Src inhibition ameliorates polycystic kidney disease

Abstract

Despite identification of the genes responsible for autosomal dominant polycystic kidney disease (PKD) and autosomal recessive PKD (ARPKD), the precise functions of their cystoprotein products remain unknown. Recent data suggested that multimeric cystoprotein complexes initiate aberrant signaling cascades in PKD, and common components of these signaling pathways may be therapeutic targets. This study identified c-Src (pp60(c-Src)) as one such common signaling intermediate and sought to determine whether Src activity plays a role in cyst formation. With the use of the nonorthologous BPK murine model and the orthologous PCK rat model of ARPKD, greater Src activity was found to correlate with disease progression. Inhibition of Src activity with the pharmacologic inhibitor SKI-606 resulted in amelioration of renal cyst formation and biliary ductal abnormalities in both models. Furthermore, the effects of Src inhibition in PCK kidneys suggest that the ErbB2 and B-Raf/MEK/ERK pathways are involved in Src-mediated signaling in ARPKD and that this occurs without reducing elevated cAMP. These data suggest that Src inhibition may provide therapeutic benefit in PKD.

Figure 1.

Developmental expression of active c-Src (pY418). (A) Western analysis of the renal expression of immunoprecipitated total/pan Src in BPK kidneys from PN0 (lane 1) to PN14 (lane 4) demonstrates little change with age or disease progression in BPK mice. (B) In contrast, renal expression of active Src (pY418) in BPK mice increases in parallel with disease progression from PN0 (lane 1) to PN14 (lane 4) in the absence of changes in total Src expression (A). (C) Western analysis of the original tissue extracts for β-actin before immunoprecipitation of pan-Src demonstrated that the original tissue extracts were accurately adjusted to equal protein concentrations, and equivalent quantities of protein were subjected to immunoprecipitation. Relative densitometry (minus background) shown below each lane were obtained by NIH Image 1.62, and both Westerns and respective densitometry data shown represent three independent, reproducible experiments.

Figure 2.

(A and B) Correlation of renal size (A) with Src activity (B). (A) Lane 3 demonstrates that large cystic kidneys from BPK mice correlate with increased expression of active Src (pY418) (B, lane 3) compared with age-matched Balb/C kidneys (A, lanes 1 and 2) and corresponding Src (pY418) activity (B, lanes 1 and 2). (B) Lane 4 demonstrates that reduction in renal Src activity with SKI-606 treatment (compared with lane 3) results in significant reduction in cystic kidney size as shown in A, lane 4. These data demonstrate that inhibition of c-Src activity (pY418) results in considerable reduction of renal growth in cystic BPK mice. (C) The original tissue extracts were accurately adjusted to equal protein concentrations, and equivalent quantities of protein were subjected to immunoprecipitation. Relative densitometry (minus background) shown below each lane was obtained by NIH Image 1.62, and both Westerns and respective densitometry data shown represent three independent, reproducible experiments.

Figure 3.

Histologic evaluation of SKI-606 treatment and immunohistochemical analysis of cyst localization. Cyst localization was studied by segment-specific lectin binding using Dolichos biflorus agglutinin (red label) as a marker for CT and Lotus tetragonolobus agglutinin (brown label) as a marker for proximal tubules. These data demonstrate that SKI-606 treatment of cystic BPK animals results in reduced size and number of renal CT cysts (B) compared with the cystic CT lesions present in PN21 untreated cystic kidneys (A). This reduction in renal CT cysts (B) occurs without morphologic evidence of renal toxicity and directly correlates with the reduction in renal Src activity and kidney size shown in Figure 2, B, lane 4, and A, lane 4, respectively. Magnification, ×40.

Figure 4.

Microscopic analysis of hepatic tissue from SKI-606 treated animals (C) reveals amelioration of BDE characteristic of untreated BPK (B) and closely resembles biliary ducts from control (A) liver. These data along with renal morphology shown in Figure 3 demonstrate that Src inhibition is effective in ameliorating PKD-associated proliferation in both renal and hepatic organs. These data also demonstrate that SKI-606 treatment at 30 mg/kg per d results in no morphologic evidence of renal (Figure 3B) or hepatic (Figure 4C) toxicity. Magnification, ×40.

Figure 5.

