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. 2012 Mar 15;27(8):3169–3175. doi: 10.1093/ndt/gfs033

ARB protects podocytes from HIV-1 nephropathy independently of podocyte AT1

Akihiro Shimizu 1,2, Jianyong Zhong 3,4,5, Yoichi Miyazaki 2, Tatsuo Hosoya 2, Iekuni Ichikawa 6,7, Taiji Matsusaka 1,3,✉
PMCID: PMC3408939  PMID: 22422866

Abstract

Background

Angiotensin I-converting enzyme inhibitors and angiotensin receptor blockers protect podocytes more effectively than other anti-hypertensive drugs. Transgenic rats overexpressing angiotensin II Type 1 (AT1) receptor selectively in podocytes have been shown to develop glomerulosclerosis. The prevailing hypothesis is that angiotensin II has a capacity of directly acting on the AT1 receptor of podocytes to induce injury. We therefore investigated the mechanism of reno-protective effect of AT1 receptor in a mouse model of HIV-1 nephropathy.

Methods

We generated transgenic mice carrying the HIV-1 gene (control/HIV-1) or both HIV-1 gene and podocyte-selectively nullified AT1 gene (AT1KO/HIV-1). In these mice, we measured urinary protein or albumin excretion and performed histological analysis.

Results

At 8 months of age, AT1KO/HIV-1 (n = 13) and control/HIV-1 (n = 15) mice were statistically indistinguishable with respect to urinary albumin/creatinine ratio (median 2.5 versus 9.1 mg/mg), glomerulosclerosis (median 0.63 versus 0.45 on 0–4 scale) and downregulation of nephrin (median 6.90 versus 7.02 on 0–8 scale). In contrast to the observed lack of effect of podocyte-specific AT1KO, systemic AT1 inhibition with AT1 blocker (ARB) significantly attenuated proteinuria and glomerulosclerosis in HIV-1 mice.

Conclusion

These results indicate that the protective effect of ARB is mediated through its receptors on cells other than podocytes, such as efferent arteriolar smooth muscle cells.

Keywords: angiotensin II, conditional gene targeting, glomerulosclerosis, HIV-1-associated nephropathy

Introduction

The histology of end-stage renal failure is typically characterized by glomerulosclerosis. A large number of studies showed that podocyte injury leads to glomerulosclerosis [1–5]. This notion is also supported by animal models of inducible podocyte-specific injury engineered by us and others [6, 7]. Accumulating evidence indicates that inhibition of angiotensin (Ang) II by Ang I-converting enzyme (ACE) inhibitors or blockers for the Ang II Type 1 (AT1) receptor (ARB) has salutary effects in attenuating progression of chronic renal failure in various forms of human kidney diseases and their corresponding experimental models. Moreover, ACE inhibitors and angiotensin receptor blockers protect podocytes more effectively than other anti-hypertensive drugs, indicating that mechanisms independent of systemic blood pressure are involved in the protection.

Among the several mechanisms thus far proposed, the prevailing hypothesis is that Ang II has a capacity of directly acting on the AT1 receptor of podocytes to induce injury. Cultured podocytes, as well as podocytes in isolated glomeruli, express AT1 protein [8–11]. In cultured cells, Ang II added to the medium increases α3(IV) collagen and vascular endothelial growth factor, decreases nephrin, heparan sulfate proteoglycans and α-actinin-4, augments reactive oxygen species production and induces redistribution of zona occludens-1 and reorganization of F-actin cytoskeleton [8, 12–17]. In an in vivo study, transgenic rats overexpressing AT1 receptor selectively in podocytes were shown to develop glomerulosclerosis [18]. In this study, podocytes constitutively express high level of AT1 without feedback regulation. The study suggested that abnormal augmentation of Ang II action, which is presumed to occur with AT1 super-expression, leads to podocyte injury and subsequently glomerulosclerosis.

Does endogenous AT1 signaling within podocytes play a role in the progression of podocyte diseases, then? In this regard, our recent study with a gene knockout mouse model revealed that the findings are somewhat inconsistent with the notion of the role of podocyte AT1. Thus, pharmacological AT1 inhibition, but not podocyte-specific AT1 disruption, attenuated podocyte injury and glomerulosclerosis in an immunotoxin-inducible podocyte injury model (NEP25) [19]. At least in this model, therefore, the protective effect of Ang II inhibition on podocytes involves AT1 not of podocytes, but instead, of non-podocyte cells. It is conceivable, however, that although NEP25 mice closely simulate the pathologic processes shared by glomerulosclerosis of a variety of causes, the finding above is unique to this model since the primary insult is artificial at best, i.e. a recombinant immunotoxin.

