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HIV-1 Vpr inhibits cytokinesis in human proximal tubule cells
http://www.kidney-international.org
original article
& 2008 International Society of Nephrology
HIV-1 Vpr inhibits cytokinesis in human proximal tubule cells Paul E. Rosenstiel1, Tina Gruosso1, Audrey M. Letourneau1, Justin J. Chan1, Amanda LeBlanc1, Mohammad Husain1, Vesna Najfeld1, Vicente Planelles2, Vivette D. D’Agati3, Mary E. Klotman1 and Paul E. Klotman1 1
Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA; 2Department of Pathology, University of Utah, Salt Lake City, Utah, USA and 3Department of Pathology, Columbia University, New York, New York, USA
Transgenic mouse models of HIV-associated nephropathy (HIVAN) show that expression of HIV-1 genes in kidney cells produces collapsing focal segmental glomerulosclerosis and microcystic tubular disease typical of the human disease. HIV-1 vpr plays an important role in the glomerulosclerosis of HIVAN, especially when it is associated with nef expression in podocytes. Further, Vpr is reported to exacerbate tubular pathology. Here we determined effects of vpr expression on renal tubular epithelial cell function by transducing them with a pseudotyped lentivirus vector carrying HIV-1 vpr and control genes. Vpr expression in the cultured cells impaired cytokinesis causing cell enlargement and multinucleation. This profound in vitro phenotype caused us to reexamine the HIVAN mouse model and human HIVAN biopsies to see if similar changes occur in vivo. Both showed abundant hypertrophic tubule cells similar to the in vitro finding that represents a previously unappreciated aspect of the human disease. Additionally, multinucleated tubular cells were identified in the murine HIVAN model and increased chromosome number was detected in tubular cells of human HIVAN biopsies. Our study provides evidence of a new clinical phenotype in HIVAN that may result from the ability of Vpr to impair cytokinesis. Kidney International (2008) 74, 1049–1058; doi:10.1038/ki.2008.303; published online 9 July 2008 KEYWORDS: HIVAN; proximal tubule; HIV; Vpr; hypertrophy; cytokinesis
Correspondence: Paul E. Rosenstiel, Department of Medicine, Mount Sinai School of Medicine, Box 1118, One Gustave L. Levy Place, New York, New York 10029, USA. E-mail: [email protected] Received 12 November 2007; revised 19 March 2008; accepted 23 April 2008; published online 9 July 2008 Kidney International (2008) 74, 1049–1058
Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) was first reported in 1984 when a small group of Haitian and African-American HIV patients were found to have decreased renal function independent of other risk factors.1 Currently, HIVAN prevalence in the black HIVinfected population is between 3.5 and 12%,2,3 affecting an estimated 1–3 million people worldwide. HIVAN is associated with direct HIV infection of the glomerular and tubular compartments and subsequent viral genome transcription.4,5 The classical histopathologic features of HIVAN are collapsing focal segmental glomerulosclerosis and focal microcystic (3 normal diameter) dilation of the tubular segments with epithelial flattening.6–9 Apoptosis, hyperplasia, pseudocrescent formation, and several other pathologic findings have also been reported.9–12 Grossly, HIVAN kidneys are typically enlarged. Transgenic models expressing either single HIV-1 genes or deletion mutants of HIV-1 have been useful in mapping the HIV-1 genes pathogenic for HIVAN. HIV-1 nef has been associated with podocyte dedifferentiation and proliferation.13–15 HIV-1 vpr has been found to contribute to overall HIVAN pathogenesis in both podocytes and tubule epithelial cells.16 Mice expressing only vpr manifested some microcystic tubule dilation, whereas those expressing HIV without vpr did not show this phenotype. When HIVDvpr mice were crossed with vpr-expressing mice, the offspring showed widespread microcystic dilation typical of HIVAN. These data suggest that the tubular cystic phenotype involves vpr expression, but the exact mechanism of how Vpr contributes to HIVAN pathogenesis remains unknown. Although HIV-1 vpr function has not been investigated in renal epithelial cells, it has been extensively studied in other cell systems. In activated lymphocytes, and many cell lines, Vpr induces G2 arrest followed by apoptosis. Expression of vpr in other cell types, however, causes nuclear anomalies, including hyperploidy, micronuclei formation, and the evidence of impaired cytokinesis.17–21 Conversely, in macrophages, Vpr does not induce an abnormal nuclear phenotype or apoptosis, but may instead be important for HIV replication by facilitating nuclear import of the HIV preintegration complex.22,23 1049
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The precise mechanism(s) by which Vpr alters cell function is incompletely understood, but recently multiple studies have identified DDB1–Cullin 4A Associated Factor 1 (DCAF1) as an important Vpr binding partner.24–28 DCAF1 binds an E3 ubiquitin ligase complex likely functioning as a substrate specificity module. Vpr binding to DCAF1 is proposed to dysregulate and usurp the E3 complex. This binding appears to be the critical upstream event mediating Vpr-induced G2 arrest. Downstream, several events have been reported, notably the presence of stalled DNA replication forks in primary CD4 þ lymphocytes.29 This DNA stress then triggers the ataxia telangiectasia mutated related (ATR) response pathway that causes G2 arrest and also initiates apoptosis.30 The mechanism by which the interaction between Vpr and the E3 ubiquitin ligase complex leads to this phenotype in lymphocytes is a topic of investigation. Despite several studies implicating a role for HIV-1 vpr in HIVAN, the mechanism underlying its role in the tubular disease is unknown. Given that Vpr is linked to a number of
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Determining the effect of HIV-1 vpr expression in human proximal tubule epithelial cell (PTECs) required an efficient transduction system. Therefore, a pseudotyped lentivirus vector with a broadly tropic VSV.G envelope was used to deliver the expression cassettes to PTEC (Figure 1a). All transfer plasmids contained either a green fluorescent protein (GFP) or RFP reporter gene to monitor infection efficiency by both microscopy and fluorescence-activated cell sorting (Figure 1b). The pHR-GFP vector transduced 95.5% of cells, and this level of efficiency is representative for all plasmids.
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pathologic phenotypes that are cell type specific, we aimed to determine the effect of Vpr on the renal tubular epithelium. In this study, we examined the effect of HIV-1 Vpr on human tubular epithelial cells in vitro and correlated these findings in vivo to reveal new aspects of HIVAN pathology.
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Figure 1 | Expression of HIV-1 vpr in PTECs causes cell enlargement and inhibits cell growth. (a) Schematic representation of the HIV-1 provirus deletion construct and pHR constructs that include green or red fluorescent protein reporters (GFP or RFP). LTR – HIV-1 Long terminal repeat promoter. Psi, packaging sequence; RRE, Rev response element; CMV, cytomegalovirus immediate early promoter; IRES, internal ribosome entry site. (b) VSV.G pseudotyped lentivirus generated with pHR-GFP efficiently transduced HK2 cells. Flow cytometry was used to determine the fraction of GFP-expressing cells at 48 h after transduction. (c) Normal sized (left panel) and enlarged HK2 cells (center and right panels) at 48 h after transduction with pseudotyped lentivirus containing pHR-GFP or pHR-Vpr vectors as indicated. (d) Growth restriction of HK2 cells after transduction with pseudotyped lentivirus containing pHR-Vpr or pNL4-3Dgag/pol (HIV-1) vectors. Untransduced HK2 cells or HK2 cells transduced with pseudotyped lentivirus containing pHR-GFP, pHR-Vpu, or pHR-Vif proliferated equally. Fold growth was obtained by counting cells manually in the same random field over 3 days. The mean and s.d. of fold growths for four random fields are plotted. *Indicates t-test significance of Po0.0001 compared both with the uninfected control. 1050
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P Rosenstiel et al.: Vpr in HIVAN pathogenesis
HIV-1 Vpr causes cell enlargement and restricts proliferation in the HK2 proximal tubule cell line
When HK2 cells expressed vpr, the cells became visibly enlarged compared with the pHR-GFP control (Figure 1c). Some HIV-1Dgag/pol expressing HK2 cells also increased in size, although the majority became smaller undergoing apoptosis as has been reported.31 Cells infected with pHRVpu and pHR-Vif expression vectors were also not enlarged (data not shown). pHR-Vpr-transduced PTECs also had altered cell growth. Renal proximal tubule cell proliferation rates were determined over 3 days in cells transduced with lentiviral vectors. HIV-1 vpr expression was associated with a loss of cell proliferation, whereas pHR-GFP-transduced cells proliferated at a rate comparable to uninfected cells (Figure 1d). HIV1Dgag/pol also blocked HK2 cell proliferation over this time period. HIV-1 vpu and vif did not have any effect on proliferation and served as additional controls given their size and viral origin. Notably, HIV-1 vif has been reported to be cytostatic and/or cytotoxic in other cell systems demonstrating the importance of cell type.32 To rule out a trans effect mediated by Vpr protein expression in the lentivirus producer 293T cells, viral supernatants
G1: 64.8 S: 6.67 G2: 23.5 >G2 (8N ): 4.68
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G1: 56.9 S: 6.99 G2: 30.5 >G2 (8N): 5.12
were collected and filtered using a 100 kDa protein filter that would remove virus but not soluble proteins. When the filtered supernatant was applied to HK2 cells, no GFPpositive cells were observed and no growth restriction or cell size increase was seen (data not shown). This result indicated that lentivirus transduction and subsequent intracellular vpr expression, and not any other soluble factor, is responsible for growth restriction. In conclusion, these studies demonstrate that the expression of HIV-1 vpr prevents proliferation and causes the enlargement of PTECs in vitro. HIV-1 vpr causes cell cycle dysregulation in HK2 cells
HIV-1 Vpr has been reported to induce G2 arrest in other cell types. Therefore, we determined if cell cycle arrest was associated with the restriction of HK2 cell proliferation. Cells were transduced as described and analyzed for cell cycle changes 48 h later. A majority of HIV-1 vpr-transduced cells were in the G2 or hyperploid state, whereas GFP transduction had no effect on the cell cycle. Furthermore, full-length HIV-1 also induced hyperploidy and G2 accumulation but Vif alone or Tat alone did not (Figure 2a). The greater fraction of cells in G2 seen with full-length HIV-1 transduction may result from Vpr synergism with other viral proteins.
