9. Conclusiones
9.2. De la masificación a la particularización
apical staining is observed whereas in antagomir-132 treated mice however,
$43LVPDLQO\ORFDWHGLQWUDFHOOXODUO\)LJXUH&6XEVHTXHQWO\ZHVRXJKW WRGHWHUPLQHLI6DQG6SKRVSKRU\ODWLRQRI$43KDGEHHQDIIHFWHG DVWKLVKDVEHHQGHVFULEHGWREHLQYROYHGLQWKHORFDOL]DWLRQRI$4327. As shown in Figure 4C-E, no differences were observed.
MiR-132 silencing suppresses sodium-hydrogen exchanger 3 (NHE3) gene expression
Unexpectedly, despite the increased osmolality we did not measure a difference in plasma sodium levels. However, urinary sodium showed a trend towards increased levels, suggesting antagomir-132 partly inhibits active sodium reabsorption. Therefore we investigated gene expression of four main sodium transporters along the tubular system, sodium-hydrogen exchanger 3 (NHE3), sodium-chloride symporter (NCC), Na-K-Cl cotransporter
Figure 3. Blocking miR-132 results in a strong change in osmolality while not in sodium levels. A and B, plasma osmolality (A) increased after miR-132 silencing, while urine osmolality decreased (B). C and D, plasma (C) and urine (D) sodium levels were not
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(NKCC2) and the amiloride-sensitive epithelial sodium channel (ENaC). Interestingly, a striking decrease in NHE3 was found (Supplementary Figure 2).
Silencing of miR-132 increases urinary PGE levels
Since we demonstrated that miR-132 could directly target Cox2, we hypothesized that silencing of miR-132 should result in a de-repression
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determined renal prostaglandin production (which is also used as a measure
Figure 4. miR-132 silencing results in altered AQP2 localization. A and B, representative
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IRU&R[DFWLYLW\E\PHDVXULQJWKLVLQWKHXULQHDVXULQDU\3*(H[FUHWLRQis mainly derived from medullary synthesis28,QGHHGZHIRXQGVLJQLÀFDQWO\ LQFUHDVHG3*(OHYHOVLQXULQHRIPLFHWUHDWHGZLWKDQWDJRPLU)LJXUH $ ,Q FRQWUDVW FLUFXODWLQJ 3*( OHYHOV KDG D WHQGHQF\ WR EH GHFUHDVHG Figure 5.Increased renal PGE2 production while no increase in AVP.$XULQDU\3*( OHYHOV LQFUHDVHG GXH WR PL5 VLOHQFLQJ % 3ODVPD 3*( OHYHOV ZHUH QRW VLJQLÀFDQWO\
altered but showed a trend towards reduced levels. C and D, representative microscopic images of renal Cox2 shows interstitial (C) and tubular epithelial (D) cell positive staining.
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(Figure 5B). To determine where miR-132 could affect Cox2 we performed a staining to assess where Cox2 is expressed. Although some controversy exists about the location of constitutive Cox2 expression, we found it to be expressed in interstitial cells, cortical tubular structures (based on morphology
SUHGRPLQDQWO\ GLVWDO GDWD QRW VKRZQ DQG LQ VSHFLÀF PHGXOODU\ WXEXODU HSLWKHOLDOVWUXFWXUHV)LJXUH&'7KHODWWHUZHLGHQWLÀHGWREHFROOHFWLQJ GXFWV DV GHWHUPLQHG E\ FRVWDLQLQJ ZLWK $43 (Figure 5E). This pattern highly corresponds to miR-132 localization, consistent with a functional link between the miR and Cox2.
MiR-132 blocking attenuates hypertonicity-induced increase in blood AVP levels
In normal physiology increased plasma osmolality stimulates drinking and secretion of AVP, resulting in increased renal water reabsorption, more concentrated urine, and less concentrated blood plasma. We assessed
YDVRSUHVVLQOHYHOVLQWKHXULQHZKLFKUHÁHFWV$93OHYHOVLQWKHEORRG29, and surpisingly found no increase in AVP levels (Figure 5F). In fact, we see a trend towards decreased levels of vasopressin. Alternatively, or additionally, the observed effect of increased diuresis, may be due to reduced central
YDVRSUHVVLQ SURGXFWLRQ DQG UHOHDVH SRVVLEOH WKURXJK 3*( DV ZHOO DV 3*(KDVEHHQLPSOLHGLQFHQWUDO$93SURGXFWLRQDQGUHOHDVH14. To assess this hypothesis we excised the hypothalamus of the animals and determined AVP gene expression by qPCR. We found decreased levels of AVP gene expression in the hypothalamus in mice that received antagomir-132 (Figure
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AVP production providing an additional explanation for the (trend towards) lower urinary AVP levels.
