1University of Melbourne Departments of Medicine St Vincent's Hospital Fitzroy Melbourne Victoria Australia 2The Haartman Institute Division of Bacteriology and Immunology University of Helsinki Finland Email: gilbert{at}medstv.unimelb.edu.au
Sir,
Proteinuria is a cardinal manifestation of kidney disease directly contributing to its progression by inducing tubulointerstitial pathology [1]. The effectiveness of blockade of the reninangiotensin system (RAS) in slowing the progression may be, at least in part, due to the powerful anti-proteinuric actions of both angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs). However, the mechanisms whereby blockade of the RAS reduces the transglomerular passage of protein is multi-factorial and incompletely understood.
Recent studies have implicated the podocyte slit pore protein, nephrin, in the pathogenesis of acquired proteinuric diseases as well the Finnish type congenital nephrotic syndrome, NPHS1 [2]. Furthermore, in addition to showing a reduction in glomerular nephrin, several studies have also shown attenuation of these changes in both experimental and human kidney disease by RAS blockade [3,4]. These findings, in conjunction with the presence of functional receptors for angiotensin II on glomerular podocytes [5], suggest that angiotensin II may directly contribute to the pathogenesis of proteinuria by modulating nephrin expression. We therefore sought to examine the effects of angiotensin II infusion on nephrin expression in the in vivo setting.
Thirteen male SpragueDawley rats aged 13 weeks were randomly assigned to receive either normal saline or angiotensin II (dose 200 mg/kg/min) by osmotic mini-pump. Systolic blood pressure was measured in pre-warmed conscious rats by tail cuff plethysmography [6]. On day 9 of the study, animals were individually housed in metabolic cages. An aliquot of urine (5 ml) was collected from the 24-h urine sample and stored at -70°C for subsequent analysis of albumin. Following the completion of the 24-h period in the metabolic cages, animals were killed by lethal injection. Both kidneys were then excised and decapsulated. Glomeruli were isolated by serial sieving of the left kidney. Glomeruli were then frozen in liquid nitrogen and stored at -80°C for subsequent RNA extraction and determination of nephrin gene expression by real time PCR, as described previously [7]. The right kidney was fixed in neutral-buffered formalin and quantitative in situ hybridization (QISH) autoradiography performed, also as described previously [8].
Rats receiving angiotensin II by mini-pump developed hypertension in association with proteinuria (Table 1). Both real-time PCR and quantitative in situ hybridization demonstrated a significant increase in nephrin gene expression in angiotensin II infused animals compared with control animals (Table 1, Figure 1).
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The findings of the present study suggest that despite increased expression of nephrin with blockade of the RAS [4,7,11,12], angiotensin II does not directly decrease nephrin expression. Indeed, rather than the expected decrease in nephrin mRNA with angiotensin II, the present study, using two different methods of assessment, documented an 2-fold increase in gene expression in response to continuous infusion. The mechanisms whereby angiotensin II leads to increased nephrin expression are unknown. However, angiotensin II is a potent activator of protein kinase C [13], a key intracellular signalling system in the regulation of nephrin expression [14].
We therefore suggest that the previously documented effects of ACE inhibition and ARBs reflect the actions of these agents in preserving renal structure and function, rather than a direct effect of RAS blockade on podocyte nephrin transcription. We further suggest caution in interpreting the findings of studies reporting modulation of nephrin expression in acquired renal disease and the effects of therapeutic intervention.
Acknowledgments
This work was supported in part by grants from the National Health and Medical Research Council of Australia and the Juvenile Diabetes Research Foundation International. Darren J. Kelly is a recipient of a career development award from Juvenile Diabetes Research Foundation. The authors are indebted to Mariana Pacheco for her excellent technical assistance.
Conflict of interest statement. None declared.
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