G1: 51.1 S: 6.75 G2: 33 >G2 (8N): 9.10
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Figure 2 | HIV-1 Vpr causes cell cycle dysregulation in PTECs. (a) Cell cycle analysis of HK2 cells at 48 h after transduction with pseudotyped lentivirus containing pNL4-3Dgag/pol, pHR-Vpr, pHR-Tat, or pHR-Vif as indicated. The gates for G1, S, G2, and 4G2 are shown for each panel. The percentage of cells within those gates is indicated in the upper right corner of each panel. (b) Propidium iodide-stained nuclei of HK2 cells 72 h after transduction with pseudotyped lentivirus containing pHR-Vpr (upper panel) or pHR-GFP (lower panel) vectors. Examples of multinucleated cells (arrows) and cells with single enlarged nuclei (arrowheads) are shown. The original magnification was 400. (c) Cell cycle analysis of HK2 cells 24 h after treatment with dihydrocytochalasin B or DMSO or transduced with pseudotyped lentivirus containing the pHR-Vpr vector as indicated. Kidney International (2008) 74, 1049–1058
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Examining the nuclei of vpr-transduced HK2 cells revealed some mononuclear cells with large nuclei and possibly increased DNA content (Figure 2b). Also prevalent were multinucleated cells, suggesting that the cause of the increased 4N and 8N populations was nuclear division in the absence of cytokinesis rather than checkpoint-mediated G2 arrest. HK2 cell fusion was not visualized, although we cannot rule out that it contributed to HK2 multinucleation. Lastly, HK2 cells were treated with the microtubule depolymerizing agent dihydrocytochalasin B to independently impair cytokinesis. The morphology and cell cycle profiles of dihydrocytochalasin B-treated and vpr-expressing cells were similar and both showed increased G2 and 8N populations (Figure 2c). This result indicates that the cell cycle dysregulation caused by Vpr in PTECs is consistent with impaired cytokinesis. This phenotype is similar to that described in studies by Shimura et al.19–21 in a stable vpr inducible cell line. HIV-1 vpr expression induces apoptosis in HK2 cells
Because full-length HIV-1 induces apoptosis in PTEC and tubule cell apoptosis is seen in HIVAN, we determined if Vpr could cause PTEC apoptosis.31,33 By two independent methods, sub-G1 peak analysis and Annexin V staining, a
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modest but significant increase in apoptosis was seen in cells expressing HIV-1 vpr compared with control (Figure 3a and b). A time course following vpr transduction revealed that apoptosis was highest after 4 days and followed the onset of the cell cycle changes (Figure 3b). Although Vpr contributes to HIV-1-induced apoptosis in PTECs, it is probably not the sole cause. Apoptosis of renal tubule cells is a feature of the pathology seen in both human HIVAN biopsies and the murine model of HIVAN.34,35 Vpr-R80A and Vpr-Q65R do not impair PTEC cytokinesis
Single amino acid Vpr variants were used to determine the molecular domain of Vpr involved in impaired cytokinesis in PTEC. Other Vpr mutagenesis studies have implicated domains in the basic C terminus as particularly important for the induction of G2 arrest in lymphocytes. However, no Vpr mutagenesis studies had been performed to investigate the domains involved in the inhibition of cytokinesis. Therefore, it was unclear if the same domain of Vpr would mediate both phenotypes. We examined the variants VprR80A, Vpr-Q65R, Vpr-R77Q, and Vpr-I74A for their ability to induce cell cycle changes. Consistent with their activities in checkpoint-mediated G2 arrest, neither Vpr-R80A nor VprQ65R inhibited cytokinesis in PTECs, whereas Vpr-R77Q
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Figure 3 | HIV-1 Vpr causes apoptosis in PTECs. (a) Quantification of apoptotic HK2 cells at 96 h after transduction with pseudotyped lentivirus containing pHR-Vpr or pHR-GFP vectors. Representative (left and center panels) and averaged (right panel) annexin V staining and PI counter-staining are shown. *Indicates t-test significance of Po0.0005. (b) Time course of cell cycle and apoptosis changes in HK2 cells transduced with pseudotyped lentivirus containing pHR-Vpr or pHR-GFP. The sub-G1 peak was used as a second assay of apoptosis in this experiment. Cell cycle analysis including this sub-G1 peak is shown at 4 days after infection for pHR-GFP and pHR-Vpr-infected HK2 cells (offset right). 1052
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P Rosenstiel et al.: Vpr in HIVAN pathogenesis
G1: 70.3 S: 6.39 G2: 18 >G2 (8N): 4.52
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Figure 4 | HIV-1 Vpr variants R80A and Q65R are inactive for cell cycle dysregulation in PTECs. (a) Cell cycle analysis of HK2 cells at 48 h after transduction with pseudotyped lentivirus containing pHR-GFP, pHR-Vpr, pHR-Vpr-I74A, pHR-Vpr-R77Q, pHR-Vpr-Q65R, or pHR-Vpr-R80A as indicated. (b) HK2 cells were untransduced or transduced with pseudotyped lentivirus containing pHR-Vpr as indicated. At 48 h after transduction, caffeine was added in the indicated concentrations. HK2 cells were harvested and analyzed for cell cycle at the time indicated (shown as hours after the pseudotyped lentivirus transduction with pHR-Vpr). The normalized G1 fraction was defined as (G1% at t0, t48, or t96 h)/(G1% at t0) and plotted as indicated.
and Vpr-I74A had a phenotype similar to wild-type Vpr (Figure 4a). Vpr-R80A and Vpr-Q65R expressions were indistinguishable from the control vector with regard to proliferation and cell morphology (data not shown). Because the same amino acid substitutions abrogate the Vpr-induced phenotype in both T cells and proximal tubule cells, the same upstream protein-binding event may be important in both settings. Vpr inhibition of PTEC cytokinesis is not abrogated by the ATR/ataxia telangiectasia mutated inhibitor caffeine
In lymphocytes, ATR is believed to be the critical mediator of the DNA damage response, which results after vpr expression and leads to G2 arrest.36 Therefore, caffeine, an inhibitor of ATR, is commonly used as an in vitro inhibitor of Vpr activity. PTECs already expressing vpr were treated with caffeine in an attempt to restore cytokinesis. PTECs were first infected with pHR-Vpr or control pHR-GFP lentivirus vector and at 48 h treated with 1–3 mM caffeine. No significant change in the cell cycle profile was observed in cells treated with caffeine compared with those untreated (Figure 4b). These results suggest that the Vpr impairment of cytokinesis is independent of the ATR pathway. Although this conclusion differs from Vpr studies in lymphocytes, it is consistent with other results where a similar phenotype was also resistant to caffeine.17 A possible explanation for why Vpr shows cell type specificity is the expression of ATR itself. Although lymphoKidney International (2008) 74, 1049–1058
cytes, HeLa cells, and many tissue types express ATR, cDNA from human kidney shows almost no ATR expression at all.37 Increased HK2 cell protein correlates with the increase in cellular DNA
HIV-1 vpr expression in PTECs is associated with cell enlargement and multinucleation with impaired cytokinesis. To determine whether the cell enlargement observed was proportional to an increase in genomic DNA, HK2 cells were infected with either pHR-Vpr or control virus and at 48 h, DNA and protein content were measured and normalized to cell number (Table 1). As expected, we found vpr-expressing cells had, on average, a 55.8% increase in DNA content and a 65.2% increase in protein content. However, these values resulted in no statistically significant difference in the DNA/ protein ratio (the molecular measure of hypertrophy) of vprtransduced cells compared with control cells.38 Therefore, the PTEC enlargement most likely results from a physiologic size increase prior to cytokinesis. HIV-1 induces hypertrophy and multinucleation in vivo in the murine model of HIVAN
HIV-1 vpr expression in PTECs in vitro caused apoptosis and impaired cytokinesis, leading to enlarged cells with multiple nuclei. Apoptosis is described in the HIVAN murine model and human biopsies. However, neither tubular cell hypertrophy nor multinucleation has been noted. Therefore, we 1053
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Control
Murine HIVAN
× 200 Control
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Murine HIVAN
Murine HIVAN
× 200 Murine HIVAN
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Figure 5 | Tubular cell hypertrophy and multinucleation in the murine HIVAN model. (a) Control murine kidney compared with murine HIVAN kidney at an original magnification of 200. A dilated tubule (arrowhead) and sclerotic glomeruli (arrow) are shown. (b) Tubular cell hypertrophy in murine HIVAN. Control and murine HIVAN sections were compared at an original magnification of 400. General hypertrophy can be appreciated and examples of individual cell hypertrophy and nucleus enlargement are shown (arrows). (c) Examples of control murine kidney tubules and multinucleated tubule cells in murine HIVAN. The original magnification was 600. All sections are PAS stains.
