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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2003  |  Volume : 14  |  Issue : 3  |  Page : 398-405
Gene Therapy of Inherited Renal Tubular Diseases

Renal Unit, Department of Medicine, Faculty of Medicine, Siriraj Hospital, Bangkok, Thailand

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How to cite this article:
Shayakul C. Gene Therapy of Inherited Renal Tubular Diseases. Saudi J Kidney Dis Transpl 2003;14:398-405

How to cite this URL:
Shayakul C. Gene Therapy of Inherited Renal Tubular Diseases. Saudi J Kidney Dis Transpl [serial online] 2003 [cited 2021 Mar 4];14:398-405. Available from: https://www.sjkdt.org/text.asp?2003/14/3/398/33019

   Introduction Top

Over the last two decades, we have expe­rienced significant progress in understanding the molecular mechanisms and genetic bases of many inherited renal tubular diseases. The observation that most disorders are caused by inheritance of a single, functionally defe­ctive gene (monogenic disease) generates the concept of gene-based therapy as a definitive treatment. Conceptually, therapeutic delivery of a normal gene-copy into the affected cells may correct the deficiency/dysfunction of that protein. Indeed, the scope of gene therapy has been expanded from 'gene supplemen­tation' to 'gene inhibition' by delivery of reagents designed to inhibit target mutant gene transcription and translation (such as ribozymes, antisense RNA or 'decoy' DNA and RNA), and to 'gene correction' by targeting a dysfunctional exon (or gene) and directly substituting it with the correct copy.

The development of more sophisticated techniques in molecular biology has allowed us to go rapidly from theory to application, as demonstrated by more than 500 ongoing clinical gene transfer trials at the end of 2001. [1] Although the promise of gene therapy to 'cure' human diseases is far from being realized, steady progress is clearly evident and it has become an established concept in medicine. As for inherited renal tubular disease, gene therapy is still at an experimen­tal stage and no clinical studies have been published so far. However, significant advances have been made in the last few years. This review highlights the salient achievements in the field of gene transfer into the renal tubule and potential application of gene the­rapy to treat human hereditary renal diseases using the transgenic and knockout animals as the models.

   Strategies for Gene Transfer into The Renal Tubule Top

Successful gene therapy in renal tubular disease must fulfill several minimal require­ments in order to achieve site-specific, high level, long-term and potentially controllable transgene expression. [2],[3],[4] In addition, the delivered material must not trigger apoptosis, immune or inflammatory responses. While the simplest approach of intravenous injection of oligonucleotides (ODN) could result in accumulation of ODN in the proximal tubular cells and should be considered as a candidate strategy for anti-sense approach targeting the proximal tubule, [5] direct injection of naked DNA only resulted in limited transgene expression in the injected area. Moreover, combination of plasmid DNA injection and electroporation to create cell membrane pore is efficient in gene transfer into the glomeruli, but not the renal tubular cells. [6]

A number of vectors have been developed to efficiently deliver genetic material into the renal tubular cells, though progress in the field is lagging far behind the discovery of therapeutic genes. Since most renal tubular cells are non-dividing and terminally diffe­rentiated, the ideal vector should efficiently transduce quiescent cells in vivo. Recently, several investigators have taken up these experimental challenges. Many of the technical problems associated with vector, delivery systems and targeting have been characterized, and some partially resolved. Vectors and stra­tegies of gene transfer into the renal tubule are listed in [Table - 1], and are broadly divided into non-viral and viral types.

Non-viral Vectors

Cationic liposome is a particularly attractive vector for issues of safety despite its low efficiency and transient gene expression. Liposome-mediated gene transfer has been shown to be effective in delivery of transgene into the proximal tubular cells via either intra­renal artery injection in the rat or renal pelvic injection in the mouse. [7],[8] Modified proteolipo­somes produced by integrating reconstituted viral envelope glycoproteins into the lipid bilayer, thus enhancing cellular uptake of the lipo­plexes, remain to be studied for its efficiency of gene transfer into the renal tubular cells.

Viral Vectors

Most vectors currently in use for kidney gene transfer are based upon viruses, such as retrovirus, lentivirus, adenovirus and adeno­associated virus (AAV). Retroviral vectors are difficult to produce in high titers, require cell replication to achieve expression, and randomly integrate into the host genome. Attempts have been made for transgene expre­ssion in the renal tubule by administration of the cytotoxic reagent folic acid prior to retroviral gene transfer. [9] This, however, resulted in limited expression of the gene product in the injured proximal tubules for only three weeks. Another approach using retroviral vectors has been ex vivo gene transfer with cultured tubular epithelial cells. [10]

Lentiviral vectors efficiently transduce many quiescent cell types in vivo, appear to be rela­tively non-inflammatory, and may provide persistent transduction through stable integra­tion into the host genome. However, concerns have been raised about the use of HIV-based vector systems in humans. Recent studies successfully demonstrated that lentiviral vectors transduced kidney cells in vivo. [11] Following retrograde ureteral infusion, promi­nent expression of the reporter transgene was observed in the proximal straight tubules while delivery through the renal artery or vein resulted in weaker transgene expression in the inner medullary collecting ducts. No appa­rent histological abnormality was observed after virus administration and the transgene expression persisted for the whole three-month study period.

