Genetic Knockout of Cingulin-like 1 Reduces Renal Injury and Blood Pressure in Dahl Salt-Sensitive Rats

Chronic kidney disease (CKD) has been estimated to impact 37 million people, ranking kidney diseases to be the ninth leading cause of death in the United States. Hypertension is very common in patients with CKD, and is a risk factor for the progression of kidney disease. Previous genetic studies involving the Dahl salt-sensitive (SS) rat identified multiple genetic loci linked to kidney injury, including a region on chromosome 8. To confirm the genetic association, an S.SHR(8) congenic was developed to transfer the linked chromosome 8 genomic segment from the SHR (resistant to kidney injury) onto the SS genetic background. The S.SHR(8) demonstrated significantly reduced proteinuria and improved renal function compared to SS control. Based on whole genome sequencing and bioinformatics analysis of the chromosome 8 region, cingulin-like 1 (Cgnl1) was identified as a putative gene linked to kidney injury. Cgnl1 is known to localize to adherens and tight cell-cell junctions, mediating junction assembly via regulating the activity of the small GTPases such as RhoA and Rac1. A knockout animal (SS-Cgnl1−/−) was developed to test the hypothesis that loss of Cgnl1 will result in a significant attenuation of kidney injury compared to SS-WT. A temporal analysis of cardiovascular and renal measurements was conducted on SS-WT and SS-Cgnl1−/− from 6–15 weeks of age under low or high-salt diets (0.3% or 2% NaCl). On low-salt, SS-Cgnl1−/− animals (n=6) demonstrated reduced proteinuria compared to SS-WT animals (n=6) for the entirety of the experiment (21.9 ± 0.6 versus 87.6 ± 9.3 mg/24hr at week 15, p=0.03). Similar results were seen with high-salt (28.7 ± 2.9 versus 161.4 ± 33.1 mg/24hr at week 15, p<0.0001). While high-salt fed SS-WT animals exhibited a significant increase in terminal mean arterial pressure (MAP) compared to low-salt SS-WT (156 ± 5.9 versus 133.9 ± 4.0 mmHg, p=0.0095), MAP for SS-Cgnl1−/− remained unchanged between low and high-salt groups (119.3 ± 3.2 and 124.8 ± 3.8 mmHg respectively, NS). Kidney pathology measures were all significantly improved in SS-Cgnl1−/− compared to SS-WT animals, including glomerular injury and tubulointerstitial injury (all p<0.05). RNA sequencing was performed on kidneys from SS-WT and SS-Cgnl1-/- to investigate molecular changes associated with reduced blood pressure and renal protection. Cgnl1 was identified as top down-regulated gene in the dataset, with many known genes linked with Cgnl1 observed to be differentially expressed compared to SS-WT. We are currently conducting additional studies using telemetry to monitor blood pressure differences over the time course. Additionally, we are working to develop a Cgnl1 knockout cell line using CRISPR/Cas9 gene-editing (HEK293) for more mechanistic study of Cgnl1 and pathways. The molecular work, along with the physiological characterization of the SS-Cgnl1−/−, is expected to provide insight into the role of Cgnl1 in the onset and progression of hypertensive kidney disease and identify new therapeutic targets.