Arhgef11 is a Rho-guanine nucleotide exchange factor that was previously implicated in kidney injury in the Dahl salt-sensitive (SS) rat, a model of hypertension-related chronic kidney disease. Reduced Arhgef11 expression in an SS- Arhgef11 SHR -minimal congenic strain (spontaneously hypertensive rat allele substituted for S allele) significantly decreased proteinuria, fibrosis, and improved renal hemodynamics, without impacting blood pressure compared with the control SS (SS-wild type). Here, SS- Arhgef11 −/− and SS-wild type rats were placed on either low or elevated salt (0.3% or 2% NaCl) from 4 to 12 weeks of age. On low salt, starting at week 6 and through week 12, SS- Arhgef11 −/− animals demonstrated a 3-fold decrease in proteinuria compared with SS-wild type. On high salt, beginning at week 6, SS- Arhgef11 −/− animals demonstrated >2-fold lower proteinuria from weeks 8 to 12 and 30 mm Hg lower BP compared with SS-wild type. To better understand the molecular mechanisms of the renal protection from loss of Arhgef11 , both RNA sequencing and discovery proteomics were performed on kidneys from week 4 (before onset of renal injury/proteinuria between groups) and at week 12 (low salt). The omics data sets revealed loss of Arhgef11 (SS- Arhgef11 −/− ) initiates early transcriptome/protein changes in the cytoskeleton starting as early as week 4 that impact a number of cellular functions, including actin cytoskeletal regulation, mitochondrial metabolism, and solute carrier transporters. In summary, in vivo phenotyping coupled with a multi-omics approach provides strong evidence that increased Arhgef11 expression in the Dahl SS rat leads to actin cytoskeleton-mediated changes in cell morphology and cell function that promote kidney injury, hypertension, and decline in kidney function.

Chronic kidney disease (CKD), which can ultimately progress to kidney failure, is influenced by genetics and the environment. Genes identified in human genome wide association studies (GWAS) explain only a small proportion of the heritable variation and lack functional validation, indicating the need for additional model systems. Outbred heterogeneous stock (HS) rats have been used for genetic fine-mapping of complex traits, but have not previously been used for CKD traits. We performed GWAS for urinary protein excretion (UPE) and CKD related serum biochemistries in 245 male HS rats. Quantitative trait loci (QTL) were identified using a linear mixed effect model that tested for association with imputed genotypes. Candidate genes were identified using bioinformatics tools and targeted RNAseq followed by testing in a novel in vitro model of human tubule, hypoxia-induced damage. We identified two QTL for UPE and five for serum biochemistries. Protein modeling identified a missense variant within Septin 8 (Sept8) as a candidate for UPE. Sept8/SEPTIN8 expression increased in HS rats with elevated UPE and tubulointerstitial injury and in the in vitro hypoxia model. SEPTIN8 is detected within proximal tubule cells in human kidney samples and localizes with acetyl-alpha tubulin in the culture system. After hypoxia, SEPTIN8 staining becomes diffuse and appears to relocalize with actin. These data suggest a role of SEPTIN8 in cellular organization and structure in response to environmental stress. This study demonstrates that integration of a rat genetic model with an environmentally induced tubule damage system identifies Sept8/SEPTIN8 and informs novel aspects of the complex gene by environmental interactions contributing to CKD risk.

Patients with hypertension or diabetes are at higher risk for developing chronic kidney disease (CKD). Low nephron number has been linked with susceptibility to develop hypertension and CKD, but a clear connection between nephron number and hyperglycemic renal injury has not been established. Thus, we seek to investigate the association between nephron deficiency and the development of diabetic kidney disease using a unique model of nephron deficiency, the HSRA rat. Offspring of HSRA are born with a single kidney 50-75% of the time with the remaining pups born with two kidneys. This provides the advantage of being able to directly compare congenital one-kidney, nephron-deficient rats (HSRA-S, ~20,400 nephrons) with nephrectomized two-kidney rats (HSRA-UNX, ~25,100 nephrons) and two-kidney control rats (HSRA-C, ~50,000 nephrons). Previous work has shown that HSRA-S develops increased renal dysfunction with age compared to HSRA-UNX and HSRA-C, which is greatly exacerbated in the presence of DOCA hypertension. Our hypothesis is that a secondary stressor of hyperglycemia in the context of low nephron number will similarly cause greater decline in renal function in HSRA-S compared to HSRA-UNX and HSRA-C. Streptozotocin (STZ) was administered at 9 weeks of age in all three groups and animals were followed until week 24. Despite overt hyperglycemia (350-450 mg/dl), diabetic groups did not develop increased proteinuria compared to their non-diabetic counterparts. Notably, a significant increase in kidney weight was observed in HSRA-S+STZ compared to HSRA-S (kidney weight to body weight ratio [mg/g] 4.8 ± 0.3089 for HSRA-S vs 7.8 ± 1.204 for HSRA-S+STZ, p=0.02), suggesting that hyperglycemia has a deleterious impact on kidney function via hyperfiltration and hypertrophy. Despite no significant effect on proteinuria after an “early insult,” we are investigating the impact of hyperglycemia after overt injury (“late insult”) is observed in HSRA-S (week >24). We will also revisit an “early insult” of hyperglycemia with the addition of modest hypertension (130 mmHg) via DOCA, which represents patients with both diabetes and hypertension . Through this research, we hope to contribute to the growing knowledge of the relationship between nephron number and disease.

