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Renal Transfer of Genetically Engineered Cells

Department of Medicine, University College Medical School, University College London, Jules Thorn Institute, Middlesex Hospital, London, United Kingdom.

Correspondence to Dr. Masanori Kitamura, Department of Medicine, University College Medical School, University College London, 7th Floor, Jules Thorn Institute, Middlesex Hospital, Mortimer Street, London W1T 3AA, UK. Fax: 44-20-7636-9941; E-mail:m.kitamura{at}medicine.ucl.ac.uk

	Abstract

Top Abstract Introduction Current Experience Perspective References

 
Abstract. For many years, ex vivo gene transfer has been used for genetic manipulation of various organs. In the kidney, ex vivo gene transfer was reported using mesangial cells and macrophages. In rats, cultured cells injected into the renal artery are accumulated selectively in the glomerulus. With this approach, it is possible to transfer genetically engineered cells to normal and diseased glomeruli. The transfer of genetically engineered cells to glomeruli can be used for several purposes. With the use of resident glomerular cells engineered in vitro, it is possible to examine how the cells that overexpress certain genes behave differently in normal and diseased glomeruli. Both gain-of-function and loss-of-function strategies are useful for this purpose. For the latter, stable expression of antisense cDNA, ribosomes, or dominant-negative mutants is available. By transfer of engineered cells producing secretory, recombinant proteins, it is possible to modify glomerular microenvironment in vivo. Transfer of genes encoding therapeutically relevant molecules could be useful for therapeutic intervention. Transfer of engineered leukocytes to the glomerulus also allows investigation of cross talk between leukocytes and resident cells. Transfer of stimulated leukocytes is useful for investigation of the pathologic actions of infiltrating cells on glomerular structure and function. Leukocytes in which certain gene functions are selectively reinforced or deleted would be useful for elucidation of the exact functions of leukocyte-associated genes in glomerular diseases. This article summarizes current experience with the adoptive transfer of engineered cells to the glomerulus for investigation of and therapy for glomerular diseases.

	Introduction

Top Abstract Introduction Current Experience Perspective References

 
Ex vivo gene transfer has been used for genetic manipulation of various organs. In this approach, cells are cultured from biopsy tissues, introduced with exogenous genes in vitro, and implanted back into the original organs. In the kidney, several ex vivo gene transfer approaches, using tubular cells, glomerular cells, and metanephric mesenchyme, have been reported (1).

An ex vivo approach has been developed for the purpose of gene transfer to glomeruli, using mesangial cells as a vector for gene delivery (2). In rats, cultured mesangial cells injected into the renal artery are accumulated selectively in the glomerulus (3). This site-selective accumulation is achieved not only by mesangial cells but also by other cell types, including macrophages (4) and fibroblasts (M. Kitamura, unpublished observations). Using this method, it is possible to transfer genetically engineered cells to normal and diseased glomeruli.

	Current Experience

Top Abstract Introduction Current Experience Perspective References

 
Transfer of Genetically Engineered Mesangial Cells
In general, the diameter of rodent cells in suspension is less than that of the glomerular afferent arteriole in rats. For example, the diameter of cultured rat mesangial cells is between 15 and 25 µm. Because the diameter of the glomerular capillaries in rats ranges from 5 to 25 µm (5), cells injected into the renal artery can pass through the afferent arteriole but are entrapped within the glomerulus.

To evaluate cell transfer efficiency, reporter cells were created by transfection of rat mesangial cells with a -galactosidase (-gal) gene. Established reported cells were transferred into the rat glomeruli by renal artery injection. Histochemical analysis demonstrated that the injected vector cells were accumulated in glomeruli (3). Sixty percent of glomeruli contained the reporter mesangial cells. In the glomeruli, the majority of transferred cells were located within the glomerular capillaries. The sustained presence of transferred cells was observed in vivo for at least 4 wk (3).

Modulation of the Glomerular Microenvironment. To examine the feasibility of using this method for the site-specific supply of secretory proteins, mesangial cells were transfected with a 105-kD gelatinase gene. Established stable transfectants (vector cells) were transferred to normal rat glomeruli via the renal artery. Gelatin zymography revealed that the recombinant gelatinase was abundantly secreted from the glomeruli transferred with the vector cells (6). This result indicated the feasibility of using this method to manipulate the glomerular microenvironment in vivo.

