Hyperglycemia and Renin-Dependent Hypertension Synergize to Model Diabetic Nephropathy
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BRIEF COMMUNICATION www.jasn.org Hyperglycemia and Renin-Dependent Hypertension Synergize to Model Diabetic Nephropathy † † † Bryan R. Conway,* Jillian Rennie,* Matthew A. Bailey, Donald R. Dunbar, † † † Jonathan R. Manning, Christopher O. Bellamy, Jeremy Hughes,* and John J. Mullins* *MRC Centre for Inflammation Research and †University of Edinburgh/British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, Scotland ABSTRACT Rodent models exhibit only the earliest features of human diabetic nephropathy, in a number of hypertensive rodent which limits our ability to investigate new therapies. Hypertension is a prerequisite models.8–10 for advanced diabetic nephropathy in humans, so its rarity in typical rodent models The renin-dependent hypertensive may partly explain their resistance to nephropathy. Here, we used the Cyp1a1mRen2 (mRen-2)27 rat has been extensively rat, in which the murine renin-2 gene is incorporated under the Cytochrome P4501a1 use to model DN9; however, it is limited promoter. In this transgenic strain, administration of low-dose dietary indole-3-carbinol by the development of malignant phase induces moderate hypertension. In the absence of hypertension, streptozotocin- hypertension.11,12 To determine how induced diabetes resulted in a 14-fold increase in albuminuria but only mild changes hyperglycemia and hypertension inter- in histology and gene expression despite 28 weeks of marked hyperglycemia. In the act at a molecular level, we used the presence of induced hypertension, hyperglycemia resulted in a 500-fold increase in Cyp1a1mRen2 rat, which harbors the albuminuria, marked glomerulosclerosis and tubulointerstitial fibrosis, and induction of murine Ren2 cDNA under the control many of the same pathways that are upregulated in the tubulointerstitium in human of the cytochrome P4501a1 promoter,13 diabetic nephropathy. In conclusion, although induction of diabetes alone in rodents such that hypertension may be induced has limited utility to model human diabetic nephropathy, renin-dependent hyperten- by dietary supplementation with indole- sion and hyperglycemia synergize to recapitulate many of the clinical, histological, and 3-carbinol (I-3-C). Unlike the constitu- gene expression changes observed in humans. tive (mRen-2)27 rat, hypertension can be induced after the onset of diabetes to J Am Soc Nephrol 23: ccc–ccc, 2012. doi: 10.1681/ASN.2011060577 mimic the natural history of human DN and the I-3-C dose may be titrated to avoid malignant phase hypertension. Diabetic nephropathy (DN) is the single artery stenosis there may be no evidence Cyp1a1mRen2 rats were allocated largest cause of end stage renal failure in of nephropathy in the kidney down- into four groups: controls (n=6), strep- the Western world.1 Although the devel- stream of the stenosis, despite severe ne- tozotocin-induced diabetes (DM; n=6), opment of novel therapeutic strategies phropathy in the contralateral kidney, I-3-C–induced hypertension (HTN; for DN remains a research priority, we suggesting that transmission of systemic n=7), and combined hypertension and are constrained by the fact that current hypertension to the diabetic glomerulus rodent models replicate only the earliest is a prerequisite for the development of stages of human DN.2 One potential ex- advanced nephropathy.6,7 Received June 15, 2011. Accepted October 13, 2011. planation for the resistance of rodents to Despite the crucial role of hyperten- DN is that they tend not to develop hy- sion in the pathogenesis of DN, there is J.H. and J.J.M. contributed equally to this work. pertension, which is critical for progres- a paucity of data regarding how high Published online ahead of print. Publication date sive DN in humans. Abnormalities in BP, BP and hyperglycemia interact at a mo- available at www.jasn.org. such as loss of nocturnal dipping, occur lecular level to promote nephropathy. Correspondence: Dr. Bryan Conway, Centre for early in the course of human DN3 and Whereas it is difficult to dissect the rel- Inflammation Research, Queen’s Medical Research rigorous BP control is at least as effective ative contribution of hypertension and Institute, University of Edinburgh, 47 Little France Crescent, Room W3.06, Edinburgh EH16 4TJ, UK. as glycemic control in retarding disease diabetes in humans, rodent studies may Email: [email protected] progression.4,5 Indeed, in patients with be informative as hyperglycemia and high Copyright © 2012 by the American Society of diabetes and co-existing unilateral renal BP synergize to promote nephropathy Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012 ISSN : 1046-6673/2303-ccc 1 BRIEF COMMUNICATION www.jasn.org diabetes (DN+HTN; n=8). During the There was very mild histological in- Tubulointerstitial fibrosis (TIF) and subsequent 28 weeks, blood sugar levels jury in the DM group; however, induc- inflammation are key components in