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*
Division of Nephrology, Mount Sinai Medical Center, New York, New
York
Division of Gene Therapy, Mount Sinai Medical Center, New York, New
York
Laboratory of Virology, Istituto Superiore di
Sanità, Rome, Italy
Division of Nephrology, Albert Einstein College of Medicine, Bronx, New
York.
Correspondence to Elissa J. Schwartz, Box 1243, Division of Nephrology, Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029. Phone: 212-241-6155; Fax: 212-987-0389; E-mail: schwae01{at}doc.mssm.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies with HIV-1 transgenic murine models have been critical to our understanding of HIVAN (9,10,11). We previously reported the establishment of a murine transgenic line in which a gag/pol deletion construct of HIV-1 provirus pNL4-3 was used as the transgene under the control of the viral LTR. Env and regulatory genes are expressed, but no infectious virus is produced. Yet renal disease develops in these mice identical to HIVAN in humans. HIV-1 mRNA is expressed in the exact sites of injury in HIVAN in both human and mouse, namely renal glomerular and tubular epithelium (12,13). Markers for proliferation have been localized to these cells as well (7,14). A causal role for HIV-1 in inducing proliferation of epithelial cells has been difficult to establish.
The renal epithelial cell, in particular the glomerular visceral epithelial cell, or podocyte, has emerged as the glomerular target of HIV-1 pathogenesis. Podocytes are terminally differentiated, highly specialized renal cells that function in the regulation of glomerular filtration. Podocyte injury is thought to play a central role in the development of focal segmental glomerulosclerosis (15), particularly the collapsing variant (16). In idiopathic collapsing glomerulopathy and HIVAN, podocytes are thought to proliferate, but in one report the hyperplastic cells are hypothesized to originate from parietal epithelial cells (17). Observed changes in podocyte structural features, the loss of differentiation markers, and the increase in proliferation markers in HIVAN patients suggest that dysregulation of the podocyte leads directly to the development of HIVAN (7). Identical alterations have been found in the transgenic murine model, including increased markers of proliferation and loss of markers of differentiation (14). The current hypothesis, then, suggests that HIV-1 gene expression in podocytes results in the deterioration of nephron architecture and loss of glomerular structure and function.
Evidence suggests that expression of viral gene products may play a central role in HIVAN pathogenesis, but no in vitro culture system has been established to permit a detailed investigation of HIV-1 gene expression in podocytes and the potential mechanism by which viral gene expression induces disease. The aim of the present study was to establish such an in vitro culture system and to determine whether HIV-1 gene expression directly causes podocyte proliferation. We found that HIV-1 gene expression induces podocyte proliferation and causes a loss of contact inhibition. The results presented here suggest that HIV-1 gene expression is causative in the podocyte proliferation seen in HIVAN in both humans and transgenic mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN-) to induce the H-2Kb promoter driving synthesis of the
temperature-sensitive (tsA58) SV-40 T antigen ("permissive
conditions"). For allowing differentiation, cells were cultured at
37°C without rIFN-, resulting in degradation of the T antigen
("nonpermissive conditions").
Cell Growth Assays
HIV-1 transgenic and nontransgenic podocytes were plated in 96-well
flat-bottom microtiter plates at various densities (1000, 10,000, 50,000, or
100,000 cells/100 µl) and assayed for [3H] thymidine
incorporation on day 3 or day 7 after 18 h of incubation. Cell growth was also
quantified on day 7 by use of the CellTiter 96 Cell Proliferation Assay
(Promega, Madison, WI) according to manufacturer's instructions and by
hemocytometric counting after trypan blue dye exclusion. To determine cell
doubling times, we seeded 500 cells of each clone in 96-well plates with 100
µl of medium and allowed them to grow for 14 d. Triplicate wells were
trypsinized and counted after trypan blue dye exclusion at 24-h intervals. All
growth rate studies were carried out in complete medium supplemented with
rIFN-, at 33°C, to allow proliferation.
