title: Characterising the mechanisms of uromodulin secretion and trafficking with salt loading from the thick ascending limb in hypertension
Creators: name: Boder, Philipp Simon
Publisher: name: University of Glasgow
Year: 2024

description: Uromodulin (UMOD) is a complex glycoprotein that is to a large extent secreted by renal epithelial cells lining the thick ascending limb (TAL) and is the most abundant protein found in the urine of healthy individuals. A number of functions have been associated with urinary UMOD, including regulation of salt transport and ion homeostasis, protection against urinary tract infections and kidney stones, kidney injury, and innate immunity. UMOD undergoes a number of conformational changes and post-translational modifications, including glycosylation, during its maturation and intracellular trafficking. UMOD secretion is polar by the fact that it can be secreted apically into the urine, or basolaterally into the serum. Importantly, altered urinary UMOD excretion and intracellular retention of UMOD have been linked to various kidney-related pathologies, including chronic kidney disease (CKD). Variants in the UMOD promoter have been associated with hypertension by genome-wide association studies. Hypertension is one of the main drivers of cardiovascular disease risk and is intricately linked to kidney function, with the kidneys playing a major role in blood pressure regulation. Increased salt intake is strongly linked to a number of detrimental cardiovascular outcomes, including renal end-organ damage and hypertension. Previously, studies have focused on the role of UMOD in salt reabsorption. However, the reverse (i.e., the effect of salt on UMOD) remained unexplored. In this thesis, the main aim was to investigate the influence of salt on UMOD excretion in order to gain a better understanding of the relationship between salt handling, blood pressure/hypertension, and kidney physiology. To model normotensive and chronic hypertensive conditions, Wistar-Kyoto (WKY) and Stroke-Prone Spontaneously Hypertensive (SHRSP) rats were utilised, respectively. These rats were administered 1% NaCl/salt in their drinking fluid, which is at a physiological concentration, thereby more closely replicating human conditions.

Initial dissection of the role of dietary salt in renal UMOD excretion rate was achieved by exposing WKY and SHRSP rats to a 3-week continuous 1% salt loading phase. Salt loading in the chronic hypertensive SHRSP led to increased blood pressure and expression of kidney injury markers kidney injury marker-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Excretion of urinary UMOD was decreased by 26 % in WKY rats and 55% in SHRSP rats. It was hypothesized that the addition of anti-hypertensive drug nifedipine (calcium channel blocker) would assist in separating the blood pressure component from salt-induced effects on UMOD urinary excretion rate. Nifedipine treatment of salt-loaded rats did not inhibit the reduction in urinary UMOD excretion rate in SHRSP, even with reduced blood pressure. Following this, the aim was to gain insights into the kidney physiology and intracellular mechanisms that may occur with salt loading. Examination of total kidney lysates from salt loaded WKY and SHRSP revealed a lack of UMOD protein and mRNA levels. Immunofluorescence and co-localization studies revealed increased intracellular UMOD retention in both WKY and SHRSP, specifically in the endoplasmic reticulum (ER). This was further corroborated by ex-vivo incubations with isolated medullary TAL (mTAL) tubules, the region with a high UMOD content in rats, incubated with mannitol (an osmotic control) and salt, whereby salt-incubated TAL showed greater UMOD retention, especially in the SHRSP. This highlighted the importance of salt in regulating urinary UMOD excretion and trafficking, and potential exacerbations in chronic hypertension and kidney injury. However, the dynamics and exact mechanisms behind the influence of salt on UMOD excretion and trafficking were not clear.

To explore the dynamics of salt loading on UMOD excretion rate, WKY rats were exposed to an intermittent salt loading regime whereby they received 1% salt in an on-off-on pattern over 3 weeks (1 week of salt loading, 1 week of normal water, 1 week of salt loading). The normotensive model was chosen to remove hypertension as a confounding factor and therefore provide mechanistic insight into a normal physiological setting. It was hypothesized that the salt-induced lowering of UMOD excretion rate would be reversible, and this would be apparent in UMOD trafficking. Intermittent salt loading resulted in an acute lowering of UMOD urinary excretion rate and a reversal to almost normal levels upon salt removal. Although there were differences in electrolyte levels, especially sodium and chloride levels, there was no change in intracellular UMOD accumulation in the normotensive background. This provided further proof of the acute influence of salt on UMOD excretion and suggested that UMOD accumulation occurs over a longer timescale.

Given that salt loading lowers UMOD excretion acutely and results in significant intracellular accumulation in the chronic hypertensive model, the next aim was to characterize the effects of long-term salt loading on UMOD excretion and trafficking. It was hypothesized that long-term salt loading would result in a “chronic hypertensive-like” phenotype in the normotensive models, with intracellular accumulation of UMOD and a change in renal physiology. Salt loading over a period of 3 months showed increased intracellular accumulation of UMOD in the ER of the epithelial cells in the TAL region. Moreover, this was paralleled by the upregulation of ER stress marker Binding Immunoglobulin Protein (BiP). This novel finding suggests a mechanism whereby long-term salt loading may trigger early deleterious processes in the kidneys of normotensive WKY. These lead to accumulation of UMOD, which may contribute to renal injury as seen in the chronic hypertensive SHRSP. Only after continued salt exposure over a longer period of time, in combination with ER accumulation, is an ER stress response triggered. Whether this is related to increased UMOD processing times in the ER and the exact mechanistic contributions of UMOD to blood pressure, and ultimately the chronic hypertensive phenotype, remains to be characterized.

Given the evidence presented of the profound effect of salt on UMOD urinary excretion and trafficking, a suitable model was required to untangle the molecular mechanisms involved in the process. This would also assist in the discovery of novel therapeutic targets for the treatment of hypertension, as well as other UMOD-related diseases such as CKD, especially with regard to UMOD retention and ER stress. Previous models include primary mTAL cells. However, these lose UMOD expression during subculturing and undergo significant changes in morphology with salt incubation. To overcome this, a transgenic cell model is presented in this thesis expressing UMOD with a Yellow Fluorescent Protein tag (UMOD-YFP 293 FT cells). This model offers the advantage of inducible UMOD expression via a tetracycline promoter at a user-defined time point. Moreover, the YFP-tag would enable live cell imaging, which would help with understanding the kinetics of UMOD trafficking, to immunoprecipitation assays, which would greatly assist in identifying the interactors of UMOD and by extension the signalling pathways involved with salt loading. This thesis also therefore offers a novel resource for future molecular studies of UMOD.

Taken together, this thesis provides novel evidence for the salt-driven lowering of UMOD urinary excretion in both normotensive and chronic hypertensive models. In the chronic hypertensive model particularly, there is an increase in intracellular retention of UMOD and ER stress, paralleled by kidney injury. In the normotensive model, this accumulation and retention of UMOD in the medullary TAL only occurs after long-term salt loading, which provides a novel avenue of investigation: does salt induce ER stress which leads to retention of UMOD, or does the retention of UMOD induce ER stress? And importantly, how does this tie in with renal end-organ damage and the progression of hypertension? Further molecular characterisation is required in this regard and may eventually inform new therapies for the treatment of hypertension.