Table 1 | The protein content and DNA content of HK2 cells at 48 h after transduction with lentivirus containing pHR-Vpr or pHR-GFP as indicated pHR-VPR/pHR-GFP
Average s.d. (s)
Protein/DNA
DNA/cell
Protein/cell
1.06 0.11
1.56 0.16
1.65 0.03
The ratios shown indicate the mean and s.d. of a single experiment performed in triplicate. This experiment was representative of three experiments.
examined the Tg26 transgenic mouse, a model of HIVAN that expresses all of the HIV genes except gag and pol and shows classic HIVAN pathology (Figure 5a). The kidney sections from nephrotic (3 þ proteinuria) mice were inspected for enlarged and multinucleated tubule cells. Surprisingly, frequent examples of enlarged cells and multinucleated cells were seen (Figure 5b and c). Tubular cell hypertrophy was evident when comparing control with murine HIVAN at the same magnification. For each comparison, the HIVAN kidneys always had larger tubules appearing as if they were examined at a higher magnification (Figure 5b). These results resemble the phenotype seen when vpr is expressed in PTECs in vitro resulting in enlarged and multinucleated cells (Figure 2b). Tubular cell hypertrophy and increased chromosome number is observed in HIVAN
The examination of human HIVAN biopsy samples also revealed widespread hypertrophy similar to the murine 1054
HIVAN model (Figure 6). HIVAN biopsies had larger tubules compared with control human kidney biopsies with individual tubule cell hypertrophy also being apparent. In these HIVAN biopsies, multinucleation could not be conclusively demonstrated. However, many tubular cell nuclei were enlarged mimicking vpr expression in vitro. To determine if these nuclei had increased DNA content, we applied fluorescence in situ hybridization to the centromeric regions of chromosomes 8, X, and Y. Nuclei exceeding a diploid signal were indicative of increased DNA. The technique required 2 mm sections, much thinner than the diameter of a typical cell nucleus. Therefore, the signal number underestimated the actual chromosome number in the large majority of nuclei. This effect can be appreciated in a field view of the control kidney sample (Figure 7a upper panel). The arrow depicts a diploid tubular cell but neighboring cells show fewer hybridization signals. In contrast to the control, the HIVAN sample showed many tubular cell nuclei with an increased number of centromere hybridization signals (Figure 7a lower panel—arrowed, Figure 7b, and c). These results demonstrate a previously unrecognized aspect of HIVAN pathology that correlates with Vpr activity in vitro. DISCUSSION
The microcystic distortion of the tubulointerstitium is one of the most striking features of HIVAN. We now know that HIV-1 infects tubular cells in patients with HIVAN. In situ hybridization and laser capture microscopy combined with PCR have shown that HIV-1 mRNA is expressed in tubular cells and replicates within the renal compartment in HIVAN Kidney International (2008) 74, 1049–1058
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P Rosenstiel et al.: Vpr in HIVAN pathogenesis
Control PAS
HIVAN PAS
HIVAN PAS
×400
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Figure 6 | Tubule cell hypertrophy in human HIVAN biopsies. Compared with normal adult control kidney, the HIVAN kidneys display increased tubular diameter of noncystic tubules and diffuse hypertrophy of individual tubular epithelial cells. The original magnification of control and HIVAN sections was 400. All sections are PAS stains.
Control L
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Figure 7 | Increased chromosome number in tubule cells in human HIVAN biopsies. (a) Renal FISH staining on a female human control (upper panel) and male human HIVAN biopsy (lower panel). L, tubular lumen; arrows, cell of normal ploidy (control) or increased ploidy (HIVAN). Centromeric enumeration probes (CEP) are Aqua, chromosome 8; red, chromosome X; and green, chromosome Y. The original magnification of control and HIVAN sections was 1000. (b) Selected examples of tubular cells with increased copy number for X and/or Y chromosomes in human HIVAN biopsies. (c) Selected examples of tubular cells with increased copy number of chromosome 8 in human HIVAN biopsies.
patients.4,5 HIV expression within transgenic mouse kidney produces the characteristic features of the disease that are identical to those observed in humans.39 As reported previously, tubular cell apoptosis is common and epithelial dedifferentiation is widespread as evidenced by changes in expression levels and patterns of characteristic cellular markers. The viral genes responsible for the tubular disease have not been defined nor has the mechanism been well established. Previous work has provided some insights; however, Dickie et al.40 reported increased tubular pathology Kidney International (2008) 74, 1049–1058
in transgenic mice expressing HIV-1 vpr, suggesting that Vpr has an important role in this aspect of HIVAN pathogenesis. HIV-1 vpr expression has already been shown to induce G2 cell cycle arrest and apoptosis of CD4 þ T cells. Thus, we hypothesized that Vpr might induce similar pathology in the renal tubule epithelium (RTE) of HIVAN patients and transgenic mice, although, ultimately, we found a different mechanism was responsible. To explore the possible role of Vpr in producing disease in tubular cells in HIVAN, we used an in vitro model of the 1055
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human proximal tubule cell and efficiently expressed HIV-1 vpr using a lentiviral vector. This approach allowed a detailed phenotypic analysis of the vpr-transduced cells and provided insight into Vpr’s pathologic mechanisms. In this study, we found that Vpr induced significant apoptosis in tubular cells in vitro. More prominently, however, the vpr expressing tubular cells became enlarged with enlarged or multiple nuclei. As this phenotype was not previously described in vivo, we examined kidney sections from the murine HIVAN model and human HIVAN biopsies. Surprisingly, RTE hypertrophy was striking in both mouse and human. Both RTE cell size and nuclei were significantly larger in both transgenic and HIVAN kidneys when compared with controls. We also found multinucleated RTE cells in transgenic mice. Although terms such as ‘reactive’ epithelial cells have been used before to describe some of these findings, the extent of cellular changes observed in both mouse and human is striking. In addition, the tubule cell hypertrophy may contribute to the gross pathologic kidney enlargement in HIVAN. On the basis of these findings in vitro and in vivo, we conclude that Vpr contributes to HIVAN tubular pathology by interfering with and disrupting tubule epithelial cell division. However, the examination of vpr transgenic murine models for hypertrophy and multinucleation will determine whether HIV-1 vpr expression is sufficient to cause these changes in vivo or it works in conjunction with other HIV proteins. Normally, the renal tubule epithelium is in a quiescent, non-proliferative state (G0). In response to acute injury, however, RTE cells have the capacity to proliferate.38 Previously we reported that a large fraction of tubule cells in the kidneys of HIV transgenic mice are positive for the proliferation marker Ki-67.35 This finding indicates that many tubule epithelial cells have re-entered the cell cycle. The impetus for cells exiting G0 is not entirely clear but could be in response to the concurrent apoptosis induced by HIV, epithelial injury, or a viral gene product such as Nef, which is known to induce proliferation.15 Whatever the cause, Vpr appears to interfere with tubule cell division leading to cell enlargement and multinucleation. The mechanism of Vpr-induced enlargement and multinucleation was investigated in vitro and appears to result from a specific defect in cell cytokinesis. HK2 cells expressing vpr continued to cycle through S phase, often multiple times, without cytokinesis occurring. This phenotype is distinct from Vpr-induced G2 arrest in lymphocytes where the cell cycle stops prior to mitosis through a classical checkpoint pathway. Differentiated proximal tubular epithelial cells display a Vpr phenotype similar to what has been described in various cell lines, primary HUVECs, and yeast.17,18,20,41 Vpr functions as an adaptor protein between an unknown target protein and DCAF1, a substrate specificity module in certain Cullin 4-based E3 ubiquitin ligase complexes. Proteasomal degradation of this putative target protein is proposed to trigger G2 arrest, apoptosis, and potentially other phenotypes induced by HIV-1 vpr expression. The 1056
