In the present study we investigated the potential renoprotective effects of the therapeutic association of LOS + MMF + TAM on an experimental model of hypertensive nephrosclerosis, based on the chronic inhibition of NO synthesis, induced by L-NAME administration. The main aim of our research was to verify whether the combination of TAM, a selective estrogen receptor modulator, recommended for the treatment of positive estrogen receptor (ER+) breast cancer, to the currently employed conservative CKD treatment, here represented by RAAS blockade and immunosuppression, would promote additional anti-inflammatory and/or antifibrotic beneficial effects, when compared to the respective monotherapies.
Corroborating previous data, rats submitted to the L-NAME model of CKD developed severe hypertension, probably caused by glomerular and systemic vasoconstriction due to the lack of physiological vasodilatory effects of NO. Systemic hypertension and CKD are closely related conditions with an intricate cause/effect relationship. The decline of kidney function usually leads to high blood pressure, due to both a decreased ability of the kidneys to remove salt from the bloodstream, and an increased release of renal vasoconstrictive hormones. On the other hand, systemic hypertension sustained for long periods leads to the damage of multiple target organs, including the kidneys [7, 8]. Hypertensive nephrosclerosis is one of the main causes of end-stage renal failure. High blood pressure also contributes to the aggravation of CKD, regardless of its etiology [1]. Accordingly, the clinical management of hypertension is currently one of the most employed strategies to control CKD progression [7, 8]. According to our results, systemic hypertension induced by L-NAME administration was only partially reduced by the monotherapies with both LOS or MMF, and equally by the association of LOS + MMF + TAM. There was no synergistic effect of the combination of drugs as regards lowering blood pressure. The poor hemodynamic effect of therapeutic schemes may have limited the potential effects of the therapeutic association on the maintenance of renal function.
Along with the hypertension, NAME animals also exhibited a markedly increased urine albumin excretion rate, a clear evidence of renal impairment. Because of its strong predictive power for cardiovascular and renal events, albuminuria is one of the most important biomarkers of CKD progression, particularly in patients with hypertension or diabetes mellitus. Moreover, the reduction of albuminuria is the most important goal to prevent the progression of kidney disease in CKD patients. Usually, in this regard, significant benefits are achieved by the therapeutic treatment with RAAS inhibitors. According to our results, although all the tested monotherapies significantly limited the development of albuminuria in NAME animals, the antiproteinuric effect obtained with the combined treatment was noteworthy. Is spite of the severe sustained hypertension, LOS + MMF + TAM association completely averted the development of albuminuria in this CKD model.
The increased urinary albumin excretion is directly related to the disruption of one or more components of the glomerular filtration barrier, and with glomerular structural damage, generally caused or worsened by renal inflammation. Histological glomerular alterations are a common feature in most human and experimental nephropathies. Accordingly, severe glomerulosclerosis and glomerular collapse were observed in untreated NAME animals, and the association of LOS + MMF + TAM significantly prevented the development of glomerular histological damage, probably reflecting the effects of LOS (for glomerulosclerosis) and MMF (for collapsed glomeruli), thus evidencing that TAM did not exerted antagonism, blockade or inhibition upon the pharmacological effects of both LOS and MMF, and did not diminish the renoprotective effects observed with these drugs.
