Abstract 163: The Role of Genetic Variants in Arhgef11 via the Rho-rock Pathway on Kidney Injury in the Dahl S Rat

Abstract

Hypertension plays an important role in the development of chronic kidney disease. The Dahl salt-sensitive (S) rat is a widely studied model of salt-sensitive hypertension that develops concurrent kidney injury and progressive decline in kidney function. Genetic studies using the S rat, along with the kidney injury resistant, but hypertensive SHR (spontaneously hypertensive rat) have identified many genomic regions associated with kidney injury. Through positional cloning approaches, Arhgef11 , a Rho guanine nucleotide exchange factor has been identified as a strong candidate gene involved in the kidney injury. The S rat exhibits increased expression of Arhgef11 , up-regulation of Rho-ROCK pathway, and coding sequence variants that may alter protein function compared to the SHR. Inhibition of the Rho-ROCK pathway (using fasudil) in the S resulted in an attenuation of proteinuria, providing evidence the pathway could influence kidney injury. To better understand the role of Arhgef11 in the Rho-ROCK pathway, genetic knockdown in cell culture was achieved in HEK293 and NRK cell lines using lentiviral shRNA- Arhfgef11 constructs. Transduced cells achieved ∼70% knockdown of Arhgef11 protein. RhoA activity was significantly decreased ∼40% (p<0.01) in both transduced cell lines compared to scramble control. Gene expression and protein levels of Arhgef11 , RhoA, ROCK1, MLC, Cofilin were all significantly down-regulated 40-65% in transduced HEK293 and NRK (p<0.01). Reduced expression of Arhgef11 in transduced cells showed a different cellular distribution and less stress fiber formation and elongation compared to control (2-fold reduction in length/width ratio, p<0.01) by immunofluorescence. Genetic knockdown demonstrated a similar effect as fasudil for both stable cell-lines. Microarray studies have also identified other novel pathways influenced by genetic knockdown of Arhgef11 . Current studies are investigating the specific effect of the S or SHR Arhgef11 allele (using lentiviral overexpression) on the Rho-ROCK pathway and stress fiber formation. In summary, our promising data suggests that the biological and functional significance of genetic variants in Arhgef11 could be of major importance in development of kidney injury in the S rat.