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critical binding site of Vpr has been mapped by mutagenesis of the key amino acids between positions 65 and 80. The mutation of the Q65 residue prevents DCAF1 binding and impairs the ability of Vpr to induce G2 arrest.26,38 Other mutations that retain binding to DCAF1 can also inhibit the ability of Vpr to induce G2 arrest, possibly because the ability to bind to the unknown target protein is lost. Examples of such mutations include R73A, G75A, C76A, R80A, and R90A. In our study we examined four mutants, and the results were consistent with previous studies. Mutants I74A and R77Q acted as wild type, whereas both R80A and Q65R were completely inactive. Although there is a strong correlation between the in vitro Vpr phenotype in PTEC and pathology seen in the murine HIVAN model and HIVAN biopsies, hypertrophy, irregular cell division, and apoptosis are not the only tubular pathologies associated with HIVAN. Microcystic dilation remains an unexplained hallmark of HIVAN. We have shown previously that the cells lining the microcysts of transgenic mice are flattened with less brush border and no longer support transcription of HIV-1.35 One possibility is that microcyst formation is an end-stage result that arises after an initial hypertrophic expansion, and hypertrophied HIV-1expressing cells have died or transformed. In conclusion, we report that Vpr impairs cytokinesis in proximal tubular epithelial cells leading to cell enlargement and often multinucleation. The examination of kidney sections from human and mouse showed extensive tubular hypertrophy, multinucleation, and evidence of increased chromosome number. These in vivo results reveal new aspects of HIVAN pathology. Given Vpr’s central role in HIVAN pathogenesis, we anticipate it may provide a good molecular target for future therapeutic intervention. MATERIALS AND METHODS Cell culture system The HK2 line of human proximal tubule cells was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Defined Keratinocyte Serum-Free Media (Gibco, Carlsbad CA, USA, 17005-042) with 5 ng/ml of recombinant epidermal growth factor and 0.05 mg/ml of bovine pituitary extract at 37.0 1C, 5% CO2 to preserve phenotype. Ryan et al.42 characterized this line as phenotypically similar to freshly isolated PTECs including Na þ -dependent sugar transport. 293T Cells were used to produce pseudotyped virus and cultured in Dulbecco’s modified Eagle’s medium with fetal bovine serum (10%), penicillin (10,000 U/ ml), and streptomycin (10 mg/ml). Pseudotyped lentivirus vector infection A pseudotyped lentivirus vector was used to efficiently transduce the desired genes into the HK2 cell line. This virus was produced by cotransfection (Invitrogen Lipofectamine 2000, Invitrogen (Carlsbad, CA, USA)) of 293T cells with three separate plasmids: pVSV.G expressing the VSV envelope, the packaging plasmid pHIVgag/pol expression gag and pol in trans, and the specific viral or pHR plasmid expressing the package RNA as previously described.15 Forty-eight hours after plasmid transfection, the supernatant was collected and filtered using a 0.45 mm filter (Millipore, Billerica, MA, Kidney International (2008) 74, 1049–1058
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USA). Virus was titered by infection of 293T cells, and an multiplicity of infection of 3 was used to infect PTECs to achieve maximum infection. Proliferation assay The cell proliferation assay was performed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA). Forward scatter (FSC þ ) side scatter (SSC þ ) events were counted for a set interval at flow speeds low, medium, and high. All values are at subconfluence. Error bars reflect the intra-assay variability and are representative of a minimum of two independent experiments. Cell cycle assay and analysis Cell cycle analysis was performed by flow cytometry (FACSCalibur and FacScan, BD Biosciences, San Jose, CA, USA). Cells were fixed and permeabilized by the addition of 20 1C ethanol to a final concentration of 50%. The fixed cells were treated with 100 mg/ml of propidium iodide and 0.5 mg/ml of RNAse A for 3 h prior to cytometry. FloJo (version 6.4.7) was used for all analysis. Microscopy Cells were fixed in cold 100% ethanol and stained with propidium iodide (3 mM) for 10 min at room temperature and examined. Representative fields were photographed in fluorescent and monochrome light and merged for publication. Apoptosis assays Quantification of the sub-G1 peak in cell cycle analysis was measured as a fraction of a FSC þ SSC þ -gated population. Annexin V staining was performed using Annexin V-APC antibody (Molecular Probes, Carlsbad CA, USA, A35110) at 5 ml per 1 106 cells. Propidium iodide co-staining was performed at final concentration of 1.5 mg/ml.
Vysis Paraffin Pretreatment Kit III protocol (Vysis/Abbott Molecular, Abbott Park, IL, USA). Denaturation of the target DNA on slides and probes was performed simultaneously at 73 1C for 6 min on a Thermobrite System (Vysis/Abbott Molecular). Hybridization was carried out in a humidified chamber at 37 1C overnight using chromosome enumeration probe 8 (CEP-8, Abbott Molecular, 32-131008) and/or CEP-X/CEP-Y (Abbott Molecular, 30-161050). 40 -6-Diamidino-2-phenylindole (Vysis/Abbott Molecular) was used as a counterstain. The slides were analyzed and imaged with the Cytovysion 3.92 program (Applied Imaging/Genetix, New Milton, UK) and Axioplan II microscope (Zeiss, Oberkochen, Germany). DISCLOSURE
The authors declare no competing financial interests. ACKNOWLEDGMENTS
This study was funded by the HIVAN Program Project Grant: NIH NIDDK-P01DK56492-01, principal investigator: Paul E. Klotman. INFORMED CONSENT AND ETHICS The research involving human subjects and animals was performed in accordance with the ethical standards of the responsible institutional committees on human subjects research under the approved Institutional Review Board protocol number 98-953 and the Institutional Animal Care and Use Committee, approved protocol number 98-0953. REFERENCES 1.
2.
3. 4.
Drug treatments Caffeine (Fisher Scientific, Hampton, NH, USA) was dissolved in water applied to PTECs at a final concentration of 1–3 mM. Dihydrocytochalasin B (Fisher Scientific) in dimethyl sulfoxide solution was applied to PTECs at a final concentration of 10 mM. DNA and protein quantification HK2 cells were infected as described and resuspended 48 h later. DNA and protein were harvested from equal volumes of resuspended cells in triplicate. DNA and protein concentrations were measured by spectrophotometry using the NanoDrop ND1000 Thermo Scientific, Wilmington, DE, USA. Values were normalized to cell number as measured by flow cytometry. The data shown are representative of two independent experiments. Histology HIV transgenic (Tg26 murine line) FVB/N mice with 3-plus proteinuria were killed and examined. Comparisons were made to wild-type FVB/N mice. Human HIVAN biopsies were compared with normal control biopsies. Representative areas were chosen for publication.
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Fluorescence in situ hybridization Human HIVAN biopsies were fixed in paraffin and cut to 2 mm sections on positively charged silanized glass slides. Deparaffinization and pretreatment of the slides were performed according to the Kidney International (2008) 74, 1049–1058
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Rao TK, Filippone EJ, Nicastri AD et al. Associated focal and segmental glomerulosclerosis in the acquired immunodeficiency syndrome. N Engl J Med 1984; 310: 669–673. Shahinian V, Rajaraman S, Borucki M et al. Prevalence of HIV associated nephropathy in autopsies of HIV-infected patients. Am J Kidney Dis 2000; 35: 884–888. Ahuja TS, Borucki M, Funtanilla M et al. Is the prevalence of HIVassociated nephropathy decreasing? Am J Nephrol 1999; 19: 655–659. Marras D, Bruggeman LA, Gao F et al. Replication and compartmentalization of HIV-1 in kidney epithelium of patients with HIV-associated nephropathy. Nat Med 2002; 8: 522–526. Bruggeman LA, Ross MD, Tanji N et al. Renal epithelium is a previously unrecognized site of HIV-1 infection. J Am Soc Nephrol 2000; 11: 2079–2087. Kopp JB, Klotman ME, Adler SH et al. Progressive glomerulosclerosis and enhanced renal accumulation of basement membrane components in mice transgenic for human immunodeficiency virus type I genes. Proc Natl Acad Sci USA 1992; 89: 1577–1581. Ross MJ, Bruggeman LA, Wilson PD et al. Microcyst formation and HIV-1 gene expression occur in multiple nephron segments in HIV-associated nephropathy. J Am Soc Nephrol 2001; 12: 2645–2651. D’Agati V, Appel G. HIV infection and the kidney. J Am Soc Nephrol 1997; 8: 138–152. D’Agati V, Suh JI, Carbone L et al. Pathology of HIV-associated nephropathy: a detailed morphologic and comparative study. Kidney Int 1989; 35: 1358–1370. Barisoni L, Kriz W, Mundel P et al. The dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy. J Am Soc Nephrol 1999; 10: 51–61. Barisoni L, Mokrzycki M, Sablay L et al. Podocyte cell cycle regulation and proliferation in collapsing glomerulopathies. Kidney Int 2000; 58: 137–143. Weiss MA, Daquioag E, Margolin EG et al. Nephrotic syndrome, progressive irreversible renal failure, and glomerular ‘collapse’: a new clinicopathologic entity? Am J Kidney Dis 1986; 7: 20–28. Kajiyama W, Klotman PE, Dickie P et al. Glomerulosclerosis and viral gene expression in HIV-transgenic mice: role of nef. Kidney Int 2000; 58: 1148–1159. Husain M, D’Agati VD, He JC et al. HIV-1 Nef induces dedifferentiation of podocytes in vivo: a characteristic feature of HIVAN. AIDS 2005; 19: 1975–1980.
