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Allelic variants in Arhgef11 via the Rho-Rock pathway are linked to epithelial-mesenchymal transition and contributes to kidney injury in the Dahl salt-sensitive rat

PLoS ONE (2015) 10(7)
DOI: 10.1371/journal.pone.0132553
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Abstract

Previously, genetic analyses identified that variants in Arhgef11 may influence kidney injury in the Dahl salt-sensitive (S) rat, a model of hypertensive chronic kidney disease. To understand the potential mechanism by which altered expression and/or protein differences in Arhgef11 could play a role in kidney injury, stably transduced Arhgef11 knockdown cell lines as well as primary cultures of proximal tubule cells were studied. Genetic knockdown of Arhgef11 in HEK293 and NRK resulted in reduced RhoA activity, decreased activation of Rho-ROCK pathway, and less stress fiber formation versus control, similar to what was observed by pharmacological inhibition (fasudil). Primary proximal tubule cells (PTC) cultured from the S exhibited increased expression of Arhgef11, increased RhoA activity, and up regulation of Rho-ROCK signaling compared to control (small congenic). The cells were also more prone (versus control) to TGFβ-1 induced epithelial-mesenchymal transition (EMT), a hallmark feature of the development of renal interstitial fibrosis, and characterized by development of spindle shape morphology, gene expression changes in EMT markers (Col1a3, Mmp9, Bmp7, and Ocln) and increased expression of N-Cadherin and Vimentin. S derived PTC demonstrated a decreased ability to uptake FITC-albumin compared to the small congenic in vitro, which was confirmed by assessment of albumin re-uptake in vivo by infusion of FITC-albumin and immunofluorescence imaging. In summary, these studies suggest that genetic variants in the S form of Arhgef11 via increased expression and/or protein activity play a role in promoting kidney injury in the S rat through changes in cell morphology (Rho-Rock and/or EMT) that impact the function of tubule cells.

