cells.

Figure 1.

Hoxb7Cre;Tgfbr2^flox/flox mice develop normally but sustain greater injury after UUO. (A) β-gal staining of Hoxb7Cre;Tgfbr2^flox/flox mice with the ROSA26 reporter demonstrates Cre expression in the collecting system. (B) Tissue lysates of renal papillae from adult mice are immunoblotted with antibodies directed against TβRII. Each blot shows a representative kidney from a total of three mice per genotype. (E through L) Kidney tissue from uninjured mice (C and D) and mice injured by UUO (E through L) is stained with hematoxylin and eosin. Three days after UUO injury, hematoxylin and eosin staining reveals tubular dilation (E and F) as well as increased epithelial flattening (G and H) and tubular casts (black arrow). Seven days after UUO, tubular damage worsens and fibrosis develops (I and J); this is further exacerbated at 14 days (K and L; black arrow). Magnifications: ×100 in C through F, I, and J; ×400 in G, H, K, and L

Figure 2.

Hoxb7Cre;Tgfbr2^flox/flox mice have increased collagen I expression that is not attributed to changes in apoptosis or inflammation. (A through D) Fibrillar collagen is detected 7 (A and B) and 14 (C and D) days after UUO by Trichrome staining. (E) Lysates of corticomedullary tissue of obstructed kidney are immunoblotted for collagen I. (F) Collagen I bands from four mice per genotype are quantified by densitometry, normalized to focal adhesion kinase (FAK), and reported as means ± SEM. *P < 0.05. (G and H) TUNEL staining shows apoptotic nuclei (brown) in Hoxb7Cre;Tgfbr2^flox/flox and Tgfbr2^flox/flox mice 3 days after UUO. CD localization of apoptosis is determined by co-staining with AQP2 (red). (I) TUNEL-positive cells from 10 high-powered fields per mouse are counted in a blinded manner using four mice per genotype. *P < 0.05. (J and K) F4/80 staining is performed for macrophage infiltration. (L) F4/80-positive cells are quantified and expressed as described for TUNEL staining. Magnifications: ×100 in A through D, J, and K; ×400 in G and H.

Figure 3.

Hoxb7Cre;Tgfbr2^flox/flox mice have increased active TGF-β compared with Tgfbr2^flox/flox mice. (A) pSmad2 immunoblots are performed on obstructed kidney lysates from Hoxb7Cre;Tgfbr2^flox/flox and Tgfbr2^flox/flox mice 3 days after UUO. TGF-β–treated cells are used as a positive control. Three representative blots per genotype are shown, and the vertical white line separates samples that are rearranged from two blots performed simultaneously and developed for equivalent times. (B) Band intensity is quantified as a ratio of pSmad2 to total Smad2 using densitometry. Data are derived from five mice per genotype and reported as the mean ± SEM. *P < 0.05. (C) Active TGF-β is determined by the PAI/L assay, as described in the Concise Methods section. Composite values for six mice per genotype at 3 days after UUO are reported as the mean in random luciferase units (RLU) ± SE. *P < 0.05. (D) Total TGF-β levels from kidney lysates 3 days after UUO are measured by an ELISA assay. The data represent the average values of TGF-β (pg) per mg of protein for five mice in each genotype.

Figure 4.

Conditioned media from TβRII^−/− CD cells have greater levels of active TGF-β than TβRII^flox/flox CD cells. (A) TβRII^−/− and TβRII^flox/flox CD cell lysates are immunoblotted with a TβRII antibody. (B) TβRII^flox/flox and TβRII^−/− CD cells are stimulated with TGF-β (5 ng/ml) for various periods, and then immunoblots for pSmad2 are performed. (C and D) Total and active levels of TGF-β from media of TβRII^flox/flox and TβRII^−/− CD cells collected over 72 hours are determined by ELISA (C) and PAI/L assays (D). Total TGF-β (C) is reported as pg of TGF-β per mg of protein, and active TGF-β (D) is measured by random luciferase units (RLU). The values reported in C and D reflect averages ± SE from three separate experiments. *P < 0.05.

Figure 5.

Conditioned medium from TβRII^−/− CD cells stimulates increased collagen I production by interstitial cells. (A) Fibroblasts are incubated with conditioned medium from both TβRII^flox/flox and TβRII^−/− CD cells and immunoblotted for collagen I expression in the presence or absence of 2G7 (a blocking pan–TGF-β antibody at 20 μg/ml). Fibroblasts are incubated with serum-free (SF) medium as a negative control. The white lines separating samples represent where the order of samples within the same blot was changed. (B) Densitometry of collagen I/focal adhesion kinase (FAK) is performed using three separate experiments and reported as means ± SEM. *P < 0.05. (C) Conditioned medium from CD cells with and without TβRII is placed on fibroblasts in the presence or absence of 2G7 (blocking pan–TGF-β antibody) for varying periods, after which the fibroblasts are lysed and immunoblotted for pSmad2. The vertical white line separating samples indicates where the order of samples from the same experiment on two gels developed for equivalent times is changed.

Figure 6.

Increased TGF-β activation in TβRII^−/− CD cells is mediated by integrin αvβ6. (A) Effects of the protease inhibitors tranexamic acid (plasmin, plasminogen), pepstatin A (aspartyl), E-64 (cysteine), and GM6001 (matrix metalloproteinase) on TGF-β activation by TβRII^−/− CD cells are determined by the PAI/L assay. The levels of active TGF-β in A, C, and D are expressed as a percentage of active TGF-β in TβRII^flox/flox CD cell serum-free (SF) conditioned medium. (B) TSP-1 levels in conditioned medium from TβRII^flox/flox and TβRII^−/− CD cells are determined by immunoblotting. (C) LSKL, a TSP-1–blocking peptide, as well as a scramble peptide are added to conditioned medium to assess the effect on active TGF-β levels. (D) Blocking integrin αv and αvβ6 antibodies are added to the conditioned medium of TβRII^−/− and TβRII^flox/flox CD cells. (E) Integrin αvβ6 expression on TβRII^flox/flox and TβRII^−/− CD cells is measured by FACS analysis. Experiments in A through E were performed three times with data reported as means ± SEM. *P < 0.05. The concentrations used in these experiments are as follows: 10 mM tranexamic acid, 10 μM GM6001, 1 μg/ml integrin αv, 100 μg/ml integrin αvβ6, 10 μM TSP-1 blocking and scramble peptides, 100 μg/ml E-64, and 2.5 μg/ml pepstatin A.

Figure 7.

TβRII^−/− CD cells have increased active RhoA. (A) Rhodamine phalloidin staining of actin cytoskeleton in TβRII^flox/flox CD and TβRII^−/− CD cells. (B) Rho activity in cell lysates is measured as described in the Concise Methods section, and a representative experiment (a total of three performed) is shown. (C) A ratio of the densitometry of active Rho to total Rho from these three experiments is expressed as mean ± SEM. *P < 0.05. (D) Lysates of CD cells transfected with either pooled RhoA siRNA or control siRNA are immunoblotted with an antibody to RhoA. (E) Active TGF-β in the conditioned medium of cells transfected with RhoA siRNA is measured using the PAI/L assay and expressed as a percentage of active TGF-β measured in control siRNA-treated CD cells. This experiment was performed three times with error bars depicting the SE. *A significant (P < 0.05) drop in active TGF-β in RhoA siRNA-treated TβRII^−/− compared with TβRII^flox/flox CD cells.