Angiotensin II-induced proteinuria and expression of the podocyte slit pore membrane protein, nephrin

Robyn G. Langham1, Darren J. Kelly1, Alison J. Cox1, Renae M. Gow1, Harry Holthofer2 and Richard E. Gilbert1

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 renin–angiotensin 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 Sprague–Dawley 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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Table 1. Clinical characteristics of rats in study 1

 


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Fig. 1. In situ hybridization autoradiograph of nephrin mRNA. Intense, punctate hybridization was noted in the renal cortex consistent with an exclusively glomerular pattern of distribution. A statistically significant increase in the intensity of gene expression was noted between animals receiving saline (left) and those treated with angiotensin II (right). Magnification x10.

 
A number of mechanisms have been shown to underlie the pathogenesis of angiotensin II effects on the transglomerular passage of protein. These include modulation of efferent arteriolar tone, intraglomerular pressure and glomerular plasma flow as well as changes in the ultrafiltration co-efficient and size-dependent barrier functions [9,10]. Whilst abrogation of the loss of nephrin expression by RAS blockade has been demonstrated in the long-term injury of acquired proteinuric renal disease, it is unknown whether this represents a direct effect of angiotensin II on the podocyte or a secondary effect in preserving the structural integrity of the glomerulus. Indeed, our group has recently shown that in diabetes, the long-term structural changes, rather than short-term effects of hyperglycaemia were associated with the reduction in nephrin in that disease [7].

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.

References

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  4. Langham RG, Kelly DJ, Cox AJ et al. Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects of angiotensin converting enzyme inhibition. Diabetologia 2002;45: 1572–1576[CrossRef][ISI][Medline]
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  8. Kelly DJ, Gilbert RE, Cox AJ, Soulis T, Jerums G, Cooper ME. Aminoguanidine ameliorates overexpression of prosclerotic growth factors and collagen deposition in experimental diabetic nephropathy. J Am Soc Nephrol 2001;12: 2098–2107[Abstract/Free Full Text]
  9. Remuzzi A, Perico N, Sangalli F et al. ACE inhibition and ANG II receptor blockade improve glomerular size-selectivity in IgA nephropathy. Am J Physiol 1999;45: F457–F466
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  11. Bonnet F, Cooper ME, Kawachi H, Allen TJ, Boner G, Cao Z. Irbesartan normalises the deficiency in glomerular nephrin expression in a model of diabetes and hypertension. Diabetologia 2001;44: 874–877[CrossRef][ISI][Medline]
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  14. Wang SX, Mene P, Holthofer H. Nephrin mRNA regulation by protein kinase C. J Nephrol 2001;14: 98–103[CrossRef][ISI][Medline]




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