Using this approach, the in vivo function of transforming growth factor-1 (TGF-1) in glomeruli was examined. A cDNA encoding the active form of TGF-1 was introduced into cultured mesangial cells, and stable transfectants were transferred to normal rat glomeruli. Isolated glomeruli containing transfectants exhibited production of active TGF-, reduced mitogenic activity, and depressed responses to interleukin-1 (IL-1) (6). The mitogenic response of these chimeric glomeruli to in vivo inflammatory stimuli was then tested. Control transfectants and TGF- transfectants were transferred to the glomeruli of kidneys with anti-Thy 1 glomerulonephritis. Compared with unmodified or control cell-treated glomeruli, the in vivo mitogenic response of glomeruli harboring the TGF- transfectants was significantly attenuated (6). This result suggested the possibility that TGF-1 functions as an inhibitor of glomerular cell proliferation in vivo.

Application for Therapeutic Intervention. Using a similar strategy, it is possible to inactivate certain pathogenic mediators in glomeruli. IL-1 is known to be a crucial mediator in glomerulonephritis. Selective inactivation of local IL-1 is a possible strategy for therapeutic intervention. For this purpose, rat mesangial cells were stably transfected with an expression plasmid encoding IL-1 receptor antagonist (IL-1ra). In vitro, the established vector cells secreted recombinant IL-1ra proteins and exhibited blunted responses to IL-1 (7). The established cells were then transferred into rat glomeruli via the renal circulation, and isolated chimeric glomeruli were assayed for IL-1 responses. Compared with either unmodified glomeruli or glomeruli containing mock transfectants, glomeruli treated with the IL-1ra vectors exhibited attenuated responses to IL-1 (7).

Infiltrating leukocytes, especially macrophages, play a crucial role in the generation of glomerular injury. As an alternative approach to therapeutic intervention in glomerulonephritis, the introduction of genes coding for macrophage-deactivating cytokines would be useful. Toward this end, Fouqueray et al. (8) investigated the usefulness of IL-10 for the treatment of anti-glomerular basement membrane-induced glomerulonephritis in rats. Mesangial cells were stably transfected with IL-10 cDNA and transferred into nephritic glomeruli. The left kidneys, which had been injected with IL-10-transfected cells, produced 10-fold higher levels of IL-10, compared with unmodified right kidneys. This increase was associated with modest suppression of proteinuria (8).

In Vivo Regulation of Transgene Activity. An ideal in vivo gene transfer system should be competent for site-selective, long-term, high-level transgene expression. However, in some situations, modifiable expression of transgenes is also required. To create a reversible on/off system for site-specific control of glomerular transgene activity in vivo, a tetracycline-controlled transactivation system was used (9). In this method, a tetracycline-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription via binding to a tTA-responsive promoter that is present in a response plasmid. Tetracycline inhibits this tTA-dependent transactivation via its affinity for tTA (10). In double-transfected cells, therefore, the activity of the transgene can be controlled by tetracycline.

Cultured rat mesangial cells were co-transfected with a regulator plasmid encoding tTA and a response plasmid introducing a -gal gene. In vitro, the stable double-transfectants exhibited no -gal activity in the presence of tetracycline. However, after withdrawal of tetracycline from the culture medium, the expression of -gal was induced. When tetracycline was again added, expression was resuppressed. Low concentrations of tetracycline (50 to 100 ng/ml) were observed to be sufficient to maintain the silent state of the tTA-dependent promoter (9). The established cells were then transferred into normal rat glomeruli by renal artery injection. When tetracycline-pretreated cells were transferred into the glomeruli of untreated rats, -gal expression was induced in vivo. Oral administration of tetracycline in drinking water dramatically suppressed this in vivo transgene activation (9). This result suggested the utility of engineered cells combined with the tetracycline regulatory system for strict control of transgene expression in glomeruli.

Automatic Sensing of Glomerular Inflammation. In gene transfer-based therapies for inflammatory disorders, tight control of transgene expression, depending on disease activity, is essential. Exogenous anti-inflammatory molecules should be expressed in response to the initiation of disease, and expression must be switched off after recovery from the diseased state. Toward this goal, one possible approach would be to generate a local sensor that recognizes endogenous pathologic stimuli and allows subsequent control of transgene activity. Regulatory elements of particular genes that are activated under pathologic conditions would be useful for this purpose.

-Smooth muscle actin is normally undetectable in glomeruli but is markedly induced in mesangial cells in various glomerular diseases. The regulatory element of this gene would be ideal as a molecular sensor of glomerular injury. The 5'-flanking region of the -smooth muscle actin gene contains CArG box elements that are necessary and sufficient for induction of this gene. By combining the cell transfer system with the CArG box elements, it may be possible to create an intraglomerular cytosensor that allows automatic sensing of glomerular inflammation and subsequent control of transgene expression.

