Phosphorylation of threonine-788/789 sites of β1-integrin specific to juxtaglomerular cells
To investigate β1-integrin phosphorylation during DN progression, specific antibodies against phosphorylated threonine-788 and threonine-789 of β1-integrin were applied to kidney tissue sections from various stages of streptozotocin-treated rats (STZ rat) to study the distribution and phosphorylation of β1-integrin. Both antibodies showed intensive reactions at the juxtaglomerular apparatus in the kidney of STZ rats and controls (Fig. 1a). Since stained cells seemed to exist around afferent arterioles, double staining with anti-renin antibody was performed, and these cells were confirmed to be renin-secreting juxtaglomerular cells (Fig. 1b).
A negative correlation between the number of cells containing threonine-788/789 phosphorylated β1-integrin and the renin expression
The changes in the phosphorylation of threonine-788/789 of β1-integrin specific to juxtaglomerular cells during DN progression were investigated in the kidneys of STZ rats, along with their relationship with the renin production. The production of renin gradually increased after the onset of DN, peaking in the second month but then decreasing and returning to the original level in the sixth month. In contrast, the number of juxtaglomerular cells in which threonine-788/789 phosphorylation of β1-integrin was detected gradually decreased after the onset of DN, peaking in the second month and then increasing further before ultimately returning to the original level (Fig. 2). These results showed that there was a negative correlation between the renin production and threonine phosphorylation of β1-integrin during DN progression in STZ rats (correlation coefficient = 0.61).
Activation/inhibition of protein kinase Cε reproduced the negative correlation between threonine-788/789-phosphorylated β1-integrin and the renin expression in vitro
As a negative correlation between the renin production and threonine-788/789 phosphorylation of β1-integrin in juxtaglomerular cells was observed in kidneys of STZ rats, the relationship between them was examined in cultured cells in vitro. As4.1 cells, a line of mouse juxtaglomerular cells, were used to examine the changes in renin production after the addition of PMA/BIM-1, an activator/inhibitor of PKCε, which is a subtype of PKC that phosphorylates threonine-788/789 of β1-integrin . As TGF-β1 also reportedly promotes the threonine-788/789 phosphorylation of β1-integrin specifically , we also investigated the effect of TGF-β1 addition to the culture.
The results showed that renin production in As4.1 cells after 24 h of drug addition was inhibited by PKC activation, and conversely, renin production was enhanced when PKC was inhibited, confirming a negative relationship between these two, similar to that in vivo (Figure. 3a). TGF-β1 also inhibited renin production and supported this relationship. When we monitored the changes in renin production up to 24 h after PMA/BIM-1 treatment, we found that the renin production showed severe changes, including a rapid rise and fall in the early period up to three hours after treatment, but thereafter there was a gradual suppression and steady increase respectively until the end of the experiment (Fig. 3b). Western blotting for phosphorylated threonine-788/789 of β1-integrin showed results compatible with the above findings, wherein the acceleration of phosphorylation by PMA occurred 3 h after drug addition and was sustained for 24 h. BIM-1 suppressed the phosphorylation of the sites, and TGF-β1 promoted the phosphorylation but not as much as PMA (Fig. 3c).
Knockdown of the β1-integrin expression results in out-of-control renin expression, as does knockdown of the connexin-40 expression
Since PMA and BIM are effective across a broad spectrum of PKC subtypes, we cannot ignore the possibility that the changes in renin production seen in Fig. 3 are a combination of effects from other phosphorylation pathways in addition to the phosphorylation of β1-integrin by PKCε. In order to clarify the effect of β1-integrin on renin production, we performed knockdown of β1-integrin in As4.1 cells and examined its effect on renin production. The results showed that knockdown of β1-integrin enhanced renin production in As4.1 cells to about twice that in controls. Knockdown of Cx40, a putative pressoreceptor, also increased the renin production to the same extent, suggesting that these renin production levels represent an uncontrollable state of renin production of juxtaglomerular cells (Fig. 4).