Preparation of Conditioned Medium
HIV-1 transgenic cells were plated at high density, and culture supernatant
was removed after 36 h. Conditioned medium was then centrifuged for 10 min to
remove cellular debris, filtered, and frozen at -80°C until use. For
growth studies, 50,000 cells were plated in flat-bottom 96-well culture plates
either in conditioned medium with fresh medium in a 1:1, 1:4, or 1:10 dilution
or in fresh medium alone (as controls). All media were complete and
supplemented with rIFN-. On day 4, medium was removed and replenished
with freshly prepared conditioned medium in identical concentrations. On day
7, cells were assayed for [3H] thymidine uptake after 18 h of
incubation.
Flow Cytometry
HIV-1 transgenic and nontransgenic cells were grown in flasks at 33°C
to 90 to 95% confluence. Cells (106) were trypsinized, washed with
1x phosphate-buffered saline and fixed with ice-cold 80% ethanol with
gentle agitation. After incubation at -20°C for 30 min, cells were
pelleted, resuspended in 1x phosphate-buffered saline, and passed
through a 25-G needle to create a single cell suspension. RNase (1 mg/ml) and
propidium iodide (400 µg/ml) were added, and cells were incubated for 30
min at 37°C. Cell cycle analysis was performed on a FACSCalibur flow
cytometer, and results were analyzed with CellQuest software (Becton
Dickinson, Mountain View, CA). Doublet exclusion was performed by pulse shape
analysis (height versus area of the fluorescence pulse). The
percentage of cells in the proliferative (S/G2/M) and resting
(G0/G1) phases of the cell cycle was determined. Cells
with subdiploid DNA content were considered apoptotic.
Reverse Transcription and PCR
To confirm HIV-1 gene expression in podocyte clones, we extracted total
cellular RNA with TRIZOL Reagent (Life Technologies BRL, Grand Island, NY)
from HIV-1 transgenic and nontransgenic clonal cultures grown to 90 to 100%
confluence at 33°C according to manufacturer's protocol. Extracted RNA was
DNase-treated (Life Technologies BRL) and stored at -80°C until use.
Alternatively, total cellular RNA was isolated by use of the RNeasy Mini Kit
with the RNase-Free DNase Set (QIAGEN, Valencia, CA) as per manufacturer's
protocols. RNA (5 µg) was reverse-transcribed with random primers by use of
the Reverse Transcription System (Promega). One fourth of the first-strand
cDNA synthesis reaction product was used as template for PCR amplification by
use of AmpliTaq DNA Polymerase (Perkin Elmer, Branchburg, NJ) and 5
ng/µl each of HIV-1 env-specific primers QS-Env-2s (TGT CCA AAG
GTA TCC TTT GAG CCA ATT CC) and QS-Env-2as (AGT AGA AAA ATT CCC CTC CAC AAT
TAA). Mouse G3PDH primers (Clontech Laboratories, Palo Alto, CA) were used as
positive controls. After a 3-min hot start at 94°C, 40 cycles of
amplification were carried out with the following parameters: denaturation at
94°C for 1 min, annealing at 62°C for 1 min, and synthesis at 72°C
for 30 s, with a 5-s increment extension per cycle. A 7-min extension at
72°C was included at the end of the last cycle. PCR products were resolved
on a 1% agarose gel and visualized with ethidium bromide staining.
After soft agar assays, aggregates were removed from soft agar with a pipette, boiled at 56°C for 10 min in TRIZOL Reagent or RNeasy Buffer RLT, and centrifuged for 5 min to remove residual agar. Total cellular RNA then was extracted as described.
Growth in Soft Agar
HIV-1 transgenic and nontransgenic podocytes were grown in soft agar in
sextuplet in 6-cm plates. Cells (10,000 or 100,000) were suspended in 5 ml
0.2% agar (Fisher Scientific, Fair Lawn, NJ) in complete medium with
rIFN-, overlaid on 5-ml presolidified 0.4% agar in the same medium, and
incubated at 33°C for up to 8 wk. Normal complete medium with rIFN-
(1 ml) was layered gently over the cultures every 5 d. Results were scored by
three independent observers, who counted all planes of view of 10 fields each,
10x power. Viability of cells was ascertained by staining with vital dye
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride for 48 h.