To examine this possibility, rat mesangial cells were stably transfected with an expression plasmid introducing a -gal gene under the control of CArG box elements. Under low-serum culture conditions, the established sensing cells expressed only low levels of -gal activity. However, after stimulation with serum, -gal activity was upregulated within 24 h (11). To examine whether the established sensing cells were able to automatically control transgene activity in vivo, serum-stimulated or unstimulated vector cells were transferred into normal or nephritic rat glomeruli by renal artery injection. In normal glomeruli treated with serum-stimulated vector cells, expression of -gal was automatically switched off. In contrast, when unstimulated vector cells were transferred into glomeruli with acute anti-Thy1 glomerulonephritis, transgene expression was substantially induced in vivo (11). These data indicated the utility of the CArG box element as a molecular sensor for glomerular inflammation and the feasibility of using glomerular cells combined with an inflammation-responsive promoter for automatic in vivo regulation of transgene activity.

Use of Autologous Cells as Gene Transfer Vectors. In ex vivo gene transfer approaches, rejection of implanted cells is a crucial problem. To overcome this problem, autologous cells would be useful. To examine this possibility, mesangial cells were cultured from renal biopsy specimens (approximately 100 mg), transfected with a -gal gene, and transferred back into the glomeruli of identical animals. One week after the injection of cells, 30% of glomeruli were positive for -gal (12). The use of autologous mesangial cells from biopsy tissues is thus possible and would be useful to obviate the risk of rejection with this approach.

Transfer of Genetically Engineered Macrophages
One of the most common pathologic features of glomerular disease is infiltration of leukocytes. These are mainly monocytes/macrophages, with neutrophils and T lymphocytes being present in smaller numbers. Infiltrating macrophages supposedly play a crucial role in the generation of glomerular injury. However, this hypothesis is largely based on histopathologic observations. It is currently unclear whether macrophages alone induce certain molecular/cellular events in normal glomeruli and, if they do, what type of intracellular machinery is responsible for the pathologic actions. It is also unclear how resident cells modulate the activity of infiltrating macrophages in glomeruli. To answer these questions, there are two major hurdles, i.e., macrophages must be accumulated in normal glomeruli, and certain functions of macrophages must be selectively reinforced or deleted. A possible approach to achieving these goals would be to manipulate macrophages at the genetic level and transfer them into glomeruli. To examine the feasibility of this idea, rat macrophages were transduced with a replication-incompetent retrovirus that introduces a -gal gene. Established reporter macrophages were transferred to the glomeruli of rats by renal artery injection. In the injected kidneys, 80% of isolated glomeruli contained the engineered macrophages (4).

Evaluation of Pathogenic Actions of Macrophages on Normal Glomeruli. The matrix metalloproteinase stromelysin is induced in isolated normal glomeruli in response to macrophage-derived proinflammatory cytokines (13). Using this molecule as an indicator, we investigated whether activated macrophages affect the function of resident glomerular cells. Lipopolysaccharide (LPS)-stimulated reporter macrophages were transferred into normal rat glomeruli. After the injection of cells, both kidneys were removed and processed for glomerular isolation. Isolated glomeruli were incubated ex vivo for 24 h, and Northern blot analysis of the expression of stromelysin was performed. Immediately after cell transfer, stromelysin transcripts were not detectable in isolated glomeruli. After ex vivo incubation of these glomeruli, the expression of stromelysin was dramatically induced in macrophage-treated glomeruli, but only modest induction was observed in glomeruli from untreated contralateral kidneys (4). The expression of other cytokine-inducible molecules, including gelatinase B, was also induced in macrophage-treated glomeruli (14).

If both resident cells and transferred macrophages are able to produce certain molecules, it is necessary to identify the cell type responsible for molecular expression. For this purpose, the "neomycin subtraction method" was developed (4). Unmodified glomerular cells are susceptible to the neomycin analogue G418, whereas reporter macrophages expressing neo are resistant to this drug. Using this difference, it is possible to subtract the contribution of resident cells from the total responses of macrophage-containing, chimeric glomeruli (4). LPS-stimulated reporter macrophages were transferred to normal glomeruli. Isolated glomeruli were incubated ex vivo in the absence or presence of G418 and were subjected to Northern blot analysis. After ex vivo incubation of these glomeruli, stromelysin expression was substantially induced in the absence of G418. However, this induction was completely abolished in the presence of G418 (4). This result indicated that activated macrophages recruited into normal glomeruli stimulated resident cells to express stromelysin. The transfer of genetically engineered macrophages thus provides a useful tool to elucidate the pathogenic effects of macrophages on glomeruli.