Diabetes remains one of the main culprits of end‐stage kidney disease in the US. Investigation into the role of nephron number in chronic kidney disease (CKD) alone or with hypertension has revealed a strong inverse relationship (i.e., lower nephron number ‐‐> increased susceptibility to hypertension and CKD); however, not much is known about the connection between nephron number and diabetic kidney disease. To address this gap in knowledge we utilize the HSRA rat, a novel inbred genetic model of nephron deficiency and chronic kidney disease. HSRA rats exhibit failure of one kidney to develop (HSRA‐S, ~20,400 nephrons) in 50–75% of offspring, while remaining offspring are born with two kidneys (HSRA‐C, ~50,000 nephrons). HSRA‐S rats can be directly compared to nephrectomized HSRA‐C rats (HSRA‐UNX, ~25,100 nephrons), which provides insight into the impact of nephron number differences across the three groups. HSRA‐S rats develop significant renal injury with age when compared with HSRA‐C and HSRA‐UNX; the difference in injury is significantly exacerbated in the presence of DOCA+salt hypertension. The current study sought to address the hypothesis that nephron deficiency in the HSRA rat is a risk factor for renal injury, secondary to hyperglycemia (similar to what is seen with hypertension). Streptozotocin (STZ) was used to induce a diabetic phenotype in HSRA‐S, HSRA‐C, and HSRA‐UNX at nine weeks of age, and animals were followed for 15 weeks. All animals exposed to STZ developed robust hyperglycemia (random glucose averages: 415mg/dL – 456 mg/dL), but in contrast to the response to hypertension, neither HSRA‐S nor HSRA‐UNX developed overt proteinuria, nor were there significant differences between groups at the conclusion of the experiment. There was however, a significant increase in kidney weight to body weight ratio in STZ/hyperglycemic groups versus controls (e.g., kidney weight to body weight ratio [mg/g] 4.8 ± 0.3089 for HSRA‐S vs 7.8 ± 1.204 for HSRA‐S+STZ, p=0.02). STZ/hyperglycemic groups also exhibited significant decreases in creatinine clearance (e.g., creatinine clearance [μL/min/g] 719.0 ± 44.81 for HSRA‐S vs. 455.2 ± 72.91 for HSRA‐S+STZ, p=0.009). Targeted RNA sequencing was performed on isolated glomeruli using a custom panel of 42 genes involved in maintenance of glomerular function. In support of the physiological data stated above, few genes were observed to be dysregulated; however, Nphs1 and Nphs2 genes (associated with the protein nephrin) were increased in two‐kidney rats (HSRA‐C and HSRA‐C+ STZ) versus either one kidney group, regardless of glycemic status. In total, the data indicates that hyperglycemia does not have a significant impact on the onset and progression of injury in young HSRA‐S animals. We are currently investigating a mixed model of hypertension + hyperglycemia in the HSRA, which represents a large cohort of patients that are susceptible to CKD. Animals will be provided a low dose of DOCA+salt (35 mg) to induce moderate hypertension at ten weeks of age, along with 45mg/kg ...

Around 30 million people of all ages in the United States are affected by chronic kidney disease (CKD), with hypertension being a major cause of patients progressing to kidney failure. Previous genetic analyses 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. Protein levels of Cgnl1 (via western blot analysis) were found to correlate with increased kidney injury in the SS rat. We have developed a genetic knockout animal (SS‐Cgnl1−/−) to test the hypothesis that loss of Cgnl1 will result in a significant attenuation of hypertension and 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). All statistical analysis was performed using one‐way ANOVA/Tukey multiple correction. On low‐salt, SS‐Cgnl1−/− animals (n=6) demonstrated reduced proteinuria compared to SS‐WT animals (n=6) for the entirety of the experiment (e.g., 21.9 ± 0.6 versus 87.6 ± 9.3 mg/24hr at week 15, p=0.03). Similar results were seen with high‐salt; SS‐Cgnl1−/− animals (n=6) demonstrated reduction in proteinuria compared to SS‐WT (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 in 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). On both low and high‐salt diets SS‐Cgnl1−/− animals demonstrated less kidney hypertrophy and lower heart weight compared to SS‐WT, supporting the proteinuria and blood pressure measurements. For example, high‐salt fed SS‐Cgnl1−/− kidney weight to body weight ratio (mg/g) = 9.0 ± 0.26 compared to 10.3 ± 0.45 for the SS‐WT, p=0.03; high‐salt SS‐Cgnl1−/− heart weight to body weight ratio (mg/g) = 3.64 ± 0.04 compared to 4.46 ± 0.19 for the SS‐WT, p=0.0003. In addition, 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). In total, the physiological characterization of the SS‐Cgnl1−/− suggests that Cgnl1 plays an important role in the onset and progression of kidney disease in the Da...