Construction of Lentiviral Vectors and Infection
For production of pseudotyped virus containing the HIV-1 transgene, 293T
cells were contransfected with transcomplementing vector pCMV
R9.1
(provided by Dr. Didier Trono, University of Geneva, Geneva, Switzerland),
HIV-1 gag/pol deletion construct pNL4-3 d1443, and vesicular stomatitus virus
G (VSVG) envelope vector pMD.G. For production of pseudotyped control virus
expressing only green fluorescent protein (GFP) but no HIV-1 viral
transcripts, 293T cells were contransfected with transcomplementing vector
CMVR8.2 (provided by Dr. Inder Verma, The Salk Institute for Biological
Studies, La Jolla, CA), pHR'-CMV-GFP (constructed by inserting eGFP
plasmid [Clontech Laboratories] into the vector pHR'-CMV-Lucif [22]
between the sites BamHI and XhoI), and pMD.G. VSV G envelope
pseudotyping allows highly efficient entry in a wide variety of cell types.
Nontransgenic podocytes (60,000) were grown to 50 to 80% confluence in 6-well
plates and infected by these lentiviral vectors. Green fluorescence was seen
in control-GFP—infected cells after 3 d. Subsequently, cells were
suspended in soft agar as described.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of HIV-1 and Podocyte Markers
To confirm HIV-1 gene expression in podocyte clones, we performed reverse
transcription-PCR (RT-PCR) on total cellular RNA extracted from the HIV-1
transgenic clone and the nontransgenic clone grown at subconfluence and at
confluence at 33°C. Primers were specific for HIV-1 env and for
the housekeeping gene G3PDH, as a control. As shown in
Figure 2, a 541-bp fragment of
HIV-1 env was amplified from the transgenic clone grown at both
subconfluence and confluence. Nontransgenic cells did not express HIV-1
env. All cells expressed G3PDH.
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The expression of podocyte markers WT-1, synaptopodin, and podocalyxin was confirmed by RT-PCR (for WT-1) and Northern blot analysis (for synaptopodin and podocalyxin) in both the HIV-1—transgenic and -nontransgenic clones (data not shown). Consistent with the loss of differentiation markers seen in both humans (7) and the transgenic murine model (14), expression of all three markers was downregulated in the nontransgenic podocytes infected with the HIV-1 deletion construct, as described below (data not shown).
Cell Growth Rate Studies
A sigmoidal growth curve was observed for both transgenic and nontransgenic
podocytes. Both clones grew at comparable rates until they reached confluence
on day 8. At confluence, nontransgenic cell growth plateaued as expected. The
transgenic clone, however, continued to proliferate
(Figure 3). The differences in
cell number were not statistically significant before confluence (P
> 0.5), but they were significant after confluence (P < 0.001).
Before confluence, the mean doubling time for both nontransgenic and
transgenic podocytes was 0.7 d. After confluence, however, nontransgenic
podocyte doubling time was 8.1 d, whereas transgenic podocyte doubling time
was 4.5 d. To explore the apparent differences in growth properties between
the transgenic and nontransgenic cells, we performed the following studies at
95 to 100% confluence at 33°C.
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Tritiated Thymidine Incorporation
Incorporation of [3H] thymidine was measured in transgenic and
nontransgenic podocytes. Cells were plated at the same density, grown under
permissive conditions, and measured for [3H] thymidine uptake after
7 d. Independent of initial cell density, the transgenic clone incorporated
more [3H] thymidine, compared with the nontransgenic clone
(Figure 4). The increase in
[3H] thymidine was two- to threefold. The same results were
obtained at 3 or 7 d, at 33°C or 37°C, and with or without
collagen-coated plates. No difference was seen in SV-40 T antigen expression
with the HIV-1 transgene (data not shown).
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Identical results also were seen when podocyte growth patterns were assessed by use of a proliferation assay that measures cellular metabolism and by enumeration of cells after trypan blue dye exclusion (data not shown). Growth patterns seen with the [3H] thymidine incorporation assay and proliferation assay were consistent with those found measuring cell growth rates and doubling times (Figure 3).
