Transfer of SS Bone Marrow into Irradiated TLR4 Knockout Recipients Restores Salt-Sensitive Hypertension and Associated Renal Disease

Previous work has revealed that toll-like receptor 4 (TLR4), a component of innate immune activation, is upregulated in macrophages infiltrating the kidneys of Dahl Salt-Sensitive (SS) rats fed high salt. Furthermore, we have identified TLR4 in immune cells in diseased human kidney tissue utilizing a CyTOF mass cytometry approach (Cytometry by Time Of Flight), with CD14+CD16- classical monocytes exhibiting the greatest expression. Genetic deletion of TLR4 on the SS background (SSTLR4-/-) attenuates mean arterial pressure (MAP) (SSTLR4-/-: 147.1 ± 3.5 vs. SS: 162.5 ± 4.9 mmHg, p< 0.0001, n=10/group) and albuminuria (170.6 ± 27.1 vs. 321.3 ± 62.7 mg/day, p< 0.05, n=10/group) in males compared to parental SS male rats after three weeks on a high salt diet. Furthermore, peritoneal macrophages isolated from the SSTLR4-/- produce significantly less IL-1β compared to SS macrophages upon stimulation with lipopolysaccharide (LPS). Since a single cell RNA-seq analysis of SS kidneys illustrated widespread cellular distribution of TLR4 in the kidney, with the strongest expression in innate immune cell population, we therefore examined the specific effects of TLR4 deletion in immune cells via a total body irradiation/bone marrow transfer approach. The present study tested the hypothesis that TLR4 specifically in hematopoietic cells is required for the full development of male Dahl SS hypertension and renal damage. At 6 weeks of age, Dahl SSTLR4-/- male recipients were irradiated (11Gy) then received either SS or SSTLR4-/- bone marrow transplantation via tail vein injection (~15-20 million cells) to yield chimeric rats with (+SS, n=6) or without (+SSTLR4-/-, n=5) intact TLR4 in immune cells with peripheral TLR4 absent in both groups. Following recovery, rats (maintained on a low salt chow (LS, Dyets AIN-76A 0.4% NaCl) underwent telemeter implantation. Animals were switched to a high salt diet (HS, Dyets AIN-76A 4.0% NaCl) following recovery and baseline blood pressure recordings. Urine samples were collected once during the LS period and weekly throughout a 3-week HS challenge. Upon euthanasia, kidneys were flushed and harvested. MAP was not found to be different between groups during the LS baseline period (+SS: 103.1 ± 0.8 vs. +SSTLR4-/-: 102.2 ± 1.0 mmHg). By the end of the HS challenge, +SS rats demonstrated a significant increase in MAP compared to +SSTLR4-/- (HS day 21: +SS: 151.3 ± 5.6 vs +SSTLR4-/-: 134.2 ± 6.4, p=0.0042). Urinary albumin excretion increased in both groups from LS to HS, but our preliminary data did not reveal a significant difference in albuminuria between groups during the HS challenge (+SS: 505.3 ± 42 vs. +SSTLR4-/-: 306.3 ± 105.3 mg/day, p=0.1). Immune cells were isolated from the blood and kidneys of these animals and characterized by flow cytometry. There was no difference in the number of circulating leukocytes (+SS: 2.8x106 vs. +SSTLR4-/-: 2.6x106 CD45+ cells per ml blood) indicating comparable reconstitution of immune cells in both groups. We also surprisingly found no difference in renal infiltrating leukocytes (+SS: 1.7x106 vs. +SSTLR4-/-: 1.8x106 CD45+ cells per kidney). Together, these data are consistent with the protective effects of whole-body TLR4 knockout and suggest that TLR4 in hematopoietic cells may be responsible for promoting Dahl SS hypertension and renal damage in males. Further studies are needed to assess the inflammatory status of immune cells. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.