Similarly recombinant adenoviral vectors can transfect non-proliferating cells with significant advantage of delivering relatively large gene, and transduction of the highest expression among all available vectors. Adeno­viral vectors have been shown to transduce renal tubular cells in vivo both in the kidney cortex (mainly proximal tubular cells) and kidney medulla by intra-renal-arterial and retrograde injection, respectively. [12] Adenovirus polylysine DNA complex has also been used in the first trial of ex vivo gene transfer into the human kidney with significant transgene expression in the proximal tubular cells. [13] Nevertheless, adenoviral vectors provide only transient expression, produce intense infla­mmation, and trigger a significant immune response. It is conceivable that recently developed "gutless" vectors created by removal of all adenoviral genes and replacing with substitutes of helper viruses may be non-immunogenic, and produce no virtual toxicity. [14] This possibility requires further studies to define the safety limits and efficacy for adenovirus-mediated gene therapy.

Recombinant AAV is rapidly becoming the vector of choice for small gene transfer (less than 4.5 kb) to the kidney because of its broad tropism for renal tubular cells, low immnogenicity and inflammation. Moreover, AAV vectors have the ability to efficiently transduce both dividing and non-dividing cells and to integrate into the host genome resulting in long-term gene expression. Direct injection of AAV vectors into renal paren­chyma can deliver reporter genes to renal tubular cells including proximal tubule, thick ascending limb of Henle and collecting tubule. [1] One other study has shown that reporter genes are expressed in the collecting tubule and corticomedullary junction for over one year following intra-ureteral injection. [16] With the recent technological advances in producing large quantities of purified virus, [17] AAV vector has emerged as the most potential gene delivery system for clinical gene therapy of renal tubular diseases.

   Targeting Gene into The Renal Tubular Cell Top

One important issue in the development of gene therapy protocols is the need to target therapeutic gene delivery in order to minimize the risk of germ-line cell transduction and to prevent side effects to the surrounding healthy tissues. Moreover, targeting can reduce vector wastage, and the amount of vector stocks that need to be produced and administered in vivo.

Although holding considerable promise, there are several challenging hurdles to be overcome for achievement of cell-type­specific transgene expression in the kidney. The major obstacle is related to its unique architectural and cellular heterogeneity of more than 10 renal cell types within the nephrons and renal tubular segments. Routes of transgene delivery employed to date inclu­ding intra-renal-arterial injection, retrograde infusion into the collecting duct via the renal pelvi-calyceal system, subcapsular injection, and intra-parenchymal injection are not effi­cient in transduction of all renal cell types. Most studies have shown that intra-renal­arterial administration results in transduction of proximal tubular cells whereas retrograde injection via the renal calyx leads to detect­able expression in the medullary tubules and papilla. [2] However, there are some different results based on utilization of different vector systems as well as different animal models. The limiting factors for entry of vectors into the well-differentiated epithelial cells of renal tubule, e.g. viral tropism and anatomical barriers, remain to be investigated as have been proved for the airway epithelia. [18],[19]

An alternative approach involves intro­duction of cell-type-specific promoters in order to limit (and to regulate, in case of inducible promoters) the expression of transgene, thus avoiding unintended side effects to non-targeted cells. Some examples of potentially promising promoters for specific renal cells are listed in [Table - 2]. [20],[21] It is interesting to note that many of these promoters have been studied in mice for the reproduction of transgenic and knockout animals; they remain to be tested as reporter gene promoters in other animal species commonly used in gene transfer experiments.

