G. Luca Gusella

Replication and compartmentalization of HIV-1 in kidney epithelium of patients with HIV-associated nephropathy

HIV-associated nephropathy is a clinicopathologic entity that includes proteinuria, focal segmental glomerulosclerosis often of the collapsing variant, and microcystic tubulointerstitial disease. Increasing evidence supports a role for HIV-1 infection of renal epithelium in the pathogenesis of HIV-associated nephropathy. Using in situ hybridization, we previously demonstrated HIV-1 gag and nef mRNA in renal epithelial cells of patients with HIV-associated nephropathy. Here, to investigate whether renal epithelial cells were productively infected by HIV-1, we examined renal tissue for the presence of HIV-1 DNA and mRNA by in situ hybridization and PCR, and we molecularly characterized the HIV-1 quasispecies in the renal compartment. Infected renal epithelial cells were removed by laser-capture microdissection from biopsies of two patients, DNA was extracted, and HIV-1 V3-loop or gp120-envelope sequences were amplified from individually dissected cells by nested PCR. Phylogenetic analysis of kidney-derived sequences as well as corresponding sequences from peripheral blood mononuclear cells of the same patients revealed evidence of tissue-specific viral evolution. In phylogenetic trees constructed from V3 and gp120 sequences, kidney-derived sequences formed tissue-specific subclusters within the radiation of blood mononuclear cell-derived viral sequences from both patients. These data, along with the detection of HIV-1-specific proviral DNA and mRNA in tubular epithelium cells, argue strongly for localized replication of HIV-1 in the kidney and the existence of a renal viral reservoir.

HIV-1 Nef Induces Proliferation and Anchorage-Independent Growth in Podocytes

HIV-associated nephropathy (HIVAN) is now the third leading cause of end-stage renal disease in the African American population. HIV-1 infects renal tubular and glomerular epithelial cells or podocytes, cells that are a critical part of the filtration barrier. HIV-1 infection induces the loss of podocyte differentiation markers and increases podocyte proliferation. It has been previously shown that HIV-infection induces loss of contact inhibition. Here, the HIV-1 gene responsible for proliferative changes is identified by using cultured podocytes in vitro. The HIV-1 proviral construct, pNL4-3 was rendered noninfectious by replacing the HIV-1 gag/pol sequences with an EGFP reporter gene (pNL4-3: DeltaG/P-EGFP). This construct was then pseudotyped with VSV.G envelope to infect podocytes that were conditionally immortalized with SV-40 T antigen. In addition, mutated constructs were engineered with premature stop codons in the HIV-1 env, vif, vpr, vpu, nef, or rev genes. The parental construct and all the other mutated constructs, with the exception of nef, induced proliferation under nonpermissive conditions and anchorage-independent growth (colony formation in soft agar) under permissive conditions. In contrast, deletion of nef markedly reduced proliferation and colony formation. Although tat alone, or tat plus rev induced marginal levels of anchorage-independent growth, coexpression with nef significantly increased colony formation. Finally, stable expression of Nef in a retroviral vector, pBabe-puro, was sufficient to induce increased proliferation and colony formation. Moreover, nef induced saturation density and loss of contact inhibition. These data indicate that Nef induces multiple proliferative effects in podocytes in culture and that nef may therefore be an important gene in the pathogenesis of HIVAN in vivo.

Loss of polycystin-1 causes centrosome amplification and genomic instability

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic disease predominantly caused by alteration or dysregulation of the PKD1 gene, which encodes polycystin-1 (PC1). The disease is characterized by the progressive expansion of bilateral fluid-filled renal cysts that ultimately lead to renal failure. Individual cysts, even within patients with germline mutations, are genetically heterogeneous, displaying diverse chromosomal abnormalities. To date, the molecular mechanisms responsible for this genetic heterogeneity remain unknown. Using a lentiviral-mediated siRNA expression model of Pkd1 hypomorphism, we show that loss of PC1 function is sufficient to produce centrosome amplification and multipolar spindle formation. These events lead to genomic instability characterized by gross polyploidism and mitotic catastrophe. Following these dramatic early changes, the cell population rapidly converges toward a stable ploidy in which centrosome amplification is significantly decreased, though cytological abnormalities such as micronucleation, chromatin bridges and aneuploidy remain common. In agreement with our in vitro findings, we provide the first in vivo evidence that significant centrosome amplification occurs in kidneys from conditional Pkd1 knockout mice at early and late time during the disease progression as well as in human ADPKD patients. These findings establish a novel function of PC1 in ADPKD pathogenesis and a genetic mechanism that may underlie the intrafamilial variability of ADPKD progression.