In view of the need for verifying the role of podocyte AT1 in a setting where injury of podocytes and development of glomerulosclerosis occur as a result of naturally occurring insult, we used a transgenic strain that congenitally expresses HIV-1 regulatory and accessory genes including nef and vpr selectively in podocytes [20]. This model appears most intriguing, in that Ang II inhibition was shown to effectively attenuate podocyte injury caused by HIV-1 both in clinical and experimental settings [21–23].

Materials and methods

Adherence to guideline for animal experiments

The Animal Experimentation Committee of Tokai University School of Medicine approved the protocol, in accordance with the principles and procedures outlined in the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Podocyte-specific AT1 knockout in HIV-1 transgenic mice

The HIV-1 transgenic mice, as reported previously [20], express HIV-1 regulatory and accessory genes, including nef and vpr, selectively in podocytes. This line, originally generated in a mixed genetic background of C57BL/6 and DBA/2, was backcrossed with C57BL/6 more than ten times and mated with podocyte-specific AT1 knockout mouse line carrying Agtr1aloxP and Nephrin Cre [19]. Agtr1aloxP line was maintained on C57BL/6. Nephrin Cre line was originally generated in a mixed genetic background of C57BL/6 and DBA/2 and backcrossed with C57BL/6 more than five times.

Thirteen (five males and eight females) Agtr1aloxP/loxP/Cre(+)/HIV-1 mice (hereafter designated as AT1KO/HIV-1) and 15 (eight males and seven females) Agtr1aloxP/loxP/Cre(−)/HIV-1 (Control HIV-1) mice were followed up to 8 months of age. In these mice, 24-h urine was collected using metabolic cages before sacrifice.

Experiments with ARB

For this experiment, we used transgenic offspring at N1 generation obtained by mating between the male founder transgenic mouse (HIV13) and wild-type female FVB/N mice. A total of 14 transgenic mice were treated daily with subcutaneous injection of either losartan (30 mg/kg BW/day) (n = 7, five male and two females) or saline (n = 7, five males and two females) starting at 3 days after birth until 4 weeks of age. At 4 weeks of age, 24 h urine was collected using metabolic cages, and their kidney histology was analyzed.

Histology and morphological analysis

All staining was performed in 2-μm paraffin sections. For nephrin staining, deparaffinized sections were autoclaved at 120°C for 10 min in citrate buffer and treated with 0.03% hydrogen peroxide in methanol. The sections were then rinsed with phosphate-buffered saline and incubated with polyclonal guinea pig anti-nephrin antibody (GP-N2; Progen, Heidelberg, Germany) diluted 200-fold overnight at 4°C. Sections were incubated with biotin anti-guinea pig antibody, then with avidin-biotinylated peroxidase complex (Vector Laboratories), and signal was visualized by diaminobenzidine.

Glomerulosclerosis was semiquantified in PAS-stained sections using 0–4 scales, which represent glomerulosclerosis involving 0, 1–25, 26–50, 51–75 or >76% of the glomerulus, respectively. Mesangial expansion without adhesion was not regarded as glomerulosclerosis. The average of scores of all glomeruli on a section was calculated for each mouse and defined as glomerulosclerosis index.

For semiquantification of nephrin staining, each quadrant of a glomerulus was scored as 0 (no staining), 1 (diminished) or 2(normal), with total glomerular score ranging from 0 (complete loss) to 8 (normal). The average of scores of all glomeruli on a section was calculated for each mouse and defined as nephrin index.

Blood pressure

Conscious systolic blood pressure was measured by tail-cuff method using MK-2000 (Muromachi Kikai, Tokyo, Japan).

Urinalysis

Concentrations of total protein, creatinine and albumin in the urine were determined by the pyrogallol red, enzymatic methods and turbidimetric immunoassay, respectively, in an outside laboratory (SRL, Tokyo, Japan).

Statistical analysis

Urinary protein/creatinine and albumin/creatinine ratios (ACRs) showed logarithmic normal distribution. For these data, geographic mean and 95% confidence intervals are reported in the text. To compare these data between two groups, t-test was used after logarithmic transformation.