In addition to using cell-type-specific promoters, the timing and duration of trans­gene expression also can be modulated by employing inducible promoters that can be externally controlled. Several inducible systems have been developed, and some have been used for gene transfer experiments in the kidney, such as tetracycline ecdysone, and Cre/loxP system. [22],[23],[24] Another example of inducible promoter system is an endogenous one that becomes selectively activated during active disease. Based on the observation that the a-smooth muscle actin promoter in mesangial cells becomes activated after exposure to pro-inflammatory cytokines, it can be used as the 'cytosensor' to provide optimal control of transgene expression in inflammatory glomerular diseases. [25] Transfer of an intact gene along with its promoter should provide optimal control of transgene expression. How­ever, many intact genes of medical interest exceed in size the capacity even of gutless adenovirus vectors. One solution on the horizon is the development of mammalian artificial chromosomes, which will transfer very large segments of DNA and maintain them as stable episomal replicons. [26]

   Gene Transfer and Experimental Renal Tubular Disorders Top

Nephrogenic Diabetes Insipidus (NDI)

In the large majority of cases, congenital NDI is an X-linked recessive disorder caused by mutations in the vasopressin V2 receptor gene (V2R) rendering the renal collecting ducts insensitive to the antidiuretic actions of the hormone arginine vasopressin (AVP). Given the fact that there is only one abnormal allele on the X chromosome for the affected males, introducing a normal V2R gene as augmen­tation therapy seems reasonable. Schoneberg et al showed that adenovirus-mediated gene transfer of only the V2R-C-terminal tail gene fragment could restore function of mutationally inactivated G protein-coupled V2 receptors in CHO cells. [27] Four of the six truncated forms of V2R (E242X, 804delG, 834delA, and W284X) and one missense V2R mutant, Y280C, regained considerable functional acti­vity as demonstrated by an increase in the number of AVP surface binding sites and in AVP-stimulated adenylyl cyclase activity. Nonetheless, application of this promising rescue phenomenon has yet to be tested in vivo. The access of adenoviral vector to particular cells in the kidney collecting ducts can more likely be achieved by retrograde injection via the pelvi-calyceal system. [8],[12]

It is interesting to note that adenoviral re­ceptors in the respiratory tract are restricted to the epithelial cell basolateral surface. If this is true to the renal epithelial cells also, maneuvers to transiently violate epithelial integrity will be required for adenovirus to encounter its receptor. Alternatively, adeno­virus will require pseudotyping with ligands for luminal membrane receptors, or covalent modification of the viral surface to allow customized labeling with ligands specific for each target cell. [8]

Carbonic Anhydrase (CA) II Enzyme Deficiency and Renal Tubular Acidosis

The cytosolic enzyme CAII is responsible for catalyzing the reversible hydration of carbon dioxide during tubular fluid acidification pro­cesses of the proximal tubule, loop of Henle and collecting duct of the kidney. Deficiency of CAII in humans produces a syndrome of renal tubular acidosis, osteopetrosis, cerebral calcifications, and mental retardation. As in humans, CAII-deficient mice exhibit renal tubular acidosis manifested by growth retar­dation, low blood bicarbonate, and inappro­priately high urine pH at baseline and after acid challenge.

Lai and colleagues examined the efficacy of gene therapy in a CAII-deficient mouse strain by intra-renal pelvi-calyceal injection of cationic liposomes carrying plasmid DNA encoding human CAII cDNA driven by a cytomegalovirus promoter. [29] Successful gene transfer was demonstrated, both at the levels of CAII mRNA and protein accumulation, with the peak at day three and the expression persisted for the entire three-week duration of the study period. In parallel, urinary acidification was partially improved after ammonium chloride administration for up to three weeks but not at six weeks. Although transient, this report represents the first successful in vivo gene therapy in an experimental model of the inherited renal tubular disease. It is interesting to note that heterologous CAII gene expression was restricted to a subset of collecting duct cells concentrated at the corticomedullary and outer medullary region junction, whose identity was not fully explored, whereas those cells of the inner medullary collecting duct, presumably first exposed to the liposome-DNA complexes, were not appa­rently transfected. [30]

Aquaporin (AQP) I Deficiency and Concentrating Ability Defect

Another example of gene therapy for functional correction of renal tubular disorders is the recent report of a temporary concentra-ting ability defect in AQP1 null mice. AQP1 is a member of the pore-forming intrinsic membrane proteins (MIP) family, of which protein is located in the proximal tubule, thin descending limb of Henle and vasa recta in the mammalian kidney. Deficiency of AQP1 in humans, also known as Colton null blood group, results in modest reduction of urinary concentrating ability, while AQP1-deficient mice exhibit profound water loss with submaximal urinary concentrating ability. The defect is unresponsive to administration of vasopressin agonist dDAVP and probably results from a combination of defects in proximal tubule fluid reabsorption and countercurrent exchange mechanism.

