The spontaneously hypertensive rat (SHR) line, SHR-A3, is susceptible to hypertensive renal injury, while other SHR lines, including SHR-B2, share similar levels of hypertension, but resist injury. These two inbred SHR lines are both progeny of the same founding pair of rats and have different ancestry at only 13% of their genomes. We have studied the development of hypertensive renal injury in this model and observed its emergence in young adult animals and its progression to moderately severe levels that cause increased serum creatinine levels at 40 weeks of age. Histological analysis indicates extensive infiltration of immune cells into the kidney concurrent with development of injury. We have demonstrated that pharmacological immunosuppression prevents hypertensive renal injury and we have linked disease to functional genetic variation in genes involved in the immune response and its regulation. We have recently acquired very high quality whole genome sequence for our hypertensive renal injury prone and resistant rats. This has uncovered extreme genetic variation in the immunoglobulin heavy chain locus, an ~8Mb segment of the genome that is essential for B lymphocyte-mediated immunity and which our SHR lines have inherited from different ancestors. The genetic variation endows SHR-A3 with a pre-immune immunoglobulin repertoire that is very different from SHR-B2. Indeed, >17% of all genomic amino acid coding difference between our injury resistant and susceptible SHR lines occurs in this locus, even though it accounts for just 0.3% of the genome. We have attached function to this genetic variation including: immunoglobulin abundance in serum and; receptor binding of the Fc region of immunoglobulin. This repertoire difference may alter the recognition of neo-antigens that are present as renal injury emerges. We have also uncovered a recent single nucleotide mutation creating immune deficiency by disrupting immune regulation. The affected gene, Stim1, is the key regulator of store-operated calcium entry (SOCE). This signal links depletion of ER calcium stores to generation of a persistent calcium signal generated by Stim1 gating of extracellular calcium entry and is vital for normal T lymphocyte function. Stim1 possesses a truncation mutation in SHR-A3 and the truncated protein suffers loss of key functional residues in the C-terminal that incompletely suppresses Stim1 calcium signaling function. Using backcrossing to permit congenic line creation (in which loci containing these variations are transferred from injury-resistant SHR-B2 into the SHR-A3 genetic background) we have developed evidence that hypertensive renal injury susceptibility arises from differences in the immune response arising from these loci. The initiating damage to the kidney caused by hypertension arises from the breakdown of auto-regulation of renal blood flow as elevated blood pressure exceeds the range across which this regulation can operate. We have identified an additional locus that increases the likelihood that blood pressure in SHR-A3 will exceed the auto-regulatory threshold. The chief hypothesis our proposal will test is that these three loci account for the difference in susceptibility to renal injury between SHR-A3 and SHR-B2. We further hypothesize that it is the effect of these mutations within the immune system that drive renal injury and we will attempt to prove the tissue specificity of disease pathogenesis by transfer of immune cells from SHR-B2 to SHR-A3. Finally, studies of immune dysfunction attributable to Stim1 mutation indicate that loss of T cell function, including the role of T helper cells to guide B lymphocyte maturation and of T regulatory cells to control B lymphocyte proliferation, leads to a disease process that amplifies auto-antibody production. Development of renal injury may be attenuated by specific interventions to alter B cell mediated auto-antibody induced immunity and we propose experiments to test this hypothesis.
Project Relevance: Progressive renal disease leads to loss of renal function, ultimately proceeding to end-stage (ESRD) and requiring dialysis or renal transplantation. ESRD is costly in health terms with more patients dying from ESRD in the US than from breast and prostate cancer combined. The burden of illness among minorities, particularly African-Americans, is especially high. However, progressive renal disease has health burdens that are even more significant than ESRD and that occur while renal function is declining: even small decrements in renal function are associated with increasing blood pressure and amplification of the risk of death from all cardiovascular disease (heart attack, stroke, heart failure). This amplification effect is so strong that more patients with progressive renal disease will die of cardiovascular disease than will reach ESRD. Our work uses an animal model of high blood pressure (SHR) in which inbred lines vary in their susceptibility to renal injury in hypertension. Our work with this model indicates that genetic differences in immune signaling play a role in renal injury susceptibility. We have taken a reductionist approach to develop evidence of 3 loci making a major contribution to renal injury risk. One locus contains the sequences encoding germ-line immunoglobulin sequences. Another is a recent point mutation that affects a key protein in immune cell signaling, Stim1. The third appears to act initially though blood pressure, but the locus contains two bi-allelic genes that lie in the same pro-inflammatory immune signaling pathways as immunoglobulin and Stim1. In this project we seek to determine whether the entire phenotypic difference of renal disease risk that exists between SHR lines arises from these three loci. We also seek to understand the cellular mechanisms of disease. Our first task is to find support for the hypothesis that the three loci we are studying exert their effect on renal injury risk through their effect on immune system function. We can do this by bone marrow transfer into irradiated animals in which we confer stem cells from the bone marrow that will allow the injury resistance genotype to exist only in the immune system of animals that are otherwise susceptible to renal injury. Finally, our third task is to understand how these genetic variants work in combination. Here again we focus on the immune system where insights into the functional role of IgH and Stim1 and the immune signaling paths that link them suggest that these genes may act together to alter B lymphocyte function and that this alteration is necessary and specific in the pathogenetic mechanism or injury. We plan to investigate the role of B cells in injury using both genetic and pharmacological approaches. Ultimately these studies may have great relevance to human renal disease in hypertension. We have almost no knowledge of pathogenesis of this disease in humans. Even though heredity plays a large role, genetic studies have been of limited success. However, the presence of defects in immune function in human patients with renal failure and homologies between those defects and the defects observed in our model raise the very reasonable possibility that our model system is directly relevant to human disease and can provide new approaches to progressive renal disease by uncovering disease mechanisms and by focusing the search for relevant human genetic variation within the highly complex genetic variation found within immune genes that are constantly selected for rapidly evolving threats from pathogens.
| Dhande, Isha S; Cranford, Stacy M; Zhu, Yaming et al. (2018) Susceptibility to Hypertensive Renal Disease in the Spontaneously Hypertensive Rat Is Influenced by 2 Loci Affecting Blood Pressure and Immunoglobulin Repertoire. Hypertension 71:700-708 |
| Doris, Peter A (2017) Genetics of hypertension: an assessment of progress in the spontaneously hypertensive rat. Physiol Genomics 49:601-617 |