To compare sclerosis and nephrin staining indices between two groups, non-parametric Mann–Whitney's U-test was performed using KyPlot software. These data are presented as median.

Values were regarded as significant at P < 0.05.

Results

Effect of podocyte-specific inactivation of AT1 on HIV-1 nephropathy

To examine whether inhibition of AT1 on podocytes can protect podocyte diseases, we generated podocyte-specific AT1KO/HIV-1 mice and compared those with control/HIV-1 mice. To minimize the influence of genetic background, we performed mating on C57BL/6 genetic background on which HIV-1 mice showed mild and slowly progressive focal segmental glomerulosclerosis.

We have shown earlier that the AT1A gene, the major subtype of AT1 receptor, was homozygously nullified in 87% of podocytes in podocyte-specific AT1 knockout mice and that AT1B, the minor AT1 receptor, was negligible in the mouse glomerulus and not upregulated upon podocyte AT1A knockout [19].

At 8 months of age, urinary ACR in control/HIV-1 mice was, on average, 9.1 mg/mg, which was not statistically different from that in AT1KO/HIV-1 mice, 2.5 mg/mg (Figure 1A). Systolic blood pressure was, on average 106.9 ± 10.2 mmHg in AT1KO/HIV-1 mice, not significantly different from that in control/HIV-1 mice, 98.8 ± 4.5.

Fig. 1.

Fig. 1.

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Effect of podocyte-specific AT1 knockout on the nephropathy of HIV-1 mice. Control HIV-1 mice (n = 15, open circles) and AT1KO/HIV-1 mice (n = 13, closed circles) at 8 months of age showed no significant difference in urinary ACR (A), glomerulosclerosis (B) and downregulation of nephrin (C). The horizontal bars represent median and interquartile range.

The degree of glomerular injury was also similar in both mouse groups at 8 months of age (Figure 2). AT1KO/HIV-1 mice had median glomerulosclerosis index of 0.63, which was statistically indistinguishable from that of control/HIV-1 mice 0.45 (Figure 1B). Both types of mice had glomeruli with similarly diminished nephrin staining (median nephrin index, AT1KO/HIV-1 mice 6.90; control/HIV-1 mice 7.02) (Figure 1C). Thus, podocyte-specific inactivation of AT1 showed no impact on the progression of glomerular injury in this mouse model.

Fig. 2.

Fig. 2.

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Representative pictures of HIV-1 mice with or without podocyte-specific AT1 knockout. Both control HIV-1 and pododcyte-AT1KO/HIV-1 mice showed similar degrees of glomerulosclerosis [(A) and (B), arrows] and downregulation of nephrin [(C) and (D), arrows]. (A) and (C), (B) and (D) are from adjacent sections (×200).

ARB-attenuated podocyte injury in HIV-1 transgenic mice

We verified whether losartan, an ARB, can protect against glomerular injury in this model. In this experiment, we used HIV-1 mice with nephrosis-prone FVB/N genetic background. We treated HIV-1 transgenic mice daily with subcutaneous injection of either losartan (30 mg/kg BW/day, n = 7) or saline (n = 7) from 3 days until 4 weeks of age.

As reported previously, all HIV-1 mice without ARB treatment rapidly developed severe nephropathy. At 4 weeks of age, urinary protein/creatinine ratio in non-treated HIV-1 mice was, on average, of 55.3 mg/mg. ARB treatment significantly decreased urinary protein/creatinine ratio, on average, to 23.2 mg/mg (Figure 3A). Systolic blood pressure in HIV-1 mice treated with ARB was, on average, 110.8 ± 9.8 mmHg, which was significantly lower than that in HIV-1 mice without ARB, 136.6 ± 9.6. The observed reduction in blood pressure by ARB is consistent with the concurrently observed increased renin staining in the ARB-treated HIV-1 mice (Supplementary Figure S2). Histologically, HIV-1 mice without ARB showed prominent vacuolar degeneration of visceral and parietal glomerular epithelial cells, expansion of mesangial cells, adhesion and glomerulosclerosis along with microcystic tubular dilatation and tubular casts. In contrast, these lesions were remarkably attenuated in HIV-1 mice with ARB except for mesangial expansion, which was likewise observed in wild-type littermates that had been simultaneously treated with ARB (Figure 4). Glomerular sclerosis was semiquantified using a 0 (no sclerosis) to 4 (global sclerosis) scale. The median glomerulosclerosis index was 1.21 in the non-treated group contrasting to a markedly lower value in the ARB-treated group 0.16 (Figure 3B).