Cell Cycle Analysis
Cell counts and [3H] thymidine incorporation measurements do not
provide information on the proportion of cells in a culture that are actively
progressing through the cell cycle. To determine the percentage of cells in
culture that were in the proliferative phase of the cell cycle (S,
G2, or M), we performed flow cytometric cell cycle analysis by
using propidium iodide staining. As shown in
Figure 5, the percentage of
HIV-1—transgenic cells in the proliferating phase (39.4%) was more than
twice that of nontransgenic cells (18.2%). In addition, the percentage of
apoptotic cells in nontransgenic podocytes (1.4%) was seven times that of
transgenic podocytes (0.2%). Hence, a greater percentage of transgenic cells
were cycling, correlating with the [3H] thymidine incorporation
studies. These data indicated that HIV-1—transgenic podocytes
proliferate faster than non-transgenic podocytes at confluence in
vitro, consistent with in vivo studies that show increased
proliferation in both the murine model and humans
(14,16).
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[3H] Thymidine Incorporation with Conditioned Medium
To determine whether proliferation was due to an intracellular or
extracellular effect of HIV-1 gene expression, we performed the following
studies. Stimulation of nontransgenic podocyte growth by conditioned medium
from transgenic podocytes was tested to explore whether a secreted factor was
sufficient for increased growth of transgenic cells. Tritiated thymidine
incorporation was measured in nontransgenic cells cultured with conditioned
medium from transgenic culture supernatants. Conditioned medium was prepared
in 1:1, 1:4, and 1:10 dilutions with fresh medium. As controls, nontransgenic
cells were cultured with conditioned medium from non-transgenic cultures in
the same dilutions, and transgenic and nontransgenic cells also were cultured
with fresh medium alone, as before.
Conditioned medium from transgenic podocytes did not stimulate [3H] thymidine incorporation in nontransgenic cultures at 1:1, 1:4, and 1:10 dilutions (Figure 6). Transgenic cell [3H] thymidine uptake increased three- to fourfold over uptake in nontransgenic cells, consistent with results shown in Figure 4. These data suggest that the accelerated growth by transgenic podocytes was not due to a secreted factor.
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Growth in Soft Agar
The finding that transgenic podocytes continued to grow after reaching
confluence suggests that they were not contact inhibited. To test this
hypothesis, we grew transgenic and nontransgenic cells in a soft agar matrix,
which typically inhibits untransformed cell growth. Although nontransgenic
podocytes remained as isolated single cells suspended in soft agar
(Figure 7A), transgenic
podocytes continued to proliferate, forming aggregates
(Figure 7B). Aggregates were
first seen in transgenic cultures after 2 wk. The largest differences in
aggregate formation between transgenic and nontransgenic cultures were
observed after 4 wk. The average number of aggregates per field in transgenic
cultures (11.07) was 85 times that in nontransgenic cultures (0.13). The
number of cells in a typical aggregate in transgenic cultures was 16 to 32
cells, whereas that in nontransgenic cultures was 4 to 6 cells. Thus,
transgenic podocytes were less contact inhibited in soft agar, compared with
nontransgenic podocytes.
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Growth of Nontransgenic Podocytes Infected with Pantropic Viruses in
Soft Agar
Formation of aggregates in soft agar by transgenic podocytes may have been
the result of clonal selection. To prove that HIV-1 was causative in this
process, we introduced the deletion construct from the transgenic mouse into
the nontransgenic clone using a pantropic virus. Nontransgenic podocytes
(Figure 1A) were infected with
a VSV G-pseudotyped lentivirus that contained pNL4-3 d1443. Nontransgenic
control podocytes were infected with a VSV G-pseudotyped virus expressing only
GFP. Green fluorescence was observed in control-infected cells after 3 d.
Subsequently, infected nontransgenic cells, as well as uninfected
nontransgenic and transgenic cells as controls, were plated in soft agar as
described, 3 d after infection.
