The Genetic Basis of Impaired Myogenic Response and Renal Injury in Fawn Hooded Hypertensive Rats

The Fawn hooded hypertensive (FHH) rat is a genetic rat model of chronic kidney disease that develops mild systolic hypertension, proteinuria, glomerulosclerosis and end stage renal disease (ESRD). The development of renal disease is associated with an impaired autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR), elevated glomerular capillary pressure and impaired myogenic response in preglomerular renal arteries.;Previously, FHH rats were crossed with Brown Norway (BN) rats to create 22 consomic strains in which one chromosome at a time from the BN rat was introgressed onto the FHH genetic background. After the consomic strains were developed, it was found that the transfer of BN Chromosome 1 into the FHH background attenuated the development of proteinuria and restored the autoregulation of RBF. This led to efforts to positionally clone the region of chromosome 1 containing genes that influence RBF autoregulation by creating FHH.1BN congenics with smaller regions of the BN introgressed.;This study examined the effect of substitution of a 2.4 megabase (Mb) region of BN rat chromosome 1 (RNO1) between 258.8 and 261.2 Mb onto the genetic background of FHH rats on autoregulation of RBF, development of proteinuria and renal injury, and K+ channel activity in renal vascular smooth muscle cells (VSMCs) and then assessed the 15 predicted genes in the 2.4 Mb region for sequence and/or expression differences that may be responsible for the impaired renal myogenic response of FHH rats. Additionally, an identified candidate causal gene, Adducin gamma (Add3), was knocked down in isolated vessels to assess its role in the myogenic response.;Hypothesis: The impaired myogenic response in FHH is due to sequence variation that alters the function or expression of Add3 that alters its kinase activity or cytoskeletal function..;Specific aims of this project were to: 1) Thoroughly phenotype our 2.4 Mb FHH.1BN minimal congenic strain to confirm that substitution of the region of the genome between markers D1Rat09 and D1Rat225 alone is sufficient to rescue RBF autoregulation, reduce proteinuria and the prevent the development of chronic kidney disease. 2) Evaluate gene sequence variants and expression levels in the 2.4 Mbp region of interest in FHH compared to the 2.4 Mb FHH.1 BN minimal congenic strain to identify genes that may be responsible for the impaired myogenic response. 3) Functionally evaluate ability of sequence variants or changes in the expression of a candidate gene to alter myogenic tone or channel activity in isolated vessels or cells.;FHH rats exhibited poor autoregulation of RBF which rose 33% when renal perfusion pressure (RPP) was increased from 100 to 140 mmHg while the FHH.1BN minimal 2.4 Mb congenic strain had controlled RBF values over the same pressure increase. Protein excretion rose from 42±5 to 259±28 mg/day in 9 to 21 week FHH rats but only increased to 197±19 mg/day in the FHH.1BN minimal 2.4 Mbp congenic strain. Patch clamp experiments indicated that large conductance calcium activated K+ channel (BK) current was 5-fold greater at +100 to +120 mV in VSMCs isolated from preglomerular arterioles of FHH rats compared to VSMCs obtained from the minimal 2.4 Mb congenic strain. Single channel analysis indicates that this is due in part to an increase in the open probability of the BK channel in VSMCs isolated from FHH rats. Sequence analysis of available data on genes in the 2.4 Mb region revealed that three genes; Add3, Rbm20 and Soc-2, have exon sequence variants that could potentially alter function while two of the 15 genes in this region, Mxi1 and Rbm20, have expression differences. Of the genes identified as possible candidates, only Add3 has been reported to have a role in cardiovascular function. Rat arteries from the minimal 2.4 Mb congenic strain that were cultured with Add3 siRNA dilated in response to a pressure increase from 50 to 150 mmHg whereas vessels cultured with scrambled siRNA constricted in response to increasing pressure.;These data indicate that a gene that impairs autoregulation of RBF in FHH rats lies within a 2.4 Mb region of Chromosome 1 containing 15 predicted genes and that transfer of BN into this region restores RBF autoregulation and the myogenic response in afferent arterioles, decreases K+ current and slows the progression of proteinuria and glomerular and renal damage. Additionally, Add3 knock down experiments suggest that loss of add3 leads to an impaired myogenic response and further suggests that the sequence variants of Add3 in FHH rats causes a loss of function of Add3 which is responsible for the strain's impaired myogenic response.