Fig. 3.

Fig. 3.

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ARB-attenuated nephropathy in HIV-1 mice. (A) HIV-1 mice were treated with ARB (+ARB) or left untreated (−ARB) from 3 days of age. HIV-1 mice with ARB treatment (n = 7, closed circles) showed significantly attenuated urinary protein/creatinine ratio (U-Prot/Cr) (A), glomerulosclerosis (B) and downregulation of nephrin (C) when compared to control mice without ARB (n = 7, open circles). Wild-type mice with or without ARB treatment showed no glomerulosclerosis and normal nephrin staining pattern (Supplementary Figure S1), with sclerosis index 0 and nephrin index 8 (not shown). The horizontal bars represent the median and interquartile range. *Represents P < 0.05 compared with −ARB.

Fig. 4.

Fig. 4.

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Representative pictures from HIV-1 mice with or without ARB. HIV-1 mice without ARB treatment show severe glomerulosclerosis [(A), arrows] and marked decrease in nephrin staining [(C), arrows]. In contrast, HIV-1 mice with ARB treatment showed no glomerulosclerosis (B) and almost normal nephrin staining (D). Of note, ARB-treated mice showed mesangial expansion. Similar patterns were also observed in wild-type mice treated with ARB (Supplementary Figure S1) and whole body AT1 knockout mice, indicating that the mesangial expansion was caused by inhibition of AT1 from neonatal period. (A) and (C), (B) and (D) are from adjacent sections (×200).

The HIV-1 mice without ARB showed remarkable downregulation of nephrin staining. The median nephrin staining index in the non-treated group was 3.87. In contrast, HIV-1 mice with ARB treatment showed remarkably well-protected nephrin staining with median nephrin staining index 7.34 (Figure 3C). Similarly, ARB treatment improved synaptopodin staining (data not shown).

As previously reported, in glomeruli that were not fully injured in non-treated HIV-1 mice, podocytes often showed immature cuboidal morphology, localizing only on the surface of the glomerulus. These were contrasting to flat-shaped podocytes of wild-type littermates, which were invaginated inward. Of note, in ARB-treated HIV-1 mice, the immature morphology and the abnormal configuration of podocytes were not normalized, although podocyte were remarkably well preserved (data not shown).

Discussion

The present study revealed that systemic treatment with ARB efficiently attenuated podocyte injury and glomerulosclerosis in HIV transgenic mice with nephropathy-prone genetic background, whereas podocyte-specific AT1 knockout had no impact even on the mild nephropathy of mice with disease-resistant genetic background. Taken together, we concluded that protection of podocytes by ARB in HIV-1 model is independent of AT1 receptor on podocytes.

These observations are in parallel with those in our recent study on AT1 KO/NEP25 mice [19]. NEP25 mice express human (h) CD25 (i.e. Interleukin-2 receptor) selectively on podocytes [24]. After injection of hCD25-targeted immunotoxin, LMB2 [25], AT1KO/NEP25 mice showed proteinuria and glomerulosclerosis in degrees that were similar to those of control/NEP25 mice. In contrast, administration of ARB and ACE inhibitor, but not hydralazine, effectively attenuated podocyte injury and glomerulosclerosis in NEP25 mice. Of note, LMB2 contains a portion of pseudomonas exotoxin and universally inhibits protein synthesis by inactivating elongation factor 2 in targeted podocytes of NEP25 mice [25–27]. It was, therefore, conceivable that in the above experiments, AT1 protein itself or signaling molecules may already be suppressed in control NEP25 mice, so that knockout of AT1 gene may have had no additional impact although this possibility should have also precluded the effectiveness of ARB.