Comparison of EGFR targeted therapies and Src inhibition on active EGFR (pY1068). (A) Western analysis of immunoprecipitated total EGFR (ErbB1) in Balb/C (lane 1) and BPK (lanes 2 through 5) PN21 kidneys. (B) PN21 renal levels of active EGFR (pY1068) after immunoprecipitation of total EGFR with and without therapy as indicated. As seen in lane 5, Src inhibition is as effective as previously published EGFR targeted therapies (lanes 3 and 4) at reducing the activity of the receptor in the BPK model. (EKB is an EGFR tyrosine kinase inhibitor, and WTACE2 inhibits the processing of preproEGFR ligands, thereby reducing the availability of ligand. EKB was tested alone or in combination with WTACE2). (C) Western blot analysis of the original tissue extracts for β-actin before immunoprecipitation of total EGFR demonstrates that equivalent quantities of protein were subjected to immunoprecipitation. Relative densitometry (minus background) shown below each lane were obtained by NIH Image 1.62, and both Westerns and respective densitometry data shown represent three independent, reproducible experiments.

Figure 6.

Renal ErbB2 expression in PCK. (A) Western analysis of active ErbB2 (pY1221/1222) after immunoprecipitation of total renal ErbB2 demonstrates that active ErbB2 levels correlate with disease progression in the PCK. (B) Immunohistochemical staining of PN90 PCK kidney for active ErbB2 illustrates abnormally high expression levels of p-ErbB2 on both basolateral and apical cell surfaces of cystic epithelia. Magnification, ×40.

Figure 7.

Developmental profile of active Src and active p44/p42 MAPK (p-ERK1/2). (A and B) A developmental profile of total active Src (pY418) after immunoprecipitation of pan Src (A) and p-ERK1/2 after immunoprecipitation of total p44/p42 MAPK (B) in Sprague-Dawley and PCK kidneys. Ages are listed above each lane, with lanes 1 through 4 from Sprague-Dawley and lanes 5 through 8 from PCK. As seen in A, lanes 1 through 4, the level of active Src increases marginally in Sprague-Dawley, whereas B, lanes 1 through 4, demonstrate a developmental decrease in p-ERK1/2 in the Sprague-Dawley controls. In contrast, A, lanes 5 through 8, demonstrates a sizeable increase developmentally in Src (pY418) that correlated with a substantial increased p-ERK1/2 activity (B, lanes 5 through 8). The increasing level of active Src shown in A, lanes 5 through 8, and the increasing activity of p-ERK1/2 (B, lanes 5 through 8) correlate with progressive stages of renal cystic disease in the PCK. (C) The original tissue extracts were accurately adjusted to equal protein concentrations, and equivalent quantities of protein were subjected to immunoprecipitation.

Figure 8.

Correlation of renal size (A) with the levels of phosphorylated Src (B), ErbB2 (C), B-Raf (D), and ERK1/2 (E) after immunoprecipitation of total protein of interest. These data demonstrate that SKI-606 treatment of Sprague-Dawley (A2) did not alter kidney size compared with untreated Sprague-Dawley (A1) but decreased levels of active Src (B2) and p-ErbB2 (C2) but not p-B-Raf (D2) or p-ERK1/2 (E2). SKI-606 treatment in PCK resulted in reduced expression of all four phospho-proteins assessed (B through E, lane 4 compared with lane 3) and a decreased in whole-kidney size (A, lane 4 compared with lane 3). (F) The original tissue extracts were accurately adjusted to equal protein concentrations, and equivalent quantities of protein were subjected to immunoprecipitation.

Figure 9.

Histologic evaluation of SKI-606 treatment. (A and C) Hematoxylin-stained PCK renal sections demonstrate SKI-606 treatment of PCK results in reduction of renal CT cysts (C) compared with the cystic CT lesions present in untreated PN90 cystic kidneys (A). This reduction in renal cysts occurs without morphologic evidence of renal toxicity and directly correlates with the reduction in renal size and reduced activity of p-Src, p-ErbB2, p-B-Raf, and p-ERK1/2 activity shown in Figure 8, A through E, lane 4. (B and D) Masson's Trichrome–stained hepatic tissue from SKI-606–treated animals (D) reveals a significant reduction in biliary ductal cyst development and fibrosis when compared with untreated PCK livers (B). Magnifications: ×10 in A and C; ×20 in B and D.