The aim of our study was to determine - in contrast to most other published data that dealt with short-term effects - the long-term effects of experimental unilateral renal artery stenosis on blood pressure, kidney weight and renal renin, ETs and eNOS expression.
A significant increase in blood pressure after clipping is well known in 2K1C-rats [2, 10], making them a commonly used model of renovascular hypertension. Our data fits with other findings showing a lower, but still increased blood pressure four weeks after clipping . Furthermore, many authors also show an increase in weight of the contralateral kidney while the weight of the clipped kidney decreased with time [11–13]. In contrast some authors found no change in the weight of the clipped kidney, while they also found an increase in the contralateral kidney [14, 15]. Sham-operated rats showed as expected no difference in kidney weight [16, 17].
Our findings of appropriate changes in kidney weight and blood pressure after unilateral clipping suggest effective creation of unilateral renal artery stenosis. The apparent decrease in measured systolic blood pressure on day 20 is thought to be due to a change in cuff size for blood pressure measurement with growth of rats.
Renal hypoperfusion is well-known as a stimulator of both renal renin gene expression and plasma renin activity. Several groups including ourselves have shown an increased plasma renin activity and increased ipsilateral renal renin gene expression after induction of unilateral renal artery stenosis . Renin upregulation has generally been accepted as neurohumoral counterregulation in renal hypoperfusion to maintain post-stenotic renal perfusion and renal function.
In accordance to our findings many groups report decreasing, but still elevated plasma renin levels in the more chronic phase of renovascular hypertension [19–21], as well as of increased renin RNA-levels in the clipped kidney and suppressed levels in the contralateral kidney in the early phase of 2K1C-hypertension [14, 16, 20].
We demonstrated that renal renin gene expression is upregulated in the clipped kidney as an early and long-term adaption process to renal hypoperfusion. In parallel downregulation of the renin gene expression occurs in the unclipped kidney most presumably due to systemic hypertension again as an early- and long-term adaption process. Hypertension most probably is due to an early markedly increased plasma renin activity that declines by time but still is inadequately high when related to systemic blood pressure, and presumably goes along with sodium retention in addition to vasoconstriction.
Short-term renal hypoperfusion has resulted in an upregulation of renal ET-1 and a downregulation of renal ET-3 . Studies in patients with angioplasty of renal artery stenosis showed inconsistent results. While one report found no change of plasma ET-1 levels after repair, another report described an increased level of ET-1 [23, 24]. In a further study no difference in plasma ET-1 levels six weeks after experimental 2K1C-clipping compared to baseline were found . However, since endothelins are known to act mainly in an autocrine/paracrine manner, plasma levels of endothelin-1 may not be sufficient to judge whether the endothelin system is involved i.e. in the pathophysiology of unilateral renal artery stenosis. An additional paper showed that the ET-system seemed to be activated in low-renin, volume-dependent 1K1C Goldblatt rats , however this model differs in several aspects to the 2K1C model, used by us.
Our findings that there is no difference in the gene expression of ET-1 and ET-3 at day 28 and 70 suggest, that ETs might not to be involved in long-term adaptation processes. In contrast ET-1 gene expression being upregulated in the clipped kidney on day 7 and endothelin-3 being downregulated on day 3, suggest, that ETs might be involved in short-term adaptation processes right after renal hypoperfusion.
Focusing on overall renal eNOS gene expression we found no significant difference at any timepoint. This matches well with previous findings that there is no difference at any timepoint in eNOS protein expression in the left and right kidneys of 2K1C rats when the clip was removed after 6 weeks and eNOS protein expression was measured before and after unclipping . This study  reported in addition that there are different expression patterns in medulla (down-regulation) and cortex (up-regulation), which obviously could not be detected by us when analysing whole kidney eNOS gene expression.
The analysis of eNOS expression data in eNOS and nNOS deficient mice suggests that eNOS derived NO enhances renal renin gene expression . Barton found that renal hypoperfusion leads to a stimulation of renal eNOS gene expression . When using pharmacological NO donors as sodium nitroprusside renin secretion is increased . According to our results, overall eNOS gene expression seems not to be quantitatively involved in adaption processes of whole 2K1C-rat-kidneys, though recent data show clearly a protective role for NO in 2K1C hypertensive mice .
Since angiotensin II has been reported to stimulate the release of endothelin-1 [29, 31], unilateral renal artery stenosis with resulting upregulation of the renin system might result in an increased expression of endothelins. Furthermore the endothelin-system has an inhibitory influence on renin . This fits with our findings of the upregulation of endothelin-1 while the renin activity is high in early stenosis and decreasing renin levels after activation of the endothelin-system. Although there is clear evidence for interactions between renin and the ET-system, ETs appear not to be involved in chronic long term adaptation processes after unilateral renal clipping as discussed above.
Overall, the gene expression analysis of vasoactive mediators like the ETs and eNOS in whole kidney is a limitation of this study with regard to their differential expression and regulation in cortex and medulla [26, 33].
In conclusion, we demonstrated that the 2K1C-model in male rats leads to a long-term stimulation of the renin system with ipsilateral upregulation and contralateral downregulation. ETs and endothelium derived NO seem to play no major role in chronic adaptation processes in the kidney of 2K1C-rats, although ET-1 and ET-3 might be involved in short-term adaptation processes, but their functional relevance should be confirmed in further studies, using pharmacological agonists/antagonists and/or genetic knockout models.