The HIV-1 mice used in the present study express HIV-1 accessory and regulatory genes selectively on podocytes. Previous studies by us and others indicate that HIV-1 gene products, VPR and NEF, cooperatively damage podocytes [26–28]. Many studies reported that podocytes proliferate in human HIV-1-associated nephropathy and its experimental models [29–31]. However, in our HIV-1 mice, most proliferating cells within the Bowman's capsule are parietal epithelial cells and the re-emergence of podocytes in the cell cycle is extremely rare and, therefore, unlikely to be the major pathogenic mechanism. Instead, the morphological feature suggests that HIV-1 gene product(s) disturb cytoskeleton of podocytes. Thus, before development of glomerulosclerosis, podocytes in HIV-1 mice with FVB/N background showed immature cuboidal morphology with less extended primary processes. Indeed, HIV-1 gene product NEF has been shown to decrease actin stress fibers and increase lamellipodia via interaction with DIP and VAV2 [32]. Interestingly, ARB treatment effectively attenuated the development glomerulosclerosis and podocyte injury in the present study, but it did not normalize the immaturity in podocyte morphology. This observation, together with the lack of protection by podocyte-AT1KO, suggests that ARB has no impact on molecular insult within podocytes, but instead, indicates that the major mechanism of podocyte protection by ARB is ascribed to its effect on the AT1 on non-podocyte cells.

The present study points to the need for research into non-podocyte cells for the primary target for the Ang II to act as a podocyte toxin or podocyte injury enhancer. The fact that the effect of ARB is independent of systemic blood pressure will rule out the systemic vascular bed also [21]. Currently, our technological approach is highly limited for pinpointing the target cell since the cell-specific gene targeting technology, as one used in the present study, cannot be applied to most non-podocyte cells, e.g. glomerular endothelial cells, mesangial cells and efferent arteriolar smooth muscle cells. Under this circumstance, perhaps the efferent arteriole may be the most favored candidate in the nephrology field. Several earlier studies in rats showed that ARB dilates the efferent arterioles and reduces glomerular capillary pressure, which was often associated with protection of the glomerulus. In this regard, we have recently published a paper demonstrating that effective nullification of net ultrafiltration pressure by ureteral obstruction almost completely protects podocytes in the HIV-1 nephropathy model as well as in immunotoxin-inducible podocyte injury model [33]. This was somewhat surprising because obstructed kidneys are exposed to upregulation of local Ang II, and develop ARB-sensitive interstitial fibrosis. The results of ureteral obstruction point to the highly sensitive nature of podocytes to net ultrafiltration pressure. Ureteral obstruction nullifies net ultrafiltration pressure by increasing hydraulic pressure in the Bowman's space. Our current study and the published obstruction study collectively support the notion that ARB, through its well-known effect to lower glomerular pressure, hence net ultrafiltration pressure, protects podocytes from injury. The glomerular pressure per se may not be the final readout of its toxic effect. Serum proteins filtered into Bowman's space can be toxic to podocytes [34, 35] and their quantity is determined by the level of glomerular pressure, which affects the permselectivity of the capillary wall [36]. In that, the effect of Ang II on the efferent arteriole determines the amount of serum protein traffic into Bowman's space.

As another potential mechanism of podocyte protection by ARB, one may think ARB may suppress aldosterone. However, ARB treatment, or even angiotensinogen knockout, cannot suppress aldosterone because of alternative regulation by potassium [37, 38]. Considering this notion, it is unlikely that suppression of aldosterone is the major mechanism underlying podocyte protection by ARB.

Importantly, although podocyte damage is the key step leading to renal failure, the present study indicates that the target of therapeutic measure may not necessarily be the podocyte per se. Instead, therapies targeting on non-podocyte cells can profoundly affect the process of glomerulosclerosis.

Supplementary data

Supplementary data is available online at http://ndt.oxfordjournals.org.

Supplementary Data
supp_27_8_3169__index.html (911B, html)

Acknowledgements

This study was supported by National Institutes of Health (NIH) grants DK37868 and DK44757, the Research for the Future Program and Grant-in Aid for Scientific Research of Japan Society for the Promotion of Science, MEXT, HAITEKU, the Jikei University Research Fund and in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Parts of this study were presented in abstract form at the annual meetings of the American Society of Nephrology in 2003. We thank Ms Shiho Imai, Ms Naoko Sasaoka, Ms Suguri Niwa and Ms Chie Sakurai for technical assistance.

I.I. received research funds from Daiichi Sankyo Co., Ltd. T.M. received a research fund from Chugai Pharmaceutical Co. Ltd.

Conflict of interest statement. None declared.

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Supplementary Materials

Supplementary Data
supp_27_8_3169__index.html (911B, html)
supp_gfs033_gfs033_figS1.tif (11.7MB, tif)
supp_gfs033_gfs033_figS2.tif (11.4MB, tif)

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