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Husain M, Gusella GL, Klotman ME et al. HIV-1 Nef induces proliferation and anchorage-independent growth in podocytes. J Am Soc Nephrol 2002; 13: 1806–1815. Dickie P, Roberts A, Uwiera R et al. Focal glomerulosclerosis in proviral and c-fms transgenic mice links vpr expression to HIV associated nephropathy. Virology 2004; 322: 69–81. Chang F, Re F, Sebastian S et al. HIV-1 Vpr induces defects in mitosis, cytokinesis, nuclear structure, and centrosomes. Mol Biol Cell 2004; 15: 1793–1801. Bartz SR, Rogel ME, Emerman M et al. Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control. J Virol 1996; 70: 2324–2331. Minemoto Y, Shimura M, Ishizaka Y et al. Multiple centrosome formation induced by the expression of Vpr gene of human immunodeficiency virus. Biochem Biophys Res Commun 1999; 258: 379–384. Shimura M, Onozuka Y, Yamaguchi T et al. Micronuclei formation with chromosome breaks and gene amplification caused by Vpr, an accessory gene of human immunodeficiency virus. Cancer Res 1999; 59: 2259–2264. Shimura M, Tanaka Y, Nakamura S et al. Micronuclei formation and aneuploidy induced by Vpr, an accessory gene of human immunodeficiency virus type 1. FASEB J 1999; 13: 621–637. Connor RI, Chen BK, Choe S et al. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 1995; 206: 935–944. Heinzinger NK, Bukinsky MI, Haggerty SA et al. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci USA 1994; 91: 7311–7315. Tan L, Ehrlich E, Yu XF. DDB1 and Cul4A are required for HIV-1 Vpr-induced G2 arrest. J Virol 2007; 81: 10822–10830. Wen X, Duus KM, Friedrich TD et al. The HIV-1 protein vpr acts to promote G2 cell cycle arrest by engaging a DDB1 and Cullin4A-containing ubiquitin ligase complex using VprBP/DCAF1 as an adaptor. J Biol Chem 2007; 282: 27046–27057. Le Rouzic E, Belaidouni N, Estrabaud E et al. HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4-DDB1 ubiquitin ligase. Cell Cycle 2007; 6: 182–188. Schrofelbauer B, Hakata Y, Landau NR. HIV-1 Vpr function is mediated by interaction with the damage-specific DNA-binding protein DDB1. Proc Natl Acad Sci USA 2007; 104: 4130–4135.
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Kidney International (2008) 74, 1049–1058
original article
& 2008 International Society of Nephrology
HIV-1 Vpr inhibits cytokinesis in human proximal tubule cells Paul E. Rosenstiel1, Tina Gruosso1, Audrey M. Letourneau1, Justin J. Chan1, Amanda LeBlanc1, Mohammad Husain1, Vesna Najfeld1, Vicente Planelles2, Vivette D. D’Agati3, Mary E. Klotman1 and Paul E. Klotman1 1
Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA; 2Department of Pathology, University of Utah, Salt Lake City, Utah, USA and 3Department of Pathology, Columbia University, New York, New York, USA
Transgenic mouse models of HIV-associated nephropathy (HIVAN) show that expression of HIV-1 genes in kidney cells produces collapsing focal segmental glomerulosclerosis and microcystic tubular disease typical of the human disease. HIV-1 vpr plays an important role in the glomerulosclerosis of HIVAN, especially when it is associated with nef expression in podocytes. Further, Vpr is reported to exacerbate tubular pathology. Here we determined effects of vpr expression on renal tubular epithelial cell function by transducing them with a pseudotyped lentivirus vector carrying HIV-1 vpr and control genes. Vpr expression in the cultured cells impaired cytokinesis causing cell enlargement and multinucleation. This profound in vitro phenotype caused us to reexamine the HIVAN mouse model and human HIVAN biopsies to see if similar changes occur in vivo. Both showed abundant hypertrophic tubule cells similar to the in vitro finding that represents a previously unappreciated aspect of the human disease. Additionally, multinucleated tubular cells were identified in the murine HIVAN model and increased chromosome number was detected in tubular cells of human HIVAN biopsies. Our study provides evidence of a new clinical phenotype in HIVAN that may result from the ability of Vpr to impair cytokinesis. Kidney International (2008) 74, 1049–1058; doi:10.1038/ki.2008.303; published online 9 July 2008 KEYWORDS: HIVAN; proximal tubule; HIV; Vpr; hypertrophy; cytokinesis
Correspondence: Paul E. Rosenstiel, Department of Medicine, Mount Sinai School of Medicine, Box 1118, One Gustave L. Levy Place, New York, New York 10029, USA. E-mail: [email protected] Received 12 November 2007; revised 19 March 2008; accepted 23 April 2008; published online 9 July 2008 Kidney International (2008) 74, 1049–1058
Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) was first reported in 1984 when a small group of Haitian and African-American HIV patients were found to have decreased renal function independent of other risk factors.1 Currently, HIVAN prevalence in the black HIVinfected population is between 3.5 and 12%,2,3 affecting an estimated 1–3 million people worldwide. HIVAN is associated with direct HIV infection of the glomerular and tubular compartments and subsequent viral genome transcription.4,5 The classical histopathologic features of HIVAN are collapsing focal segmental glomerulosclerosis and focal microcystic (3 normal diameter) dilation of the tubular segments with epithelial flattening.6–9 Apoptosis, hyperplasia, pseudocrescent formation, and several other pathologic findings have also been reported.9–12 Grossly, HIVAN kidneys are typically enlarged. Transgenic models expressing either single HIV-1 genes or deletion mutants of HIV-1 have been useful in mapping the HIV-1 genes pathogenic for HIVAN. HIV-1 nef has been associated with podocyte dedifferentiation and proliferation.13–15 HIV-1 vpr has been found to contribute to overall HIVAN pathogenesis in both podocytes and tubule epithelial cells.16 Mice expressing only vpr manifested some microcystic tubule dilation, whereas those expressing HIV without vpr did not show this phenotype. When HIVDvpr mice were crossed with vpr-expressing mice, the offspring showed widespread microcystic dilation typical of HIVAN. These data suggest that the tubular cystic phenotype involves vpr expression, but the exact mechanism of how Vpr contributes to HIVAN pathogenesis remains unknown. Although HIV-1 vpr function has not been investigated in renal epithelial cells, it has been extensively studied in other cell systems. In activated lymphocytes, and many cell lines, Vpr induces G2 arrest followed by apoptosis. Expression of vpr in other cell types, however, causes nuclear anomalies, including hyperploidy, micronuclei formation, and the evidence of impaired cytokinesis.17–21 Conversely, in macrophages, Vpr does not induce an abnormal nuclear phenotype or apoptosis, but may instead be important for HIV replication by facilitating nuclear import of the HIV preintegration complex.22,23 1049
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The precise mechanism(s) by which Vpr alters cell function is incompletely understood, but recently multiple studies have identified DDB1–Cullin 4A Associated Factor 1 (DCAF1) as an important Vpr binding partner.24–28 DCAF1 binds an E3 ubiquitin ligase complex likely functioning as a substrate specificity module. Vpr binding to DCAF1 is proposed to dysregulate and usurp the E3 complex. This binding appears to be the critical upstream event mediating Vpr-induced G2 arrest. Downstream, several events have been reported, notably the presence of stalled DNA replication forks in primary CD4 þ lymphocytes.29 This DNA stress then triggers the ataxia telangiectasia mutated related (ATR) response pathway that causes G2 arrest and also initiates apoptosis.30 The mechanism by which the interaction between Vpr and the E3 ubiquitin ligase complex leads to this phenotype in lymphocytes is a topic of investigation. Despite several studies implicating a role for HIV-1 vpr in HIVAN, the mechanism underlying its role in the tubular disease is unknown. Given that Vpr is linked to a number of
HIV-1 (pNL4-3: ∆G/P-EGFP) vif
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Determining the effect of HIV-1 vpr expression in human proximal tubule epithelial cell (PTECs) required an efficient transduction system. Therefore, a pseudotyped lentivirus vector with a broadly tropic VSV.G envelope was used to deliver the expression cassettes to PTEC (Figure 1a). All transfer plasmids contained either a green fluorescent protein (GFP) or RFP reporter gene to monitor infection efficiency by both microscopy and fluorescence-activated cell sorting (Figure 1b). The pHR-GFP vector transduced 95.5% of cells, and this level of efficiency is representative for all plasmids.
env GFP
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RESULTS Efficient transduction of HK2 cells using a pseudotyped lentivirus vector
GFP
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pathologic phenotypes that are cell type specific, we aimed to determine the effect of Vpr on the renal tubular epithelium. In this study, we examined the effect of HIV-1 Vpr on human tubular epithelial cells in vitro and correlated these findings in vivo to reveal new aspects of HIVAN pathology.
