Cpxm2 as a novel candidate for cardiac hypertrophy and failure in hypertension.

Treatment of hypertension-mediated cardiac damage with left ventricular (LV) hypertrophy (LVH) and heart failure remains challenging. To identify novel targets, we performed comparative transcriptome analysis between genetic models derived from stroke-prone spontaneously hypertensive rats (SHRSP). Here, we identified carboxypeptidase X 2 (Cpxm2) as a genetic locus affecting LV mass. Analysis of isolated rat cardiomyocytes and cardiofibroblasts indicated Cpxm2 expression and intrinsic upregulation in genetic hypertension. Immunostaining indicated that CPXM2 associates with the t-tubule network of cardiomyocytes. The functional role of Cpxm2 was further investigated in Cpxm2-deficient (KO) and wild-type (WT) mice exposed to deoxycorticosterone acetate (DOCA). WT and KO animals developed severe and similar systolic hypertension in response to DOCA. WT mice developed severe LV damage, including increases in LV masses and diameters, impairment of LV systolic and diastolic function and reduced ejection fraction. These changes were significantly ameliorated or even normalized (i.e., ejection fraction) in KO-DOCA animals. LV transcriptome analysis showed a molecular cardiac hypertrophy/remodeling signature in WT but not KO mice with significant upregulation of 1234 transcripts, including Cpxm2, in response to DOCA. Analysis of endomyocardial biopsies from patients with cardiac hypertrophy indicated significant upregulation of CPXM2 expression. These data support further translational investigation of CPXM2.

Analysis of the genomic architecture of a complex trait locus in hypertensive rat models links Tmem63c to kidney damage.

Unraveling the genetic susceptibility of complex diseases such as chronic kidney disease remains challenging. Here, we used inbred rat models of kidney damage associated with elevated blood pressure for the comprehensive analysis of a major albuminuria susceptibility locus detected in these models. We characterized its genomic architecture by congenic substitution mapping, targeted next-generation sequencing, and compartment-specific RNA sequencing analysis in isolated glomeruli. This led to prioritization of transmembrane protein Tmem63c as a novel potential target. Tmem63c is differentially expressed in glomeruli of allele-specific rat models during onset of albuminuria. Patients with focal segmental glomerulosclerosis exhibited specific TMEM63C loss in podocytes. Functional analysis in zebrafish revealed a role for tmem63c in mediating the glomerular filtration barrier function. Our data demonstrate that integrative analysis of the genomic architecture of a complex trait locus is a powerful tool for identification of new targets such as Tmem63c for further translational investigation.

Regulation of podoplanin expression by microRNA-29b associates with its antiapoptotic effect in angiotensin II-induced injury of human podocytes.

BACKGROUND: Angiotensin (Ang)II is involved in induction of proteinuria, renal injury, and apoptosis and thus a major contributor to the development of chronic kidney disease. Podocytes are of major importance for the pathogenesis of several kidney diseases. Decrease of podoplanin (PDPN) in podocytes and podocyte loss has been associated with the development of proteinuria. Little is known about the regulation and biological function of PDPN in podocytes and its role in AngII-mediated kidney damage. Here, we determined the influence of AngII on the expression of PDPN, microRNA (miRNA)-29b and miRNA-497 in human podocytes. Further, we analyzed the impact of small interfering RNA-mediated downregulation of PDPN on AngII-induced apoptosis and viability. Moreover, we characterized the role of miRNA-29b and miRNA-497 in expression regulation of PDPN. METHODS: Cell viability and apoptosis were determined by functional assays. Expression analyses were done via Real-Time PCR and western blot analyses. Dual luciferase assay was performed to characterize miRNA-mediated expression control. RESULTS: AngII increased the expression of miRNA-29b and reduced PDPN. Small interfering RNA-mediated downregulation of PDPN increased proapoptotic caspase-3 activation and cytochrome C translocation, whereas cell viability and Akt phosphorylation were reduced in AngII-stimulated podocytes. In contrast to miRNA-497, transfection of cells with miRNA-29b mimics significantly decreased PDPN. Cotransfection of cells with miRNA-29b and a dual luciferase reporter vector decreased the luciferase activity compared with controls. CONCLUSION: These data demonstrate the posttranscriptional control of PDPN expression by miRNA-29b and support a role of PDPN as an antiapoptotic prosurvival factor in AngII-induced injury of human podocytes.

Novel candidate genes for impaired nephron development in a rat model with inherited nephron deficit and albuminuria.

Defects in nephrogenesis can have detrimental effects on cardiovascular and renal health in adult life. This is confirmed by observations in the Munich Wistar Frömter (MWF) rat that exhibits a congenital nephron deficit and renal failure with age. Here, we performed genome-wide transcriptome analysis in embryonic kidneys to identify candidate genes for the reduced nephron number in MWF. We compared MWF rats at embryonic day (E)15.5 with stage-matched spontaneously hypertensive rats (SHR) at E16. Microarray analysis revealed 311 transcripts representing 253 known genes with differential expression between MWF and SHR (fold change > +1.5 or < -1.5, respectively). Genes located on rat chromosome (RNO) 6 were of special interest because RNO6 carries genetic loci previously linked to the nephron deficit and renal damage in MWF. Differentially expressed genes located on RNO6 were further investigated by Real-time PCR including the late-stage of fetal kidney development, i.e. E19.0/E19.5, and week 4 of postnatal life when nephrogenesis is completed. Seven genes including Abcg5, Ab1-233, Efcab11, Fntb, Gpx2, Lrrn3, and Rtn1 were assigned on RNO6 and their differential expression was confirmed. Thus, we identified several genes that may act as crucial players in nephron development and are responsible for the nephron deficit in the MWF model.

Development of progressive albuminuria in male Munich Wistar Frömter rats is androgen dependent.

Munich Wistar Frömter (MWF) rats develop spontaneous albuminuria that is linked to autosomal genetic loci and inherit a nephron deficit in both female and male animals, respectively. However, albuminuria and kidney damage are clearly more pronounced in males. Here we tested whether androgens and the androgen receptor influence albuminuria in male MWF. We first demonstrated in a pilot study that orchiectomy (Ox) of male MWF led to a significant suppression of urinary albumin excretion (UAE), while continuous testosterone supplementation in MWF Ox led to UAE levels similar to sham-operated (Sham) MWF rats. Subsequently, we performed a comparative main study between male MWF and normal Wistar rats to evaluate the effect of the androgen receptor on UAE development in adult animals up to the age of 18 wk. MWF Sham developed a marked increase in UAE compared with Wistar Sham (48.30 ± 6.16 vs. 0.42 ± 0.08 mg/24 h, P < 0.0001). UAE was significantly lower in MWF Ox compared with MWF Sham (-55%, P < 0.0001). In MWF Ox animals supplemented with testosterone and treated with the androgen receptor antagonist flutamide (OxTF) UAE at 18 wk was even lower compared with MWF Ox (-71%, P < 0.01) and similar to age-matched female MWF. The mRNA expression of renal tubular injury markers Kim1 and NGAL was increased in MWF Sham compared with Wistar Sham (P < 0.0008, respectively) and expression decreased significantly in MWF OxTF (P < 0.0004, respectively). Thus, the sexual dimorphism in albuminuria development in MWF can be attributed to testosterone and the androgen receptor in male rats.