Effects of Resident Cells on Macrophage Function in Nephritic Glomeruli. Communication between glomerular cells and infiltrating macrophages plays a crucial role in the generation of glomerular injury. Currently, however, information regarding whether and how resident glomerular cells modulate the activity of infiltrating cells is limited. Previous studies demonstrated that cultured mesangial cells secrete a factor that impairs several functions of macrophages (15). For example, a mesangial cell-derived factor strongly inhibits macrophage adhesion and the production of proinflammatory cytokines. Using a specific inhibitor, this active entity has been identified as TGF-1 (16,17,18)

To examine whether macrophage-induced activation of glomerular cells is inhibited by endogenous TGF-1, an experimental model of anti-Thy1 glomerulonephritis was used. In this model, TGF-1 is dramatically upregulated in activated mesangial cells during the regeneration of glomeruli. Reporter macrophages that had been prestimulated with LPS were transferred into normal rat glomeruli or glomeruli with acute anti-Thy1 glomerulonephritis. After cell transfer, glomeruli were isolated, incubated ex vivo, and subjected to Northern blot analysis. Immediately after cell transfer, stromelysin mRNA was undetectable in both normal and inflamed glomeruli. After ex vivo incubation of these chimeric glomeruli, stromelysin expression was induced in macrophage-treated, normal glomeruli. However, in nephritic glomeruli producing active TGF-1, macrophage induction of stromelysin expression was suppressed, compared with normal glomeruli (13). Similar results were obtained for the expression of other cytokine-inducible molecules, including gelatinase B (14).

Transfer of "Loss-of-Function" Macrophages to Glomeruli. Macrophages are thought to be important in the generation of glomerular injury, but it is still unclear what type of cellular machinery is required for the pathogenic actions. By combining the macrophage transfer technique with a loss-of-function strategy, an experimental approach was developed to explore whether and how certain cellular machinery is required for local actions of macrophages. As a prototypic investigation, in vivo roles for nuclear factor-B (NF-B) in the effector actions of macrophages were examined. NF-B-inactive (NIKMAC^NR) macrophages were created by transduction of rat macrophages with a retrovirus encoding a super-repressor mutant of IB, i.e., IBM (19). The effector functions of NIKMAC^NR cells on resident cells were evaluated by co-culture, cross-feeding, and in vivo macrophage transfer. Rat mesangial cells co-cultured with activated control macrophages exhibited abundant expression of activation markers, including monocyte chemoattractant protein 1, stromelysin, and gelatinase B. In contrast, co-culture with activated NIKMAC^NR macrophages induced only modest gene expression. Similarly, culture medium conditioned by activated control macrophages triggered mesangial cells and isolated glomeruli to express the activation markers, whereas the stimulatory effect was not observed with medium conditioned by activated NIKMAC^NR macrophages (20). To evaluate the effector actions of NIKMAC^NR macrophages in glomeruli, control macrophages and NIKMAC^NR macrophages were transferred into normal rat glomeruli via renal artery injection. After the transfer of control macrophages, substantial induction of the activation marker stromelysin was observed in resident glomerular cells. This induction was dramatically diminished in glomeruli treated with activated NIKMAC^NR macrophages (20). Inactivation of NF-B in macrophages thus effectively disrupted paracrine stimulatory loops from macrophages to resident glomerular cells.

	Perspective

Top Abstract Introduction Current Experience Perspective References

 
As described in this article, the transfer of genetically engineered cells to glomeruli can be used for several purposes. Using resident glomerular cells engineered in vitro, it is possible to examine how the cells that overexpress certain genes behave differently in normal and inflamed glomeruli. Both gain-of-function and loss-of-function strategies are useful for this purpose. For the latter, stable expression of antisense genes, ribozymes, or dominant-negative mutants is available. Genetically modified resident cells can also be used as vectors for gene delivery. By transfer of vector cells producing secretory recombinant proteins, it is possible to modify the glomerular microenvironment in vivo. Transfer of genes encoding therapeutically relevant molecules to diseased glomeruli could be useful for therapeutic intervention.