* 1
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Figure 1 | Expression of HIV-1 vpr in PTECs causes cell enlargement and inhibits cell growth. (a) Schematic representation of the HIV-1 provirus deletion construct and pHR constructs that include green or red fluorescent protein reporters (GFP or RFP). LTR – HIV-1 Long terminal repeat promoter. Psi, packaging sequence; RRE, Rev response element; CMV, cytomegalovirus immediate early promoter; IRES, internal ribosome entry site. (b) VSV.G pseudotyped lentivirus generated with pHR-GFP efficiently transduced HK2 cells. Flow cytometry was used to determine the fraction of GFP-expressing cells at 48 h after transduction. (c) Normal sized (left panel) and enlarged HK2 cells (center and right panels) at 48 h after transduction with pseudotyped lentivirus containing pHR-GFP or pHR-Vpr vectors as indicated. (d) Growth restriction of HK2 cells after transduction with pseudotyped lentivirus containing pHR-Vpr or pNL4-3Dgag/pol (HIV-1) vectors. Untransduced HK2 cells or HK2 cells transduced with pseudotyped lentivirus containing pHR-GFP, pHR-Vpu, or pHR-Vif proliferated equally. Fold growth was obtained by counting cells manually in the same random field over 3 days. The mean and s.d. of fold growths for four random fields are plotted. *Indicates t-test significance of Po0.0001 compared both with the uninfected control. 1050
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HIV-1 Vpr causes cell enlargement and restricts proliferation in the HK2 proximal tubule cell line
When HK2 cells expressed vpr, the cells became visibly enlarged compared with the pHR-GFP control (Figure 1c). Some HIV-1Dgag/pol expressing HK2 cells also increased in size, although the majority became smaller undergoing apoptosis as has been reported.31 Cells infected with pHRVpu and pHR-Vif expression vectors were also not enlarged (data not shown). pHR-Vpr-transduced PTECs also had altered cell growth. Renal proximal tubule cell proliferation rates were determined over 3 days in cells transduced with lentiviral vectors. HIV-1 vpr expression was associated with a loss of cell proliferation, whereas pHR-GFP-transduced cells proliferated at a rate comparable to uninfected cells (Figure 1d). HIV1Dgag/pol also blocked HK2 cell proliferation over this time period. HIV-1 vpu and vif did not have any effect on proliferation and served as additional controls given their size and viral origin. Notably, HIV-1 vif has been reported to be cytostatic and/or cytotoxic in other cell systems demonstrating the importance of cell type.32 To rule out a trans effect mediated by Vpr protein expression in the lentivirus producer 293T cells, viral supernatants
G1: 64.8 S: 6.67 G2: 23.5 >G2 (8N ): 4.68
G1
G1: 56.9 S: 6.99 G2: 30.5 >G2 (8N): 5.12
were collected and filtered using a 100 kDa protein filter that would remove virus but not soluble proteins. When the filtered supernatant was applied to HK2 cells, no GFPpositive cells were observed and no growth restriction or cell size increase was seen (data not shown). This result indicated that lentivirus transduction and subsequent intracellular vpr expression, and not any other soluble factor, is responsible for growth restriction. In conclusion, these studies demonstrate that the expression of HIV-1 vpr prevents proliferation and causes the enlargement of PTECs in vitro. HIV-1 vpr causes cell cycle dysregulation in HK2 cells
HIV-1 Vpr has been reported to induce G2 arrest in other cell types. Therefore, we determined if cell cycle arrest was associated with the restriction of HK2 cell proliferation. Cells were transduced as described and analyzed for cell cycle changes 48 h later. A majority of HIV-1 vpr-transduced cells were in the G2 or hyperploid state, whereas GFP transduction had no effect on the cell cycle. Furthermore, full-length HIV-1 also induced hyperploidy and G2 accumulation but Vif alone or Tat alone did not (Figure 2a). The greater fraction of cells in G2 seen with full-length HIV-1 transduction may result from Vpr synergism with other viral proteins.
G1: 51.1 S: 6.75 G2: 33 >G2 (8N): 9.10
G1: 8.6 S: 2.8 G2: 74.1 >G2 (8N ): 14.1
G1: 21.7 S: 6.37 G2: 43.1 >G2 (8N ): 28.0
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G1: 10.5 S: 3.63 G2: 71.1 >G2 (8N): 14.8
pHR-GFP
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Figure 2 | HIV-1 Vpr causes cell cycle dysregulation in PTECs. (a) Cell cycle analysis of HK2 cells at 48 h after transduction with pseudotyped lentivirus containing pNL4-3Dgag/pol, pHR-Vpr, pHR-Tat, or pHR-Vif as indicated. The gates for G1, S, G2, and 4G2 are shown for each panel. The percentage of cells within those gates is indicated in the upper right corner of each panel. (b) Propidium iodide-stained nuclei of HK2 cells 72 h after transduction with pseudotyped lentivirus containing pHR-Vpr (upper panel) or pHR-GFP (lower panel) vectors. Examples of multinucleated cells (arrows) and cells with single enlarged nuclei (arrowheads) are shown. The original magnification was 400. (c) Cell cycle analysis of HK2 cells 24 h after treatment with dihydrocytochalasin B or DMSO or transduced with pseudotyped lentivirus containing the pHR-Vpr vector as indicated. Kidney International (2008) 74, 1049–1058
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Examining the nuclei of vpr-transduced HK2 cells revealed some mononuclear cells with large nuclei and possibly increased DNA content (Figure 2b). Also prevalent were multinucleated cells, suggesting that the cause of the increased 4N and 8N populations was nuclear division in the absence of cytokinesis rather than checkpoint-mediated G2 arrest. HK2 cell fusion was not visualized, although we cannot rule out that it contributed to HK2 multinucleation. Lastly, HK2 cells were treated with the microtubule depolymerizing agent dihydrocytochalasin B to independently impair cytokinesis. The morphology and cell cycle profiles of dihydrocytochalasin B-treated and vpr-expressing cells were similar and both showed increased G2 and 8N populations (Figure 2c). This result indicates that the cell cycle dysregulation caused by Vpr in PTECs is consistent with impaired cytokinesis. This phenotype is similar to that described in studies by Shimura et al.19–21 in a stable vpr inducible cell line. HIV-1 vpr expression induces apoptosis in HK2 cells
Because full-length HIV-1 induces apoptosis in PTEC and tubule cell apoptosis is seen in HIVAN, we determined if Vpr could cause PTEC apoptosis.31,33 By two independent methods, sub-G1 peak analysis and Annexin V staining, a
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modest but significant increase in apoptosis was seen in cells expressing HIV-1 vpr compared with control (Figure 3a and b). A time course following vpr transduction revealed that apoptosis was highest after 4 days and followed the onset of the cell cycle changes (Figure 3b). Although Vpr contributes to HIV-1-induced apoptosis in PTECs, it is probably not the sole cause. Apoptosis of renal tubule cells is a feature of the pathology seen in both human HIVAN biopsies and the murine model of HIVAN.34,35 Vpr-R80A and Vpr-Q65R do not impair PTEC cytokinesis
Single amino acid Vpr variants were used to determine the molecular domain of Vpr involved in impaired cytokinesis in PTEC. Other Vpr mutagenesis studies have implicated domains in the basic C terminus as particularly important for the induction of G2 arrest in lymphocytes. However, no Vpr mutagenesis studies had been performed to investigate the domains involved in the inhibition of cytokinesis. Therefore, it was unclear if the same domain of Vpr would mediate both phenotypes. We examined the variants VprR80A, Vpr-Q65R, Vpr-R77Q, and Vpr-I74A for their ability to induce cell cycle changes. Consistent with their activities in checkpoint-mediated G2 arrest, neither Vpr-R80A nor VprQ65R inhibited cytokinesis in PTECs, whereas Vpr-R77Q
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Figure 3 | HIV-1 Vpr causes apoptosis in PTECs. (a) Quantification of apoptotic HK2 cells at 96 h after transduction with pseudotyped lentivirus containing pHR-Vpr or pHR-GFP vectors. Representative (left and center panels) and averaged (right panel) annexin V staining and PI counter-staining are shown. *Indicates t-test significance of Po0.0005. (b) Time course of cell cycle and apoptosis changes in HK2 cells transduced with pseudotyped lentivirus containing pHR-Vpr or pHR-GFP. The sub-G1 peak was used as a second assay of apoptosis in this experiment. Cell cycle analysis including this sub-G1 peak is shown at 4 days after infection for pHR-GFP and pHR-Vpr-infected HK2 cells (offset right). 1052
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G1: 70.3 S: 6.39 G2: 18 >G2 (8N): 4.52
G1
G1: 23.9 S: 4.28 G2: 59.5 >G2 (8N ): 11.7
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pHR-Vpr I74A G1: 67.3 S: 7.64 G2: 20.7 >G2 (8N ): 3.11
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pHR-Vpr R77Q G1: 82.2 S: 5.03 G2: 10.7 >G2 (8N ): 2.01
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Figure 4 | HIV-1 Vpr variants R80A and Q65R are inactive for cell cycle dysregulation in PTECs. (a) Cell cycle analysis of HK2 cells at 48 h after transduction with pseudotyped lentivirus containing pHR-GFP, pHR-Vpr, pHR-Vpr-I74A, pHR-Vpr-R77Q, pHR-Vpr-Q65R, or pHR-Vpr-R80A as indicated. (b) HK2 cells were untransduced or transduced with pseudotyped lentivirus containing pHR-Vpr as indicated. At 48 h after transduction, caffeine was added in the indicated concentrations. HK2 cells were harvested and analyzed for cell cycle at the time indicated (shown as hours after the pseudotyped lentivirus transduction with pHR-Vpr). The normalized G1 fraction was defined as (G1% at t0, t48, or t96 h)/(G1% at t0) and plotted as indicated.