Figures

  • Fig 1. Blood pressure, proteinuria, and renal function measurements and correlation with changes in Arhgef11 expression. (A) Systolic blood pressure in S, Arhgef11-congenic (C), and SHR at week 4 and 24 (n = 6 per group). (B) Proteinuria as a measure of renal injury (n = 6–20). (C) Creatinine clearance as an indication of renal function (n = 4–6 per group). (D) Renal expression of Arhgef11 from week 2 (before phenotypic differences between strains) until week 24 (n = 6 per group/time). Arhgef11-congenic is genetically similar to the S rat, except for allelic difference in Arhgef11 (S2 Fig) and few surrounding genes. *p<0.05 versus S, †, p<0.05 versus S and C. SE are presented.
  • Fig 2. Kidney pathology in the rat and immunohistological staining of ARHGEF11 in human kidney biopsies. (A) Representative glomerular and tubulointerstitial images for S, Arhgef11-congenic, and SHR at week 24. The S rat demonstrated glomerular injury (mesangial expansion, glomerulosclerosis, etc), tubular injury, and interstitial fibrosis (tubule atrophy, immune cell infiltration, and/or fibrosis), consistent with detailed histological analysis previously described.[9] Both the Arhgef11-congenic and SHR demonstrated less glomerular and tubular injury compared to S. (B) ARHGEF11 staining in human kidney biopsymaterial from patients with various etiologies, including control (biopsy 22-no specific renal pathology; nephrectomy subsequent to renal pelvic carcinoma); biopsy 12, hematuria and changes compatible with acute tubular necrosis; biopsy 5, hematuria and non-nephrotic range proteinuria; and biopsy 4, diabetic glomerulosclerosis, interstitial fibrosis, and tubular necrosis).A representative image of staining in the glomerulus is shown in the upper panel and vessel and some tubular staining is illustrated in the lower panel.
  • Fig 3. Establishment of stably transduced Arhgef11 knockdown cell lines using shRNA lentiviral methodology. A number of shRNA lentiviral constructs (shRNA1-4) were tested for optimal knockdown of Arhgef11 in both NRK and HEK293T cells (see S2 Fig). (A) Real-time PCR confirmation of shRNA3 Arhgef11 knockdown and subsequent impact on RhoA and ROCK in NRK stably transduced cells compared to scrambled control (LVC). (B) Realtime PCR confirmation of shRNA3 Arhgef11 knockdown and impact on RhoA and ROCK in HEK293T stably transduced cells compared to scrambled control (LVC). (C) Western analysis and densitometry measurements of ARHGEF11, RhoA, and ROCK between NRK-LVC and NRK-shRNA3.(D) Western analysis and densitometry measurements of ARHGEF11, RhoA, and ROCK between HEK293T-LVC and HEK293T-shRNA3. n = 4–6 independent samples/group, *p<0.05 versus LVC, Error bar are ±SD.
  • Fig 4. Analysis of the Rho-ROCK signaling pathway and RhoA activity in stably transduced Arhgef11 knockdown cell lines.Cell-culture experiments were performed under several experimental conditions: 1) control (DMEMmedium); 2) fasudil (F) (10μg/ml) treated for 4hrs; 3) serum free (SF) for 24 hours, or 4) serum free for 24 hrs + fasudil treated for 4 hours (SF+F). (A) Real time PCR of Arhgef11, RhoA, Rock1,MLC andCofilin of HEK293-shRNA3 compared to LVC (scrambled control). (B) RhoA activity between LVC and HEK293-shRNA3 under each experimental condition. (C) Western analysis of Rho-Rock pathway in LVC and HEK293-shRNA3 between control and fasudil treatment; and (D) Western analysis of Rho-Rock pathway in LVC and HEK293-shRNA3 between control and serum free conditions. Similar results were observed for LVC and NRK-shRNA3 for control versus fasudil (S3 Fig). n = 6 independent samples per group/treatment, *p<0.05 versus LVC. Error bars are ±SD.
  • Fig 5. ARHGEF11 localization and actin (F-actin) cytoskeleton immunofluorescence. (A) Localization of ARHGEF11 in stably transduced NRK cells [-LVC (lentiviral scramble control) or–shRNA3]. ARHGEF11 is located near the nucleus and throughout the cell under control conditions for both LVC and LVC-shRNA3 cells, Under serum free conditions ARHGEF11 appears to be distributed throughout the cell and toward the cell membrane. (B) Immunofluorescence of F-actin in stably transduced NRK cells under control, serum free and fasudil treatment. Cells cultured under serum free conditions develop stress fibers and demonstrate elongation of cells. NRK-shRNA3 cells demonstrate a significant reduction in stress fibers, similar to pharmacological imbibition of ROCK by fasudil, as cell length/width ratio decreases to baseline with genetic knockdown of Arhgef11. *p<0.05, n = 3 independent samples/20 random images per slide (6–10 cells per view). Error bars are ±SD.
  • Fig 6. Primary proximal tubule cell (PTC) culture and Arhgef11-Rho-ROCK signaling pathway. (A) Representative phase-contrast images (at 20X) of isolated tubular fragments (Day 0) and outgrowth and culture of proximal tubules cells (Day 2–7). There appears no obvious morphological difference between PTC derived S and Arhgef11-congenic. (B) Real-time PCR of Arhgef11-Rho-ROCK signaling genes from kidney tissue and PTC acquired from S and Arhgef11-congenic at 4–6 weeks of age. Cultured PTCs (Day 5) exhibit a similar down-regulation of Arhgef11 and signaling pathway as kidney tissue. (C) Western analysis of Rho-Rock pathway in cultured PTC. n = 4–6 independent samples, *p<0.05 versus S, Error bar are ±SD.
  • Fig 7. Real-time PCR and western analysis of TGFß-1 induced epithelial mesenchymal transition (EMT) and impact on RhoA-Rock pathway in primary proximal tubule cell (PTC). Primary PTC cells (day 5, >60% confluent) were grown from S and Arhgef11-congenic kidney (at 4 weeks of age) and treated with TGFß-1 (10 ng/ml) for 48 hrs. (A) Real-time PCR of markers indicative of EMT. (B) Western analysis and densitometry of E-Cadherin and N-Cadherin EMTmarkers. (C) Representative phase contrast and immunofluorescence of stress fibers formation (F-Actin) and Vimentin (EMTmarkers) between S and Arhgef11-congenic PTC exposed to TGFß-1. n = 4–6 independent samples, *p<0.05 versus S, †p<0.05 versus S+ TGFß-1. Error bar are ±SD.
  • Fig 8. In vitro assessment of albumin uptake in primary proximal tubule cell (PTC). Primary PTC cells (day 5, >60% confluent) were grown from S and Arhgef11-congenic kidney (at 4 weeks of age) and incubated with FITC-Albumin (10 ug/ml) at 0, 5, 10, 15, 30, 60 min. Primary PTC were derived from fawn-hooded hypertensive (FHH) rat as comparison due to a known genetic defect that impairs tubular re-uptake of albumin/protein (i.e., low albumin uptake). PTCs from S kidney exhibited decreased uptake of FITC-albumin compared to PTC from Arhgef11-congenic, while FHH demonstrated the least ability uptake FITC-albumin. n = 3–6 independent samples, *p<0.05 versus S and FHH, †p<0.05 versus FHH. Error bars are ±SD.

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Jia, Z., Johnson, A. C., Wang, X., Guo, Z., Dreisbach, A. W., Lewin, J. R., … Garrett, M. R. (2015). Allelic variants in Arhgef11 via the Rho-Rock pathway are linked to epithelial-mesenchymal transition and contributes to kidney injury in the Dahl salt-sensitive rat. PLoS ONE, 10(7). https://doi.org/10.1371/journal.pone.0132553

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