Role of Ubiquitin-Like Protein FAT10 in Epithelial Apoptosis in Renal Disease

Dysregulated apoptosis of renal tubular epithelial cells (RTEC) is an important component of the pathogenesis of several renal diseases, including HIV-associated nephropathy (HIVAN), the most common cause of chronic kidney failure in HIV-infected patients. In HIVAN, RTEC become infected by HIV-1 in a focal distribution, and HIV-1 infection has been shown to induce apoptosis in vitro. In microarray studies that used a novel renal tubular epithelial cell line from a patient with HIVAN, it was found that the ubiquitin-like protein FAT10 is one of the most upregulated genes in HIV-infected cells. Previously, FAT10 was shown to induce apoptosis in murine fibroblasts. The expression of FAT10 in HIVAN and the ability of FAT10 to induce apoptosis in human RTEC therefore were studied. This study revealed that FAT10 expression is induced after infection of RTEC by HIV-1 and that expression of FAT10 induces apoptosis in RTEC in vitro. Moreover, it was found that inhibition of endogenous FAT10 expression abrogated HIV-induced apoptosis of RTEC. Immunohistochemical studies demonstrated increased FAT10 expression in a murine model of HIVAN, in HIVAN biopsy samples, and in autosomal dominant polycystic kidney disease, another renal disease that is characterized by cystic tubular enlargement and epithelial apoptosis. These results suggest a novel role for FAT10 in epithelial apoptosis, which is an important component of the pathogenesis of many renal diseases.

Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.

Lentiviral gene transduction of kidney

Gene transfer into kidney holds great potential as a novel therapeutic approach. We have studied the transduction of kidney in vivo after delivery of lentiviral vectors by various routes of administration. A lentiviral vector expressing the bacterial lacZ gene from the cytomegalovirus early promoter was used. The lentiviral vector was delivered into the kidneys of BALB/c mice by retrograde infusion into the ureter, by injection into the renal vein or artery, or by direct injection into the renal parenchyma. Expression of the reporter gene was achieved independently of the route of administration, although it appeared more efficient after parenchymal or ureteral administration. After parenchymal or ureteral infusion, expression of the transgene was localized to the outer medulla and corticomedullary junction. In the case of parenchymal injection, expression of the reporter gene extended to the cortex. Detection of the transgene in the renal proximal tubules was confirmed by in situ polymerase chain reaction after parenchymal or ureteral infusion. On delivery of the lentiviral vector through the renal artery or vein, expression of the reporter gene was markedly lower than was observed with parenchymal or ureteral infusion and was limited to the inner medullary collecting ducts. No apparent histological abnormality was observed after virus administration and transgene expression was stable for at least 3 months. These results provide the first evidence that lentiviral vectors can stably transduce renal cells in vivo and may be effective vehicles for gene delivery to the kidney.

Primary cilia dynamics instruct tissue patterning and repair of corneal endothelium

Primary cilia are required for several signaling pathways, but their function in cellular morphogenesis is poorly understood. Here we show that emergence of an hexagonal cellular pattern during development of the corneal endothelium (CE), a monolayer of neural crest-derived cells that maintains corneal transparency, depends on a precise temporal control of assembly of primary cilia that subsequently disassemble in adult corneal endothelial cells (CECs). However, cilia reassembly occurs rapidly in response to an in vivo mechanical injury and precedes basal body polarization and cellular elongation in mature CECs neighboring the wound. In contrast, CE from hypomorphic IFT88 mutants (Tg737orpk) or following in vivo lentiviral-mediated IFT88 knockdown display dysfunctional cilia and show disorganized patterning, mislocalization of junctional markers, and accumulation of cytoplasmic acetylated tubulin. Our results indicate an active role of cilia in orchestrating coordinated morphogenesis of CECs during development and repair and define the murine CE as a powerful in vivo system to study ciliary-based cellular dynamics.