and Vpr-I74A had a phenotype similar to wild-type Vpr (Figure 4a). Vpr-R80A and Vpr-Q65R expressions were indistinguishable from the control vector with regard to proliferation and cell morphology (data not shown). Because the same amino acid substitutions abrogate the Vpr-induced phenotype in both T cells and proximal tubule cells, the same upstream protein-binding event may be important in both settings. Vpr inhibition of PTEC cytokinesis is not abrogated by the ATR/ataxia telangiectasia mutated inhibitor caffeine
In lymphocytes, ATR is believed to be the critical mediator of the DNA damage response, which results after vpr expression and leads to G2 arrest.36 Therefore, caffeine, an inhibitor of ATR, is commonly used as an in vitro inhibitor of Vpr activity. PTECs already expressing vpr were treated with caffeine in an attempt to restore cytokinesis. PTECs were first infected with pHR-Vpr or control pHR-GFP lentivirus vector and at 48 h treated with 1–3 mM caffeine. No significant change in the cell cycle profile was observed in cells treated with caffeine compared with those untreated (Figure 4b). These results suggest that the Vpr impairment of cytokinesis is independent of the ATR pathway. Although this conclusion differs from Vpr studies in lymphocytes, it is consistent with other results where a similar phenotype was also resistant to caffeine.17 A possible explanation for why Vpr shows cell type specificity is the expression of ATR itself. Although lymphoKidney International (2008) 74, 1049–1058
cytes, HeLa cells, and many tissue types express ATR, cDNA from human kidney shows almost no ATR expression at all.37 Increased HK2 cell protein correlates with the increase in cellular DNA
HIV-1 vpr expression in PTECs is associated with cell enlargement and multinucleation with impaired cytokinesis. To determine whether the cell enlargement observed was proportional to an increase in genomic DNA, HK2 cells were infected with either pHR-Vpr or control virus and at 48 h, DNA and protein content were measured and normalized to cell number (Table 1). As expected, we found vpr-expressing cells had, on average, a 55.8% increase in DNA content and a 65.2% increase in protein content. However, these values resulted in no statistically significant difference in the DNA/ protein ratio (the molecular measure of hypertrophy) of vprtransduced cells compared with control cells.38 Therefore, the PTEC enlargement most likely results from a physiologic size increase prior to cytokinesis. HIV-1 induces hypertrophy and multinucleation in vivo in the murine model of HIVAN
HIV-1 vpr expression in PTECs in vitro caused apoptosis and impaired cytokinesis, leading to enlarged cells with multiple nuclei. Apoptosis is described in the HIVAN murine model and human biopsies. However, neither tubular cell hypertrophy nor multinucleation has been noted. Therefore, we 1053
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Control
Murine HIVAN
× 200 Control
Control
Murine HIVAN
Murine HIVAN
Murine HIVAN
× 200 Murine HIVAN
× 400
× 400
Figure 5 | Tubular cell hypertrophy and multinucleation in the murine HIVAN model. (a) Control murine kidney compared with murine HIVAN kidney at an original magnification of 200. A dilated tubule (arrowhead) and sclerotic glomeruli (arrow) are shown. (b) Tubular cell hypertrophy in murine HIVAN. Control and murine HIVAN sections were compared at an original magnification of 400. General hypertrophy can be appreciated and examples of individual cell hypertrophy and nucleus enlargement are shown (arrows). (c) Examples of control murine kidney tubules and multinucleated tubule cells in murine HIVAN. The original magnification was 600. All sections are PAS stains.
Table 1 | The protein content and DNA content of HK2 cells at 48 h after transduction with lentivirus containing pHR-Vpr or pHR-GFP as indicated pHR-VPR/pHR-GFP
Average s.d. (s)
Protein/DNA
DNA/cell
Protein/cell
1.06 0.11
1.56 0.16
1.65 0.03
The ratios shown indicate the mean and s.d. of a single experiment performed in triplicate. This experiment was representative of three experiments.
examined the Tg26 transgenic mouse, a model of HIVAN that expresses all of the HIV genes except gag and pol and shows classic HIVAN pathology (Figure 5a). The kidney sections from nephrotic (3 þ proteinuria) mice were inspected for enlarged and multinucleated tubule cells. Surprisingly, frequent examples of enlarged cells and multinucleated cells were seen (Figure 5b and c). Tubular cell hypertrophy was evident when comparing control with murine HIVAN at the same magnification. For each comparison, the HIVAN kidneys always had larger tubules appearing as if they were examined at a higher magnification (Figure 5b). These results resemble the phenotype seen when vpr is expressed in PTECs in vitro resulting in enlarged and multinucleated cells (Figure 2b). Tubular cell hypertrophy and increased chromosome number is observed in HIVAN
The examination of human HIVAN biopsy samples also revealed widespread hypertrophy similar to the murine 1054
HIVAN model (Figure 6). HIVAN biopsies had larger tubules compared with control human kidney biopsies with individual tubule cell hypertrophy also being apparent. In these HIVAN biopsies, multinucleation could not be conclusively demonstrated. However, many tubular cell nuclei were enlarged mimicking vpr expression in vitro. To determine if these nuclei had increased DNA content, we applied fluorescence in situ hybridization to the centromeric regions of chromosomes 8, X, and Y. Nuclei exceeding a diploid signal were indicative of increased DNA. The technique required 2 mm sections, much thinner than the diameter of a typical cell nucleus. Therefore, the signal number underestimated the actual chromosome number in the large majority of nuclei. This effect can be appreciated in a field view of the control kidney sample (Figure 7a upper panel). The arrow depicts a diploid tubular cell but neighboring cells show fewer hybridization signals. In contrast to the control, the HIVAN sample showed many tubular cell nuclei with an increased number of centromere hybridization signals (Figure 7a lower panel—arrowed, Figure 7b, and c). These results demonstrate a previously unrecognized aspect of HIVAN pathology that correlates with Vpr activity in vitro. DISCUSSION
The microcystic distortion of the tubulointerstitium is one of the most striking features of HIVAN. We now know that HIV-1 infects tubular cells in patients with HIVAN. In situ hybridization and laser capture microscopy combined with PCR have shown that HIV-1 mRNA is expressed in tubular cells and replicates within the renal compartment in HIVAN Kidney International (2008) 74, 1049–1058
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Control PAS
HIVAN PAS
HIVAN PAS
×400
×400
HIVAN PAS
×400
×400
Figure 6 | Tubule cell hypertrophy in human HIVAN biopsies. Compared with normal adult control kidney, the HIVAN kidneys display increased tubular diameter of noncystic tubules and diffuse hypertrophy of individual tubular epithelial cells. The original magnification of control and HIVAN sections was 400. All sections are PAS stains.
Control L
Aqua-8 Red-X
HIVAN L
Aqua-8
Figure 7 | Increased chromosome number in tubule cells in human HIVAN biopsies. (a) Renal FISH staining on a female human control (upper panel) and male human HIVAN biopsy (lower panel). L, tubular lumen; arrows, cell of normal ploidy (control) or increased ploidy (HIVAN). Centromeric enumeration probes (CEP) are Aqua, chromosome 8; red, chromosome X; and green, chromosome Y. The original magnification of control and HIVAN sections was 1000. (b) Selected examples of tubular cells with increased copy number for X and/or Y chromosomes in human HIVAN biopsies. (c) Selected examples of tubular cells with increased copy number of chromosome 8 in human HIVAN biopsies.