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increased plasma osmolality and decreased urine osmolality. The effects we observed seem to be acute, as they are present within 24 hours after injection of antagomir-132, while 48 hours after injection, no further weight loss or polyuria is found. This suggests the body responds to this interference in homeostatic conditions by a feedback mechanism, which seems appropriate as regulation of water and electrolyte balance is tightly controlled. Antagomir-132 treated mice showed a trend towards decreased food intake that corresponded with reduced feces production which is therefore no contributor to weight loss. However, these observations suggest the mice display signs of malaise.
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(NIH3T3) that blocking miR-132 results in increased Cox2 levels. In vivo, this
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that miR-132 can have ambiguous effects, depending on the site of action. We found miR-132 expression predominantly in tubular structures, with highest levels in the renal medulla and more distant regions of the tubule. In addition, we observed most Cox2 expression in distant regions of the
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reported on this subject30,31. As we demonstrated that Cox2 is a direct target of miR-132, this suggests its regulation could be mainly taking place here.
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However, also (cortical) interstitial cells are expressing Cox2, and as we have
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increase in Cox2 expression. To elucidate the exact site of action of miR-132 and its target(s) involved in diuresis further investigations are necessary.
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translocation and subsequent increased diuresis as it is known to counteract
$93LQGXFHG$43WUDQVORFDWLRQ10,11. In addition, AVP levels in the urine, UHÁHFWLQJ $93 OHYHOV LQ WKH FLUFXODWLRQ29 were slightly decreased, possibly due to decreased AVP production in the hypothalamus as we show that
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hypothalamic AVP gene expression is decreased as a results of antagonizing miR-132. These decreased levels could again be explained by an alteration
LQ3*(OHYHOV14 although the exact mechanism needs further investigation. In addition, to clarify the role of central AVP production on the observed phenomena in this study, one needs to investigate the effect of antagomir-132 while also providing synthetic dDAVP to the mice, which could rescue the phenotype if it was completely due to central AVP interference.
It has been well established that AVP also has long term effects in principal
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and promoter binding by the cyclic AMP responsive element binding protein (CREB)6,7. CREB itself is known to regulate transcription of miR-13232, providing a potential feedback loop in signaling. Furthermore, miR-132 can target p30033, which is a transcriptional co-activator that can act in concert with CREB. In addition, promoter activity of Cox2 is dependent on CREB/ p300 transcription factors34,35. These factors are adding additional layers of possible autoregulation within water maintenance. Besides, this might explain
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it has been shown that inhibition of Cox2 by indomethacin reduced renal
$43 H[SUHVVLRQ LQ UDWV LPSO\LQJ WKDW SURVWDJODQGLQV PD\ DFWXDOO\ KHOS PDLQWDLQ$43OHYHOV13, which might also explain our in vitro observations. We observed an increase in plasma osmolality suggesting the mice are hypovolemic. This would normally be accompanied by decreased blood pressure and subsequent activation of the RAAS pathway. Angiotensin II, that will be increased due to RAAS activation, is known to exert a dipsogenic effect36. The dipsogenic action of angiotensin II is thought to be mediated by the subfornical organ (SFO), because the SFO contains a high concentration of the angiotensin AT1 receptor37 and ablation of the SFO completely abolished the dipsogenic response to angiotensin II38. However, we surprisingly found the polyuric mice to drink less. It could be expected therefore that angiotensin II signaling is disturbed. Indeed, a crosstalk between miR-132 and angiotensin II has been described39. Furthermore it was shown that miR-132 can directly target Angiotensin II type 1 Receptor (AT1R)40. These data suggest that also angiotensin II signaling is disturbed in these mice by silencing miR-132. Interestingly, NHE3 is also known to be regulated by angiotensin II41, so disturbed angiotensin II signaling could also reduce NHE3 expression, which is what we observed.
Some neurons in the above mentioned SFO are osmoreceptors, being sensitive to the osmotic pressure of the blood. These neurons project to
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