An estimated 19 million Americans suffer from chronic kidney disease (CKD), which is characterized by the progressive development of renal fibrosis. Currently, outside of renal replacement therapy, no effective therapies for end stage renal disease, the most severe stage of CKD, exists, highlighting the need for further investigations into the mechanisms underlying renal fibrosis. Moreover, improvements in early stage CKD detection could provide clinicians a better opportunity to treat and reduce CKD related morbidity and mortality.
To this end, studies from our group and others have identified CD40, a receptor initially discovered to mediate humoral immune reactions, as a key player in the pathogenesis of renal disease and fibrosis. We have previously demonstrated that levels of the soluble form of the ligand to the CD40 receptor, sCD40L, were elevated in patients with renal artery stenosis. Moreover, we showed that circulating CD40 mediators were predictive of changes in renal function in subjects with renal artery stenosis. As renal artery stenosis is a common cause of CKD, we set out to determine whether circulating CD40 mediators were also associated with changes in renal function in a separate cohort of all-cause CKD subjects. Our study shows that subjects with high plasma levels sCD40L, and low levels of the circulating CD40 decoy receptor, sCD40R, have greater declines in glomerular filtration rate than subjects with low sCD40L and high sCD40R. Importantly, we show that these changes are independent of differences in age, sex, baseline GFR, systolic blood pressure SBP, history of diabetes mellitus, ACEi/ARB and aspirin use. Finally, our analysis demonstrates that baseline levels of sCD40L and sCD40R are statistically significant predictors of changes in renal function out to 4 years. Our results demonstrate an association between CD40 and CKD progression, providing evidence that the CD40/CD40L signaling axis may be an important contributor to the pathogenesis of renal fibrosis. Moreover, intriguingly, our results identify sCD40R as a potential pharmacological target to attenuate CKD progression.
Despite the growing body of literature implicating CD40 as a critical mediator of renal disease and fibrosis, little is understood about its regulation in this setting. Accordingly, we set out to evaluate the role of the Na/K-ATPase in the regulation of CD40 expression and function in renal proximal tubule epithelial cells. We demonstrate that cardiotonic steroids (CTS), ligands of the Na/K-ATPase, increase CD40 expression both in vitro and in vivo within renal parenchyma. In order to understand the underlying mechanism of Na/K-ATPase mediated regulation of CD40, we first, demonstrate that knockdown of the a1 isoform of the Na/K-ATPase causes a concomitant reduction in CD40 expression and that rescue of a1 isoform of the Na/K-ATPase restores CD40 expression. Interestingly, knock-in of the a2 isoform is unable to rescue CD40 expression. Because previous studies have established that the major difference between the a1 and a2 isoforms is the ability of the a1 Na/K-ATPase to form a signaling complex with Src, a non-receptor tyrosine kinase, we next tested whether the integrity of the Na/K-ATPase/Src signaling complex was central to the regulation of CD40 within renal parenchyma. Using a2 gain-of-Src binding and a1 loss-of-Src binding mutant cell lines, we demonstrate that this is indeed the case as a2 gain-of-Src binding restores CD40 while a1 loss-of-Src binding causes a reduction in CD40 expression. Furthermore, we verified the functional importance of this novel regulatory mechanism by showing that surface CD40 expression and downstream CD40 signaling processes are inhibited in settings where the Na/K-ATPase/Src complex is disrupted. Importantly, we demonstrate that pNaKtide, a specific Na/K-ATPase/Src signaling antagonist, is capable of attenuating CTS mediated increases in CD40 expression in vitro. Due to the fact that the Na/K-ATPase and CD40 both play critical roles in the pathogenesis of renal fibrosis, our studies have identified the Na/K-ATPase and CD40 as partners in a pro-fibrotic feed-forward loop. Successful inhibition of this loop may pay significant dividends in attenuating the progression of renal fibrosis.
Recently, microRNAs (miRNAs), important post-transcriptional regulators of virtually every physiological process, have garnered interest as potential biomarkers of a diverse array of diseases, including CKD. However, careful review of the literature reveals that despite the enthusiasm over the use of miRNAs as biomarkers, numerous methodological issues still plague the field. For instance, one reason for the excitement over the use of miRNAs as biomarkers is related to their remarkable stability in circulation. Studies have revealed that this stability is due to the fact that miRNAs can be stably transported within exosomes, microvesicular structures that can be released and taken up by recipient cells in a highly regulated fashion. However, recent studies have shown that the vast majority of miRNAs do not circulate within exosomes, but instead, are bound to miRNA-related chaperone proteins or lipids. These molecules can also facilitate the stable circulation of miRNAs. Whether exosomal derived miRNAs are simply a reflection of the overall population of circulating miRNAs or whether they represent a specific subpopulation, is still unknown. This remains a key unanswered question in the realm of miRNA biomarker research and a very limited body of literature has directly addressed this topic. Therefore, we profiled miRNAs collected from either plasma, or plasma-derived exosomes in Sprague Dawley rats that underwent sham, 5/6th Partial Nephrectomy (PNx), or 2 Kidney 1 Clip (2K1C) surgery, which are two different animal models of kidney disease our group has expertise with. We demonstrate that the average expression of miRNAs is considerably lower in plasma-derived exosomes compared to plasma, which is in line with previous reports. We also report that the changes in miRNA expression following either PNx or 2K1C in plasma-derived exosomes are not in concordance with the same measurements in plasma, suggesting that plasma and plasma-derived exosomes contain two different populations of miRNAs and therefore are not simply interchangeable with one another. These results suggest that future studies that aim to measure circulating miRNAs should carefully consider which population of miRNAs is the more appropriate population to measure for their intended study.