PARticularly Forceful: PAR1 Drives Glomerular Mesangial Cell Contractility

A Perspective on “PAR1-Mediated Non-Periodical Synchronized Calcium Oscillations in Human Mesangial Cells”

It has been 33 years since protease-activated receptor 1 (PAR1), the first member of the PAR family to be discovered, was described as a canonical thrombin receptor expressed by platelets and endothelial cells.¹ Three additional protease-activated receptors (PAR2, PAR3, and PAR4) were subsequently identified.² PARs are G-protein-coupled receptors that are now known to be widely expressed in many tissues, including the kidney.² They are activated upon proteolytic cleavage of their extracellular N-terminus, afterward the neo-N-terminus functions as an intramolecular ligand to autoactivate the receptor. An ever-growing number of proteolytic enzymes have been discovered to cleave and activate PARs.² The activating proteases, via cleavage at different amino acid loci, may reveal unique neo-N-termini, resulting in activation of various signaling pathways. In the laboratory, synthetic peptides mimicking the neo-N-terminus elicited by a proteolytic enzyme of interest are often used to selectively activate PARs. Adding to the complexity of this receptor family, PARs are known to homo- and hetero-dimerize with other PARs and other, non-PAR, receptors to regulate signaling through allosteric modulation, and some PARs are able to transactivate other PARs.² Importantly, comparative physiology plays a role in this field because rodent and primate PARs often do not behave analogously to one another and may even be functionally distinct receptors.³ Nonetheless, in all species studied to date, thrombin is considered the canonical proteolytic agonist of PAR1, PAR3, and PAR4, whereas activated factor X is the prototypical proteolytic agonist for PAR2.

PARs were originally discovered, and are most well studied, as coagulation protease receptors.² However, they are now known to be expressed by a variety of non-hematogenous, non-vascular cell types. Thus, there has been growing interest in their (patho)physiologic functions and activating proteases in these settings. A variety of studies have demonstrated that PARs play a role in the pathophysiology of several types of kidney disease.^4–9Meanwhile, although PAR expression in non-vascular kidney cells (eg, podocytes, tubular epithelium, mesangium) is preserved across species, their roles in normal renal physiology are ill-defined.⁴ Meanwhile, knockout mice for all 4 PARs have been characterized and none of them appear to have a renal phenotype, thus the reason for this conserved kidney cell expression remains mysterious. Like in other cell types, kidney cells often express more than one type of PAR. However, while a protease may activate more than one type of PAR (eg, thrombin), the resulting signals are divergent suggesting that this receptor system is more cooperative than redundant.¹⁰

Glomerular mesangial cells are one of the non-vascular renal cell types that express PARs.⁴ In the current issue of Function, Stefanenko et al. set out to study the consequences of PAR1 activation in human mesangial cells.¹¹ Thrombin has been implicated as a modulator of mesangial cell contractility and PAR1 signaling has been implicated in several types of glomerular disease. Thrombin, the canonical PAR1 activating protease, is known to induce intracellular calcium transients that drive cytoskeletal rearrangement. Therefore, the investigators stimulated mesangial cells with two different thrombin mimetic activation peptides. Thrombin receptor agonist peptide (TRAP), is identical to the neo-N-terminus sequence revealed by thrombin cleavage (SFLLRNPNDKYEPF).¹ The PAR1 neo-N-terminus mimicked by TRAP is capable of PAR2 transactivation and it is thus not surprising that TRAP can also activate PAR2.²,¹² Therefore, they also used a modification of this sequence (TFLLR), that is more specific for PAR1, and avoids simultaneous PAR2 activation. PAR1, PAR2, and PAR4 antagonists, a PAR4-specific activation peptide, and several calcium channel antagonists were utilized to reveal the patterns of PAR1-mediated calcium flux in primary human mesangial cells and to dissect which calcium channels enable these patterns.

Using live cell confocal imaging to assess calcium influx, they demonstrate PAR1-specific (TFLLR) intracellular calcium transients. A single calcium spike was observed at low concentrations of TFLLR and oscillating transients were observed at higher concentrations. Both patterns were abolished by a PAR1-specific antagonist and the oscillatory peaks were not observed when calcium was removed from the buffer, suggesting that the initial peak is driven by store-operated calcium entry (SOCE) and later peaks are driven by extracellular calcium entry. Calcium channel antagonists were utilized to show that the SOCE peak is driven by STIM1/Orai1 calcium release, likely from the endoplasmic reticulum, whereas the oscillatory peaks are driven by transmembrane TRPC3 channels following depletion of stored, intracellular calcium. Intriguingly, fast confocal 3D imaging further demonstrated that TFLLR induced significant whole glomerular volume reduction, strongly suggesting that PAR1 activation leads to glomerular contraction which may alter intraglomerular hemodynamics.

These observations have potential for broad implications in the field of glomerular biology. However, additional in vivo work will be needed to determine the (patho)physiologic relevance of these observations. On a physiologic level, PAR signaling may represent a novel regulator of glomerular hemodynamics. Is mesangial PAR signaling an important regulator of blood pressure? From a pathologic perspective, mesangial PARs may represent novel therapeutic targets for a variety of glomerular diseases. Does PAR-mediated mesangial contraction modulate filtration in nephrotic syndrome, IgA nephropathy, or diabetic nephropathy? An obvious open question is which physiologic and pathophysiologic proteases govern mesangial PAR signaling. Understanding which proteolytic enzymes interact with mesangial cells and where they cleave their PAR substrates may lead to novel discoveries in both the glomerular and PAR biology fields.

Funding

This commentary was supported by grant R01DK124549 (to B.A.K.) from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

Conflict of Interest Statement

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.