The transfer of engineered leukocytes to glomeruli allows investigation of cross-talk between leukocytes and resident cells. The transfer of stimulated leukocytes is useful for investigation of the pathologic actions of infiltrating cells on glomerular structure and function. Leukocytes in which certain gene functions are selectively reinforced or deleted would be useful for elucidation of the exact functions of leukocyte-associated genes in glomerular diseases. Current experience with the transfer of engineered cells to glomeruli has been summarized in Table 1.

	References

Top Abstract Introduction Current Experience Perspective References

 

1. Kitamura M: Strategic gene transfer into the kidney: Current status and prospects. Clin Exp Nephrol1 : 157-178,1997
2. Kitamura M: Gene delivery into the glomerulus via mesangial cell vectors. Exp Nephrol 5:118 -125, 1997[Medline]
3. Kitamura M, Taylor S, Unwin R, Burton S, Shimizu F, Fine LG: Gene transfer into the rat renal glomerulus via a mesangial cell vector: Site-specific delivery, in situ amplification and sustained expression of an exogenous gene in vivo. J Clin Invest94 : 497-505,1994
4. Kitamura M, Sütö TS: Transfer of genetically engineered macrophages into the glomerulus. Kidney Int 51:1274 -1279, 1997[Medline]
5. Remuzzi A, Brenner BM, Pata V, Tebaldi G, Mariano R, Belloro A, Remuzzi G: Three-dimensional reconstructed glomerular capillary network: Blood flow distribution and local filtration. Am J Physiol263 : F562-F572,1992[Abstract/Free Full Text]
6. Kitamura M, Burton S, English J, Kawachi H, Fine LG: Transfer of a mutated gene encoding active transforming growth factor- 1 suppresses mitogenesis and IL-1 response in the glomerulus. Kidney Int 48:1747 -1757, 1995[Medline]
7. Yokoo T, Kitamura M: Gene transfer of interleukin-1 receptor antagonist into the renal glomerulus via a mesangial cell vector. Biochem Biophys Res Commun 226:883 -888, 1996[Medline]
8. Fouqueray B, Suberville S, Isaka Y, Akagi Y, Gerard C, Velu T, Imai E, Baud L: Reduction of proteinuria in anti-glomerular basement membrane nephritis by interleukin-10 gene transfer [Abstract]. J Am Soc Nephrol 7: 1698,1996
9. Kitamura M: Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus. Proc Natl Acad Sci USA93 : 7387-7391,1996[Abstract/Free Full Text]
10. Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547 -5551, 1992[Abstract/Free Full Text]
11. Kitamura M, Kawachi H: Creation of an in vivo cytosensor using engineered mesangial cells: Automatic sensing of glomerular inflammation controls transgene activity. J Clin Invest100 : 1394-1399,1997[Medline]
12. Kitamura M, Burton S, Yokoo T, Fine LG: Gene delivery into the renal glomerulus by transfer of genetically engineered, autologous mesangial cells. Exp Nephrol 4:56 -59, 1996[Medline]
13. Kitamura M: TGF-1 as an endogenous defender against macrophage-triggered stromelysin gene expression in the glomerulus. J Immunol 160:5163 -5168, 1998[Abstract/Free Full Text]
14. Sütö TS, Fine LG, Shimizu F, Kitamura M: In vivo transfer of engineered macrophages into the glomerulus: Endogenous TGF--mediated defense against macrophage-induced glomerular cell activation. J Immunol 159:2476 -2483, 1997[Abstract/Free Full Text]
15. Kitamura M, Fine LG: The concept of glomerular self-defense. Kidney Int 55:1639 -1671, 1999[Medline]
16. Kitamura M, Sütö TS, Yokoo T, Shimizu F, Fine LG: Transforming growth factor-1 is the predominant paracrine inhibitor of macrophage cytokine synthesis produced by glomerular mesangial cells. J Immunol 156:2964 -2971, 1996[Abstract]
17. Kitamura M: Identification of an inhibitor targeting macrophage production of monocyte chemoattractant protein-1 as TGF- 1. J Immunol 159:1404 -1411, 1997[Abstract]
18. Sütö TS, Fine LG, Kitamura M: Mesangial cell-derived transforming growth factor-1 reduces macrophage adhesiveness with consequent deactivation. Kidney Int 50:445 -452, 1996[Medline]
19. Kitamura M: NF- B-mediated self-defense of macrophages faced with bacteria. Eur J Immunol29 : 1647-1655,1999[Medline]
20. Kitamura M: Adoptive transfer of NF- B-inactive macrophages to the glomerulus. Kidney Int57 : 709-716,2000[Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by KITAMURA, M. Search for Related Content PubMed PubMed Citation Articles by KITAMURA, M.