patients.4,5 HIV expression within transgenic mouse kidney produces the characteristic features of the disease that are identical to those observed in humans.39 As reported previously, tubular cell apoptosis is common and epithelial dedifferentiation is widespread as evidenced by changes in expression levels and patterns of characteristic cellular markers. The viral genes responsible for the tubular disease have not been defined nor has the mechanism been well established. Previous work has provided some insights; however, Dickie et al.40 reported increased tubular pathology Kidney International (2008) 74, 1049–1058
in transgenic mice expressing HIV-1 vpr, suggesting that Vpr has an important role in this aspect of HIVAN pathogenesis. HIV-1 vpr expression has already been shown to induce G2 cell cycle arrest and apoptosis of CD4 þ T cells. Thus, we hypothesized that Vpr might induce similar pathology in the renal tubule epithelium (RTE) of HIVAN patients and transgenic mice, although, ultimately, we found a different mechanism was responsible. To explore the possible role of Vpr in producing disease in tubular cells in HIVAN, we used an in vitro model of the 1055
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human proximal tubule cell and efficiently expressed HIV-1 vpr using a lentiviral vector. This approach allowed a detailed phenotypic analysis of the vpr-transduced cells and provided insight into Vpr’s pathologic mechanisms. In this study, we found that Vpr induced significant apoptosis in tubular cells in vitro. More prominently, however, the vpr expressing tubular cells became enlarged with enlarged or multiple nuclei. As this phenotype was not previously described in vivo, we examined kidney sections from the murine HIVAN model and human HIVAN biopsies. Surprisingly, RTE hypertrophy was striking in both mouse and human. Both RTE cell size and nuclei were significantly larger in both transgenic and HIVAN kidneys when compared with controls. We also found multinucleated RTE cells in transgenic mice. Although terms such as ‘reactive’ epithelial cells have been used before to describe some of these findings, the extent of cellular changes observed in both mouse and human is striking. In addition, the tubule cell hypertrophy may contribute to the gross pathologic kidney enlargement in HIVAN. On the basis of these findings in vitro and in vivo, we conclude that Vpr contributes to HIVAN tubular pathology by interfering with and disrupting tubule epithelial cell division. However, the examination of vpr transgenic murine models for hypertrophy and multinucleation will determine whether HIV-1 vpr expression is sufficient to cause these changes in vivo or it works in conjunction with other HIV proteins. Normally, the renal tubule epithelium is in a quiescent, non-proliferative state (G0). In response to acute injury, however, RTE cells have the capacity to proliferate.38 Previously we reported that a large fraction of tubule cells in the kidneys of HIV transgenic mice are positive for the proliferation marker Ki-67.35 This finding indicates that many tubule epithelial cells have re-entered the cell cycle. The impetus for cells exiting G0 is not entirely clear but could be in response to the concurrent apoptosis induced by HIV, epithelial injury, or a viral gene product such as Nef, which is known to induce proliferation.15 Whatever the cause, Vpr appears to interfere with tubule cell division leading to cell enlargement and multinucleation. The mechanism of Vpr-induced enlargement and multinucleation was investigated in vitro and appears to result from a specific defect in cell cytokinesis. HK2 cells expressing vpr continued to cycle through S phase, often multiple times, without cytokinesis occurring. This phenotype is distinct from Vpr-induced G2 arrest in lymphocytes where the cell cycle stops prior to mitosis through a classical checkpoint pathway. Differentiated proximal tubular epithelial cells display a Vpr phenotype similar to what has been described in various cell lines, primary HUVECs, and yeast.17,18,20,41 Vpr functions as an adaptor protein between an unknown target protein and DCAF1, a substrate specificity module in certain Cullin 4-based E3 ubiquitin ligase complexes. Proteasomal degradation of this putative target protein is proposed to trigger G2 arrest, apoptosis, and potentially other phenotypes induced by HIV-1 vpr expression. The 1056
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critical binding site of Vpr has been mapped by mutagenesis of the key amino acids between positions 65 and 80. The mutation of the Q65 residue prevents DCAF1 binding and impairs the ability of Vpr to induce G2 arrest.26,38 Other mutations that retain binding to DCAF1 can also inhibit the ability of Vpr to induce G2 arrest, possibly because the ability to bind to the unknown target protein is lost. Examples of such mutations include R73A, G75A, C76A, R80A, and R90A. In our study we examined four mutants, and the results were consistent with previous studies. Mutants I74A and R77Q acted as wild type, whereas both R80A and Q65R were completely inactive. Although there is a strong correlation between the in vitro Vpr phenotype in PTEC and pathology seen in the murine HIVAN model and HIVAN biopsies, hypertrophy, irregular cell division, and apoptosis are not the only tubular pathologies associated with HIVAN. Microcystic dilation remains an unexplained hallmark of HIVAN. We have shown previously that the cells lining the microcysts of transgenic mice are flattened with less brush border and no longer support transcription of HIV-1.35 One possibility is that microcyst formation is an end-stage result that arises after an initial hypertrophic expansion, and hypertrophied HIV-1expressing cells have died or transformed. In conclusion, we report that Vpr impairs cytokinesis in proximal tubular epithelial cells leading to cell enlargement and often multinucleation. The examination of kidney sections from human and mouse showed extensive tubular hypertrophy, multinucleation, and evidence of increased chromosome number. These in vivo results reveal new aspects of HIVAN pathology. Given Vpr’s central role in HIVAN pathogenesis, we anticipate it may provide a good molecular target for future therapeutic intervention. MATERIALS AND METHODS Cell culture system The HK2 line of human proximal tubule cells was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Defined Keratinocyte Serum-Free Media (Gibco, Carlsbad CA, USA, 17005-042) with 5 ng/ml of recombinant epidermal growth factor and 0.05 mg/ml of bovine pituitary extract at 37.0 1C, 5% CO2 to preserve phenotype. Ryan et al.42 characterized this line as phenotypically similar to freshly isolated PTECs including Na þ -dependent sugar transport. 293T Cells were used to produce pseudotyped virus and cultured in Dulbecco’s modified Eagle’s medium with fetal bovine serum (10%), penicillin (10,000 U/ ml), and streptomycin (10 mg/ml). Pseudotyped lentivirus vector infection A pseudotyped lentivirus vector was used to efficiently transduce the desired genes into the HK2 cell line. This virus was produced by cotransfection (Invitrogen Lipofectamine 2000, Invitrogen (Carlsbad, CA, USA)) of 293T cells with three separate plasmids: pVSV.G expressing the VSV envelope, the packaging plasmid pHIVgag/pol expression gag and pol in trans, and the specific viral or pHR plasmid expressing the package RNA as previously described.15 Forty-eight hours after plasmid transfection, the supernatant was collected and filtered using a 0.45 mm filter (Millipore, Billerica, MA, Kidney International (2008) 74, 1049–1058
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USA). Virus was titered by infection of 293T cells, and an multiplicity of infection of 3 was used to infect PTECs to achieve maximum infection. Proliferation assay The cell proliferation assay was performed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA). Forward scatter (FSC þ ) side scatter (SSC þ ) events were counted for a set interval at flow speeds low, medium, and high. All values are at subconfluence. Error bars reflect the intra-assay variability and are representative of a minimum of two independent experiments. Cell cycle assay and analysis Cell cycle analysis was performed by flow cytometry (FACSCalibur and FacScan, BD Biosciences, San Jose, CA, USA). Cells were fixed and permeabilized by the addition of 20 1C ethanol to a final concentration of 50%. The fixed cells were treated with 100 mg/ml of propidium iodide and 0.5 mg/ml of RNAse A for 3 h prior to cytometry. FloJo (version 6.4.7) was used for all analysis. Microscopy Cells were fixed in cold 100% ethanol and stained with propidium iodide (3 mM) for 10 min at room temperature and examined. Representative fields were photographed in fluorescent and monochrome light and merged for publication. Apoptosis assays Quantification of the sub-G1 peak in cell cycle analysis was measured as a fraction of a FSC þ SSC þ -gated population. Annexin V staining was performed using Annexin V-APC antibody (Molecular Probes, Carlsbad CA, USA, A35110) at 5 ml per 1 106 cells. Propidium iodide co-staining was performed at final concentration of 1.5 mg/ml.
Vysis Paraffin Pretreatment Kit III protocol (Vysis/Abbott Molecular, Abbott Park, IL, USA). Denaturation of the target DNA on slides and probes was performed simultaneously at 73 1C for 6 min on a Thermobrite System (Vysis/Abbott Molecular). Hybridization was carried out in a humidified chamber at 37 1C overnight using chromosome enumeration probe 8 (CEP-8, Abbott Molecular, 32-131008) and/or CEP-X/CEP-Y (Abbott Molecular, 30-161050). 40 -6-Diamidino-2-phenylindole (Vysis/Abbott Molecular) was used as a counterstain. The slides were analyzed and imaged with the Cytovysion 3.92 program (Applied Imaging/Genetix, New Milton, UK) and Axioplan II microscope (Zeiss, Oberkochen, Germany). DISCLOSURE
The authors declare no competing financial interests. ACKNOWLEDGMENTS
This study was funded by the HIVAN Program Project Grant: NIH NIDDK-P01DK56492-01, principal investigator: Paul E. Klotman. INFORMED CONSENT AND ETHICS The research involving human subjects and animals was performed in accordance with the ethical standards of the responsible institutional committees on human subjects research under the approved Institutional Review Board protocol number 98-953 and the Institutional Animal Care and Use Committee, approved protocol number 98-0953. REFERENCES 1.
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Drug treatments Caffeine (Fisher Scientific, Hampton, NH, USA) was dissolved in water applied to PTECs at a final concentration of 1–3 mM. Dihydrocytochalasin B (Fisher Scientific) in dimethyl sulfoxide solution was applied to PTECs at a final concentration of 10 mM. DNA and protein quantification HK2 cells were infected as described and resuspended 48 h later. DNA and protein were harvested from equal volumes of resuspended cells in triplicate. DNA and protein concentrations were measured by spectrophotometry using the NanoDrop ND1000 Thermo Scientific, Wilmington, DE, USA. Values were normalized to cell number as measured by flow cytometry. The data shown are representative of two independent experiments. Histology HIV transgenic (Tg26 murine line) FVB/N mice with 3-plus proteinuria were killed and examined. Comparisons were made to wild-type FVB/N mice. Human HIVAN biopsies were compared with normal control biopsies. Representative areas were chosen for publication.
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