the were 20–30 mM in both diabetic groups tion of hypertension alone promoted pathogenesis of DN; indeed, the severity with no significant difference between FSGS and a nonsignificant increase in of TIF more accurately predicts progno- the DM and DM+HTN animals (Figure the glomerulosclerosis index (GSI; Fig- sis than the glomerular findings.14 The 1A). Dietary I-3-C induced an equiva- ure 2, A and B). Concurrent diabetes and absence of overt TIF in rodent models of lent increase in tail-cuff BP in both hy- hypertension significantly increased the DN compromises their ability to effec- pertensive groups compared with their GSI compared with all other groups tively model human DN. Indeed, even in nonhypertensive counterparts (Figure (Figure 2B) and resulted in the develop- the endothelial nitric oxide synthase 1B). The tail-cuff readings were consistent ment of intraglomerular fibrin caps, knockout mouse, which develops mod- with those obtained by arterial cannula- which were rarely observed with either erate hypertension and significant glo- tion under terminal anaesthesia (mean DM or HTN alone but are typical of hu- merular pathology and is arguably the arterial pressure of 12762.3, 13662.8, man DN (Figure 2, A and C). Impor- most convincing model of DN to date, 18166.4, and 16968.7 mmHg in con- tantly, there was no histological evidence there is scant evidence of TIF.15,16 As trols, DM, HTN, and DM+HTN animals, of malignant phase hypertension, such anticipated, there was no evidence of respectively). as onion-skinning of the renal arterioles TIF after induction of diabetes alone; DM animals exhibited a modest in- in either hypertensive group. Only the however, overt TIF developed in the crease in albuminuria, with a 14-fold DM+HTN rats had a significant increase DM+HTN animals as indicated by a sig- higher median albumin/creatinine ratio in mesangial cell activation as indicated nificant increase in collagen deposition than that of controls at 28 weeks, equiv- by a-smooth muscle actin (a-SMA) (Figure 3, A and C) and myofibroblast alent to microalbuminuric levels in staining (Figure 2D). There was an in- activation (Figure 3, B and D). The in- humans (Figure 1C). Hypertension and crease in glomerular macrophage infil- nate immune system plays a major role diabetes synergized to promote albu- tration in all of the intervention groups, in the pathogenesis of DN,17 and al- minuria, such that by 28 weeks the me- which reached significance in the DM+ though this was not activated by diabetes dian albumin/creatinine ratio in the HTN animals (Figure 2E). Few glomer- alone, marked macrophage infiltration DM+HTN group was 500-fold higher ular lymphocytes were observed with no was observed in the tubulointerstitium than controls and significantly greater significant differences between the of both hypertensive groups (Figure than that in either the DM or HTN groups. groups. 3E). The role of the adaptive immune system in DN is less well characterized; however, tubulointerstitial T cell and B cell infiltration is observed in human DN18 and T cells may be pathogenic in rodent DN.19 There was an increase in tubulointerstitial T lymphocytes in the hypertensive animals, which was not ev- ident with diabetes alone (Figure 3F). In addition, focal B cell aggregates were observed solely in the DM+HTN group, often adjacent to blood vessels (Figure 3G). Lymphocyte recruitment may be mediated by the increase in chemokines and chemokine receptors observed pre- dominantly in the DM+HTN group (Supplemental Table 1). To determine the molecular signature of the interaction between hypertension and hyperglycemia we performed micro- array analysis on whole kidney tissue (n=4 per group). Remarkably, despite prolonged severe hyperglycemia in the Figure 1. Diabetes and hypertension synergise to promote albuminuria. (A) Mean (6SD) DM group, only 8 and 15 genes were sig- fi early morning nonfasting blood sugar level, (B) mean (6SD) tail-cuff systolic BP, and (C) ni cantly upregulated and downregu- median (interquartile range) albumin/creatinine ratio in the four groups of rats over the lated (corrected P,0.01), respectively, 28-week course of the experiment. ***P,0.001 versus control; #P,0.05, ##P,0.01, and versus controls. Indeed, the vast major- ###P,0.001 versus diabetic alone; $P,0.05 and $$P,0.01 versus HTN alone. ity of genes were dysregulated only by 2 Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc,2012 www.jasn.org BRIEF COMMUNICATION Figure 1), the antigen presenting cell- mediated regulation of the cell cycle (Sup- plemental Figure 2) and an extracellular matrix gene network (Supplemental Figure 3). It is, however, worth noting that because the microarray was performed on terminal tissue samples, many of the changes in gene expression will be sec- ondary to the presence of an inflamma- tory cell infiltrate or to modification of the intrinsic cells due to anchorage to a scarred extracellular matrix, rather than reflect the primary causal pathways of hyperglycemic and hypertensive dam- age.