Increased renin production in mice with deletion of peroxisome proliferator-activated receptor-gamma in juxtaglomerular cells

Abstract

We recently found that endogenous (free fatty acids) and pharmacological (thiazolidinediones) agonists of nuclear receptor Peroxisome proliferator-activated receptor (PPAR)gamma stimulate renin transcription. In addition, the renin gene was identified as a direct target of PPARgamma. The mouse renin gene is regulated by PPARgamma through a distal enhancer direct repeat closely related to consensus PPAR response element (PPRE). In vitro studies demonstrated that PPARgamma knockdown stimulated PPRE-driven transcription. These data predicted that deficiency of PPARgamma would upregulate mouse renin expression. Consistent with these observations knockdown of PPARgamma increased the transcription of a reporter gene driven by the mouse renin PPRE-like motif in vitro. To study the impact of PPARgamma on renin production in vivo, we used a cre/lox system to generate double-transgenic mice with disrupted PPARgamma locus in renin-producing juxtaglomerular (JG) cells of the kidney (RC-PPARgamma(fl/fl) mice). We provide evidence that PPARgamma expression was effectively reduced in JG cells of RC-PPARgamma(fl/fl) mice. Fluorescent immunohistochemistry showed stronger renin signal in RC-PPARgamma(fl/fl) than in littermate control RC-PPARgamma(wt/wt) mice. Renin mRNA levels and plasma renin concentration in RC-PPARgamma(fl/fl) mice were almost 2-fold higher than in littermate controls. Arterial blood pressure and pressure control of renal vascular resistance, which play decisive roles in the regulation of renin production were indistinguishable between RC-PPARgamma(wt/wt) and RC-PPARgamma(fl/fl) mice. These data demonstrate that the JG-specific PPARgamma deficiency results in increased mouse renin expression in vivo thus corroborating earlier in vitro results. PPARgamma appears to be a relevant transcription factor for the control of renin gene in JG cells.

Figure 1

Effect of PPARγ knockdown on mouse renin PPRE-like driven transcription. Calu-6 cells were transfected with nontargeting siRNA as control (siControl) or with PPARγ sequence-specific siRNA (siPPARγ) and with the mPPREmPal3 construct. A. Efficacy of the PPARγ knockdown. Representative Western blots of protein extracts probed with anti-PPARγ or antiβ-actin (used for loading control) antibodies; B. Effect of PPARγ knockdown on mPPREmPal3 activity. RLA- relative luciferase activity, n=8 from two separate experiments. *P< 0.05.

Figure 2. Recombination of PPARγ allele and PPARγ expression in RC-PPARγ^wt/wt and RC-PPARγ^fl/fl mice

A. Qualitative PCR-based screening for PPARγ-recombined transcript in different organs of RC-PPARγ^fl/fl mice; B. PPARγ recombination in renal cortex and inner medulla total RNA samples isolated from RC-PPARγ^fl/fl mice; C. PPARγ mRNA is expressed in native JG cells of wildtype mice. Lane 1, molecular weight marker; lanes 2 to 7, RT-PCR of serial dilutions of RNA isolated from single JG cells with PPARγ-specific primers; lane 8, “minus” RT, the sample was “reverse-transcribed” in the absence of transcriptase before amplification (negative control); lane 9, water was reverse-transcribed and amplified (negative control); D. PPARγ mRNA levels in primary cultures of native JG cells isolated from RC-PPARγ^wt/wt (n=4) or RC-PPARγ^fl/fl (n=4) mice. PPARγ and ribosomal L32 (internal control) mRNA levels were quantified by real-time RT-PCR. *P < 0.05; E. Single-cell RT-PCR with renin (upper panel) or PPARγ (lower panel) specific primers of total RNA extracted from seven different JG cells of RC-PPARγ^fl/fl mice. The bands at the bottom of the panels represent primer dimers. St- length standard.

Figure 3

Renin expression in RC-PPARγ^wt/wt and RC-PPARγ^fl/fl mice. A. Co-staining for renin (green) and vascular smooth muscle α-actin (red). Dashed circles show the position of glomeruli (G), arrows indicate renin immunoreactivity; B. Renal renin mRNA levels in RC-PPARγ^wt/wt (n=6) and RC-PPARγ^fl/fl (n=8) mice. Renin and ribosomal L32 (internal control) mRNA levels were quantified by real-time RT-PCR. *P < 0.05; C. Plasma renin concentration (PRC) in RC-PPARγ^wt/wt (n=8) and RC-PPARγ^fl/fl (n=8) mice. *P < 0.05.

Figure 4

Hematocrits in RC-PPARγ^wt/wt and RC-PPARγ^fl/fl mice. Blood samples were obtained through mandibular bleeding. The data are means±SD, n=8 in each group.

Figure 5. Arterial blood pressure and renal perfusion parameters of RC-PPARγ^wt/wt and RC-PPARγ^fl/fl mice

A. Systolic blood pressure (SBP) measured by tail-cuff method. Each mark represents the value (average of 5 to 8 measurements) for a single animal (n=10 for each genotype); B, C. Pressure-dependent regulation of renin secretion rate (B.) and renal vascular resistance (C.) in isolated perfused kidneys of RC-PPARγ^wt/wt and RC-PPARγ^fl/fl mice, n= 3 and 4, respectively.