Identical to the transgenic podocytes, the HIV-1 transgene-infected podocytes proliferated in the soft agar matrix, forming large aggregates (Figure 7D). The control-GFP—infected podocytes, however, did not form aggregates (Figure 7C). Uninfected transgenic cells formed aggregates as before. Aggregates were first observed after 2.5 wk and were quantified after 4.5 wk, when the maximum difference between HIV-1 transgene—infected and control-GFP—infected cultures was seen. A 136-fold difference was found between the average number of aggregates per field in transgene-infected cultures (19.00) and control-infected cultures (0.14). Aggregates were much larger than those seen previously, with a typical aggregate containing 64 to 128 cells. Thus, delivery of the HIV-1 transgene to the nontransgenic podocyte clone resulted in their growth in a soft agar matrix, demonstrating that HIV-1 directly induced a loss of contact inhibition in these cells.
Confirmation of HIV-1 Gene Expression in Soft Agar Aggregates
To confirm that the proliferating cells contained the HIV-1 construct, we
extracted aggregates from soft agar cultures. RT-PCR was performed on total
cellular RNA isolated from the aggregates. As shown in
Figure 8, a 541-bp fragment of
HIV-1 env was readily amplified from aggregates in HIV-1
transgene—infected cultures and from transgenic aggregates but not from
isolated single cells in HIV-1 transgene—infected cultures,
control-GFP—infected cultures, or nontransgenic cultures. Cells from all
cultures produced a 983-bp signal for the housekeeping gene G3PDH. It is
noteworthy that no amplified HIV-1 product was found in single cells isolated
from transgene-infected cultures, which suggests that cells uninfected by the
VSV-pseudotyped virus contained no HIV-1 env and did not proliferate,
even in transgene-infected cultures. The 983-bp G3PDH signal indicated the
presence of RNA in this sample.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine whether HIV-1 induces podocyte proliferation, we established a podocyte cell culture system that closely resembles our transgenic mouse model, because it contains the HIV-1 transgenic deletion construct. We found that transgenic cells proliferated at a faster rate than nontransgenic cells at confluence (Figure 3). This difference was not attributable to a soluble factor (Figure 6), although the role of a labile soluble factor or the requirement for both a soluble factor and cell autonomous effects induced by HIV-1 cannot be excluded. It is notable that cell synchronization was not necessary to see the differences in growth patterns between the transgenic and nontransgenic clones. SV-40 T antigen may play a role in this cell culture system, but it is not sufficient to enhance growth, because the nontransgenic cells (expressing T antigen but not HIV-1 genes) did not proliferate like the HIV-1 transgenic cells. It is striking that transgenic cells formed aggregates in a soft agar matrix, whereas nontransgenic cells did not (Figure 7). This suggests that HIV-1—transgenic cells were not contact inhibited. Furthermore, the nontransgenic podocyte clone infected with the HIV-1 transgene construct also formed aggregates (Figure 7), which confirmed that this effect was the direct result of HIV-1 gene expression and not of clonal variation.
Although direct infection of the kidney by HIV-1 has been debated during recent years, new data provide convincing evidence that HIV-1 replicates in renal epithelium. Using in situ hybridization of renal biopsy tissue from HIVAN patients, Bruggeman et al. (13) showed that HIV-1 mRNA is detectable in renal epithelial cells, including visceral and parietal glomerular epithelial cells and tubular epithelial cells, as well as in interstitial cells. The detection of extrachromosomal circularized proviral DNA by PCR, which indicates nuclear import of HIV-1 after recent infection, as well as proviral DNA by in situ PCR, confirms further that the kidney is a target for HIV-1 infection and viral replication in man.
In summary, several mechanisms may contribute to the increased growth of podocytes. The most likely explanation is that single or multiple HIV-1 gene products directly impair cell—cell or cell—extracellular matrix interactions to induce a loss of contact inhibition, which then alters cell cycle progression and results in enhanced proliferation.
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Acknowledgments |
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X174 marker; lane 2, pNL4-3 d1443 DNA positive control
(top), G3PDH DNA positive control (bottom); lane 3, negative
control (water); lane 4, HIV-1 transgenic clone, at confluence; lane 5, HIV-1
transgenic clone, at subconfluence; lane 6, nontransgenic clone, at
confluence; lane 7, nontransgenic clone, at subconfluence.
nontransgenic; 
HIV-1 transgenic. Experiments
were repeated three times.
