The present study was the first to characterize the transcriptome in a specific nephron segment in a model of hypertension and one of the first to study the transcriptome in tissues highly enriched for a single cell type in hypertension. Transcriptome analyses have been reported in nephron segments isolated from humans or animal models under physiological conditions.32,33,34,35
The mTAL is particularly relevant to the SS rat model of hypertension. Impairment of pressure natriuresis in SS rats has been attributed to elevated NaCl reabsorption in the loop of Henle.19,25
Dopamine and nitric oxide inhibits Na+
-ATPase activity or chloride reabsorption in the thick ascending limb less effectively in SS rats than in Dahl salt-resistant rats.21,22
Production of 20-hydroxyeicosatetraenoic acid, which inhibits chloride transport in mTALs, is diminished in mTALs in SS rats.18,26
NKCC2, which mediates the bulk of NaCl reabsorption in the thick ascending limb, is up-regulated in SS rats and possibly in humans with elevated blood pressure salt-sensitivity.14,15,16,17
mTALs contribute to increased production of reactive oxygen species in SS rats, which may contribute to the impairment of medullary blood flow regulation that is important for pressure natriuresis.20,23,24
In the differentially expressed genes involved in the identified Ingenuity Canonical Pathways, Cxcr4, F5, Fcgr3a, Pla2g4a, and Ptgs2 are located on rat chr. 13. These genes are involved in inflammation, fibrosis, and arachidonic acid metabolism, which are known to contribute to or exacerbate the development of hypertension. Much more work, however, would need to be done before any causal contribution of these genes to hypertension could be established. Importantly, differentially expressed genes not mapped to the consomic chromosome could still be highly relevant to the hypertension phenotype. It is unlikely that causal genes located on the consomic chromosome are the only genes involved in the development and progression of the hypertension phenotype. Instead, it is likely that the causal genes contribute to the hypertension phenotype by influencing, directly or indirectly, biological pathways involving genes located on other chromosomes, forming a tree-like regulatory network that we proposed previously.11
We previously performed a transcriptome analysis of the renal outer medulla, using the same rat strains, dietary conditions, and array platform as the current study of mTALs.8
Several inflammation-related pathways were identified as highly represented in the differentially expressed genes by Ingenuity Pathway Analysis in both studies. Some pathways, however, were identified in only one of the two studies. For example, β-adrenergic signaling and nitric oxide signaling pathways were identified only in the outer medulla study, while pathways related to fibrosis and cell cycle regulation were identified only in the mTAL study.
A novel and unexpected finding of the current study was that SS rats fed the high-salt diet had more mTAL cells that were in a proliferative state, compared to SS.13BN
rats. Moreover, the high-salt diet increased proliferative mTAL cells by several fold in both strains of rats. The vast majority of terminally differentiated cells in the kidney, including mTAL cells, are in the quiescent state of G0. Proximal tubular cells can re-enter the cell cycle following massive cell death that occurs in conditions such as severe ischemic-reperfusion injury.36
But even in ischemic-reperfusion injury, mTALs are largely spared. It is, therefore, rather unexpected that we found increased proliferative mTAL cells in rats fed the high-salt diet, especially SS rats. Interestingly, alterations of cell cycle regulation in the kidneys of SS rats were suggested not only by the current mTAL transcriptome analysis but also by our previous transcriptome analysis of the renal cortex.8
The cell cycle regulation pathway, however, was not identified in the analysis of homogenized renal outer medulla tissue.8
In the current mTAL study, the cell cycle regulation pathway was prominent in strain comparisons under both 0.4% and 8% salt conditions according to the Bayesian analysis and the top 10 ranking genes largely overlapped. Yet SS rats on the 0.4% salt diet did not show a significant difference in mTAL proliferative states compared to SS.13BN
rats. The ranking of the top genes differs between the two dietary salt conditions. Expression patterns for some of the specific genes also differ between the two conditions. In addition, Ingenuity Pathway Analysis identified cell cycle related pathways from the high-salt dataset only.
It remains to be determined what mechanisms cause the increase in proliferative mTAL cells in SS rats on the high-salt diet. Angiotensin II, at doses that do not increase proliferation of proximal tubular cells, stimulates proliferation of a murine mTAL cell line via AT1 receptors.37
The SS rat is a low-renin model of hypertension. Yet intra-renal levels of angiotensin II have been shown to be abnormally high in SS rats fed a high-salt diet.38,39
In addition, reactive oxygen species at moderate concentrations can stimulate cell proliferation.40,41
The renal medulla of SS rats is known to have higher levels of reactive oxygen species including superoxide and H2
compared to SS.13BN
rats, which contributes to the development of hypertension in SS rats.42,43
The nature of the cell cycle alteration that we observed and their functional consequences remain to be investigated. Nevertheless, the current study has provided several interesting clues. Despite increased proliferative mTAL cells in SS rats, total cells per mTAL section were not significantly different between SS and SS.13BN rats. In fact, total cells per mTAL section were not significantly different between rats on the two salt diets, even though proliferative mTAL cells increased by several fold in rats fed the high-salt diet. There are two possible explanations. One, mTAL cells were proliferating to replace lost cells. Two, the proliferative mTAL cells had exited G0, but were not able to progress through the cell cycle to complete cell division. SS rats fed the high-salt diet had more mTALs that were overtly damaged and filled with casts, as one would expect. However, we only counted cells in mTALs that appeared normal because cells in damaged mTALs were distorted, making it difficult to obtain accurate counts. Therefore, it appears that the more likely scenario is that proliferative mTAL cells were arrested at some point of the cell cycle.
G2/M arrest of proximal tubular cells following injury contributes to the development of tubulointerstitial fibrosis.36
Tubulointerstitial fibrosis in the outer medullary region is a pathological hallmark of SS rats and is significantly attenuated in SS.13BN
It would be consistent with the fibrotic phenotype if proliferative mTAL cells in SS rats were arrested in G2/M. SS rats on the high-salt diet indeed tended to have more mTAL cells that were in G2/M phases. However, very few mTAL cells overall were in G2/M phases in either strain of rats on either salt diet. It suggests that the proliferative mTAL cells that we observed might be in G1 or S phase. G1/S arrest can be a response to mild DNA damage, allowing DNA repair to take place.44,45
Failure of repair could lead to apoptosis. Apoptosis appears to increase in the injured kidneys of SS rats,46
although we were not able to detect a significant difference in apoptotic cells in morphologically normal mTALs between SS and SS.13BN
rats. A pro-inflammatory milieu, which exists in the kidneys of SS rats,38,47
could also cause G1/S arrest.48
It remains to be determined if the signal that stimulates mTAL cell proliferation in SS rats is the same signal that prevents mTAL cells from completing the cell cycle. Cellular signals for proliferation and G1/S arrest are often distinct. For example, reactive oxygen species can stimulate proliferation, while anti-oxidant treatments can lead to late-G1 arrest.40,41,49
In some cases, however, a cellular signal can cause proliferation followed by G1/S arrest (Shimi 2011).50
The study provides genome-wide insights into the mechanism of hypertension at the level of a specific nephron segment. The finding of abnormalities in cell proliferation in mTALs may stimulate a new direction of research of hypertension or hypertensive renal injury.