Mechanoregulation of intracellular Ca2+ in human autosomal recessive polycystic kidney disease cyst-lining renal epithelial cells

Mutations of cilia-expressed proteins are associated with an attenuated shear-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in renal epithelial cell lines derived from murine models of autosomal recessive polycystic kidney disease (ARPKD). We hypothesized that human ARPKD cyst-lining renal epithelial cells also exhibited dysregulated mechanosensation. To test this, conditionally immortalized cell lines derived from human fetal ARPKD cyst-lining (pool and clone 5E) cell lines with low levels of fibrocystin/polyductin expression and age-matched normal collecting tubule [human fetal collecting tubule (HFCT) pool and clone 2C] cell lines were grown in culture, loaded with a Ca(2+) indicator dye, and subjected to laminar shear. Clonal cell lines were derived from single cells present in pools of cells from cyst-lining and collecting tubules, microdissected from human kidney. Resting and peak [Ca(2+)](i) were similar between ARPKD 5E and pool, and HFCT 2C and pool; however, the flow-induced peak [Ca(2+)](i) was greater in ARPKD 5E (700 +/- 87 nM, n = 21) than in HFCT 2C (315 +/- 58 nM, n = 12; P < 0.01) cells. ARPKD 5E cells treated with Gd(3+), an inhibitor of nonselective cation channels, inhibited but did not abolish the shear-induced [Ca(2+)](i) transient. Cilia were approximately 20% shorter in ARPKD than HFCT cells, but no difference in ciliary localization or total cellular expression of polycystin-2, a mechanosenory Gd(3+)-sensitive cation channel, was detected between ARPKD and HFCT cells. The intracellular Ca(2+) stores were similar between cells. In summary, human ARPKD cells exhibit an exaggerated Gd(3+)-sensitive mechano-induced Ca(2+) response compared with controls; whether this represents dysregulated polycystin-2 activity in ARPKD cells remains to be explored.

Fluid shear stress induces renal epithelial gene expression through polycystin-2-dependent trafficking of extracellular regulated kinase.

Background: The cilium and cilial proteins have emerged as principal mechanosensors of renal epithelial cells responsible for translating mechanical forces into intracellular signals. Polycystin-2 (PC-2), a cilial protein, regulates flow/shear-induced changes in intracellular Ca2+ ([Ca2+]i) and recently has been implicated in the regulation of mitogen-activated protein (MAP) kinases. We hypothesize that fluid shear stress (FSS) activates PC-2 which regulates MAP kinase and, in turn, induces MAP kinase-dependent gene expression, specifically, monocyte chemoattractant protein-1 (MCP-1). Methods: To test this, PC-2 expression was constitutively reduced in a murine inner medullary collecting duct (IMCD3) cell line, and the expression of FSS-induced MCP-1 expression and MAP kinase signaling compared between the parental (PC-2-expressing) and PC-2-deficient IMCD3 cells. Results: FSS induces MAP kinase signaling and downstream MCP-1 mRNA expression in wild-type IMCD3 cells, while inhibitors of MAP kinase prevented the FSS-induced MCP-1 mRNA response. In contradistinction, FSS did not induce MCP-1 mRNA expression in PC-2-deficient cells, but did increase activation of the upstream MAP kinases. Wild-type cells exposed to FSS augmented the nuclear abundance of activated MAP kinase while PC-2-deficient cells did not. Conclusions: PC-2 regulates FSS-induced MAP kinase trafficking into the nucleus of CD cells.

Lentiviral gene delivery to CNS by spinal intrathecal administration to neonatal mice

Direct injection of lentivectors into the central nervous system (CNS) mostly results in localized parenchymal transgene expression. Intrathecal gene delivery into the spinal canal may produce a wider dissemination of the transgene and allow diffusion of secreted transgenic proteins throughout the cerebrospinal fluid (CSF). Herein, we analyze the distribution and expression of LacZ and SEAP transgenes following the intrathecal delivery of lentivectors into the spinal canal.