Kidney infiltration by inflammatory leukocytes has been demonstrated in a variety of non-immune mediated nephropathies, such as the hypertensive nephrosclerosis [4, 19]. The recruitment of circulating monocytes, as well as the activation of resident renal macrophages often correlates positively with the worsening of renal function loss, in both human and experimental CKD [2]. Accordingly, in the present study, NAME animals showed exuberant renal inflammation, characterized by inordinate tubulointerstitial cell proliferation and massive infiltration of kidneys by both macrophages and lymphocytes. Surprisingly, similarly to the observed with LOS and MMF monotherapies, expected to exert inhibitory effects on macrophage and lymphocyte renal infiltration, as well as on interstitial proliferation rate, TAM monotherapy also exhibited independent significant anti-inflammatory proprieties. Therefore, the association of LOS + MMF + TAM seems to combine different mechanisms of action to abrogate renal inflammation. Inhibition of macrophage activity by TAM treatment has been demonstrated in both in vitro and in vivo studies, in which Tamoxifen promoted significant reduction in the transcription of important cell surface receptors, such as the fatty acid-binding proteins (FABPs) and the scavenger receptor class B member 3 (SCARB3 / CD36), which are involved in the monocyte/macrophage activation processes, and play a pivotal role in foam cell formation and in the development of atherosclerosis [25]. The anti-proliferative effects of RAAS blockade, associated with the reduction of IL1, IL6 and IL10 macrophage release, possibly achieved with the MMF treatment, may have boosted the anti-inflammatory effects of TAM. A synergetic effect among the tested drugs could also be plausible in this case. However, additional studies focused on the intracellular mechanisms of action of each employed drug and in the possible chemical interactions among them should be carried out to speculate this hypothesis.
Along with renal inflammation, the overproduction of ECM and the renal interstitial collagen accumulation are important histological features, commonly related to the worsening of CKD. In the present study renal cortical interstitial fibrosis, evidenced by the high percentage of Masson+ interstitial staining, myofibroblasts infiltration, as well as collagen I and fibronectin interstitial deposition, accompanied the progression of hypertensive nephrosclerosis in untreated NAME rats. All the tested therapies were effective in preventing renal fibrosis and α-SMA accumulation, while only TAM and the association of LOS + MMF + TAM significantly reduced collagen I and fibronectin accumulation in this CKD model, suggesting that TAM promoted additional antifibrotic effect to the therapeutic scheme, with no impairment of renoprotective action of LOS and MMF, when associated to these drugs. The suppressive effects of tamoxifen on fibrogenesis were first described in the early nineties, when Clark and collaborators described the drug to be effective and safe in the treatment of two patients with severe retroperitoneal fibrosis. Its effectiveness for the treatment of encapsulating peritoneal sclerosis, where than demonstrated, eight years later, by Allaria and co-authors. Based on these observations, Dellê and collaborators, from our research group, showed for the first time that TAM could exert protective effects on experimental progressive chronic kidney disease, in 2003. [26, 18, 27]. More recently, TAM was described to exert important antifibrotic effects in the experimental model of unilateral ureteral obstruction (UUO) in mice. Similarly, to the observed in the present study, TAM treatment reduced the production and deposition of ECM proteins in UUO kidneys, as well as the renal deposition of fibronectin and collagen [28, 29]. Although the exact mechanisms involved in the anti-inflammatory and antifibrotic effects of TAM are still poorly known, it exerts undoubted suppressive effects on fibroblast proliferation, activation and ECM secretion, evidenced in our in vitro results, which corroborate the current literature [30]. Since MMF, employed as an immunosuppressive drug in our in vivo protocol, is in fact a prodrug, which must be ingested and then metabolized into the pharmacologically active drug (mycophenolic acid), we were not able to perform in vitro studies with the full LOS + MMF + TAM association. However, we combined LOS to TAM is our analysis and clearly demonstrated that LOS did not impaired the suppressive effects of TAM on cultured fibroblasts, thus corroborating the idea that this drug combination may be safe and effective.
In summary, although further studies employing different CKD models are still required to confirm the efficacy and safety of the association of LOS + MMF + TAM, in the present paper we provided strong evidence that this therapeutic scheme can be potentially useful to slow the progression of chronic nephropathy, since it lowered systolic blood pressure, prevented albuminuria, glomerular structural damage, and renal inflammation and promoted additional antifibrotic effect to the traditional conservative treatment of CKD, in the NAME model of hypertensive nephrosclerosis. In conclusion, our pre-clinical observations suggested that the association of TAM to the conservative treatment of CKD, employing LOS and MMF, was safe and promoted additional renoprotective, anti-inflammatory and antifibrotic effect in a model of hypertensive nephrosclerosis in rats.