Microcirculation in chronic kidney disease : from injury targets to potential therapeutics

Chronic kidney disease (CKD) is a major public health problem worldwide, and patients with end-stage kidney disease (ESKD) are at increased risk of developing cardiovascular complications. Small artery dysfunction is a common feature of CKD, which contributes to the development of early vascular ageing (EVA) and cardiovascular complications. The exact mechanisms of how small artery dysfunction contributes to these complications are not fully understood. The ultimate goal of this PhD thesis is to gain a better understanding on how small artery dysfunction contributes to EVA and cardiovascular risk in the uremic environment, and to define specific targets for potential therapeutic benefit. The methodological approaches involve both ex vivo and in vivo investigations, including biochemical marker measurements, immuno staining, as well as isolated small artery bioassays and wire myography technique together with EndoPAT to assess peripheral arterial tone.

In Paper I we investigated both in vivo and ex vivo functional properties (reactive hyperemia index [RHI], contractility, vasodilatory, stiffness) of small resistance arteries from ESKD patients and non-CKD controls. We also investigated ex vivo effects of trimethylamine Noxide (TMAO), phenylacetyl glutamine (PAG) and extracellular vesicles (EVs) from CKD-5 patients, as well as pharmacological interventions using senolytics. We assessed markers of senescence, calcification, endothelial function, and oxidative stress; these data were also correlated with functional and structural properties of resistance arteries. We observed that the uremic environment influences vascular function by changing the contribution of endotheliumderived factors (i.e. reduced nitric oxide and increased endothelium derived hyperpolarization factor) and increasing vascular stiffness in patients with ESKD; these events were further modulated by inflammation, TMAO, PAG and EVs. Moreover, the vasculature of ESKD patients was characterized by hallmarks of EVA –presence of the senescence signature, microcalcification, reduced anti-oxidant control, and decreased contractile markers which might confer the development of cardiovascular complications in this specific patient group. We also showed that senolytics could be used to target senescent cells. As Paper I comprehensively phenotypes the microcirculation from ESKD patients, this study serves as a backbone for the overall thesis.

Paper II, a complementary study of Paper I, adds more insight into endothelial function and vascular structure biology in respect to different amino acids (AA) and their metabolites. New findings include impaired AA metabolism with decreased biopterin BH4/BH2 ratio in CKD, as well as elevated asymmetric dimethylarginine levels that were associated with higher vascular stiffness and reduced NO contribution.

In Paper III, we investigated differences in the expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) receptors in resistance arteries and subcutaneous adipose tissue, alongside circulating soluble ACE2 levels in female and male ESKD patients versus non-CKD controls. Our results demonstrated that soluble ACE2 levels were higher in ESKD patients. In addition, ACE2 tissue expression was higher in ESKD patients with a higher prevalence in male subjects and was present in both the endothelium and VSMCs from arteries in peripheral microcirculation.

The aim of Paper IV was to better understand the role of prostaglandin contribution to vasoactive properties and characterize the effects of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition in the microvasculature of CKD patients. A significant reduction in adrenergic vasoconstriction and improvement in relaxation was observed following mPGES-1 inhibition. Based on our findings, it can be inferred that mPGES-1 inhibition has additional vasoactive effects in the human microcirculation beyond the shunting to prostacyclin (PGI2) pathway, i.e. a reduction in the levels of local prostaglandin E2 (PGE2), as well as influencing other vascular factors. This indicates the interaction of several pathways after mPGES-1 inhibition.

The findings of this thesis provide valuable insights into the mechanisms underlying small artery maintenance and dysfunction in ESKD patients and identify potential therapeutic targets for improving vascular function in this patient population.

List of scientific papers

I. Early vascular aging in microvasculature of chronic kidney disease: focus on function, structure and senescence signature. Samsul Arefin, Neja Mudrovcic, Sam Hobson, Federico Pietrocola, Thomas Ebert, Liam Ward, Anna Witasp, Lars Wennberg, Torbjörn Lundgren, Julia Steinmetz-Späh, Karin Larsson, Anders Thorell, Stefania Bruno, Marita Marengo, Vincenzo Cantaluppi, Peter Stenvinkel*, Karolina Kublickiene*. *Authors contributed equally. [Submitted]

II. Associations of Biopterins and ADMA with Vascular Function in Peripheral Microcirculation from Patients with Chronic Kidney Disease. Samsul Arefin, Lars Löfgren, Peter Stenvinkel, Anna B. Granqvist and Karolina Kublickiene. International Journal of Molecular Science. 2023,24,5582.
https://doi.org/10.3390/ijms24065582

III. Angiotensin-converting enzyme 2 and transmembrane protease serine 2 in female and male patients with end- stage kidney disease. Samsul Arefin, Leah Hernandez, Liam J. Ward, Angelina Schwarz, GOING-FWD Collaborators, Peter Barany, Peter Stenvinkel, Karolina Kublickiene. European Journal of Clinical Investigation. 2022;52: e13786.
https://doi.org/10.1111/eci.13786

IV. Effects of microsomal prostaglandin E synthase-1 (MPGES-1) inhibition on resistance artery tone in patients with end stage kidney disease. Julia Steinmetz-Späh*, Samsul Arefin*, Karin Larsson, Jabin Jahan, Neja Mudrovcic, Lars Wennberg, Peter Stenvinkel, Marina Korotkova, Karolina Kublickiene*, Per- Johan Jakobsson*. British Journal of Pharmacology. 2022;179(7):1433-1449; *Shared authorship.
https://doi.org/10.1111/bph.15729