Yang et al reported on gene transfer using adenoviral vectors carrying AQP1-cDNA in AQP1-deficient mice with different routes of delivery. [31] By direct intrarenal injection, they observed strong AQP1 expression only locally at the injection site, while retrograde injection from the ureter resulted in intense expression in ureteral and renal papilla with lesser and patchy expression in the cortical regions. Finally, the vector encoding AQP1 was injected into the tail vein. AQP1 protein was expressed in liver sinusoids, apical and basolateral membrane of cortical tubules, and medullary vasa recta but no expression was found in glomeruli, limb of Henle or collecting duct. Concurrently, the defect in urinary concentration partially improved and water permeability in the proximal tubule increased at one week after treatment but barely above baseline by five weeks. The results from this study suggest that adenovirus-mediated gene transfer has potential applications for the therapy of a variety of diseases related to aquaporin defect.

   Perspectives and Conclusions Top

Although gene therapy is potentially a power­ful clinical approach to 'cure' inherited renal tubular diseases, several technical hurdles remain to be overcome. Among these are development of vector and delivery systems for sustained transgene expression at suffi­ciently high level, and achievement of cell­specific and time-controlled gene targeting. It is becoming evident that there will be no perfect vector or ideal single strategy to treat the large number of inherited renal tubular diseases. The limitations of individual vector and delivery systems that have besieged us are likely to be overcome by the combination of the best characteristics of innovative experi­mentation and persistence. In addition, the degree of control and the selection of cell­type for transgene expression will vary from application to application. Thus, the design of appropriate gene therapy will require complete understanding, not only of the molecular characteristics of the gene that is being transferred, but also of the patho­genesis and clinical course of the disease that is being treated.

   Acknowledgment Top

CS was supported by grants from Thai Research Foundation and Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand.

   References Top

1.National Institutes of Health Office of Biotech­nology Advances. Clinical trials in human gene transfer: query of clinical trials. Available at orm.asp. Accessed December 31, 2002.  Back to cited text no. 1    
2.Imai E. Gene therapy approach in renal disease in the 21st century. Nephrol Dial Transplant 2001;16 Suppl 5:26-34.  Back to cited text no. 2    
3.Shayakul C, Alper SL. Gene therapy of inherited kidney diseases. In Harrison's Online of Internal Medicine. The McGraw-Hill Companies, NY. ( Updates/Editorials/edl2255.html), Posted: March 21, 2000.  Back to cited text no. 3    
4.Kelley VR, Sukhatme VP. Gene transfer in the kidney. Am J Physiol 1999;276:F1-9.  Back to cited text no. 4    
5.Rappaport JB, Hanss B, Kopp JB, et al. Transport of phosphorothioate oligonucleo­tides in kidney: implications for molecular therapy. Kidney Int 1995;47:1462-9.  Back to cited text no. 5    
6.Tsujie M, Isaka Y, Nakamura H, Imai E, Hori M. Electroporation mediated gene transfer that targets glomeruli. J Am Soc Nephrol 2001;12:949-54.  Back to cited text no. 6    
7.Baudard M, Flotte TR, Aran JM et al. Expression of the human multidrug resistance and glucocerebrosidase cDNAs from adeno­associated vectors: efficient promoter activity of AAV sequences and in vivo delivery via liposomes. Hum Gene Ther 1996;7:1309-22.  Back to cited text no. 7    
8.Lien YH, Lai LW. Liposome-mediated gene transfer into the tubules. Exp Nephrol 1997;5:132-6.  Back to cited text no. 8    
9.Bosch RJ, Woolf AS, Fine LG. Gene transfer into the mammalian kidney: direct retrovirus transduction of regenerating tubular epithelial cells. Exp Nephrol 1993;1:49-54.  Back to cited text no. 9    
10.Naito T, Yokoyama H, Moore KJ, Dranoff G, Mulligan RC, Kelley VR. Macrophage growth factors introduced into the kidney initiate renal injury. Mol Med 1996;2:297-312.  Back to cited text no. 10    
11.Gusella GL, Fedorova E, Hanss B, Marras D, Klotman ME, Klotman PE. Lentiviral gene transduction of kidney. Hum Gene Ther 2002;13:407-14.  Back to cited text no. 11    
12.Moullier P, Friedlander G, Calise D, Ronco P, Perricaudet M, Ferry N. Adenoviral-mediated gene transfer to renal tubular cells in vivo. Kidney Int 1994;45:1220-5.  Back to cited text no. 12    
13.Zeigler ST, Kerby JD, Curiel DT, Diethelm AG, Thompson JA. Molecular conjugate­mediated gene transfer into isolated human kidneys. Transplantation 1996;61:812-7.  Back to cited text no. 13    
14.Balter M. Gene therapy on trial. Science 2000;288:951-7.  Back to cited text no. 14    
15.Lipkowitz MS, Hanss B, Tulchin N, et al. Transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors. J Am Soc Nephrol 1999;10:1908-15.  Back to cited text no. 15    
16.Stern AS, Klotman ME, Ioannou YA, et al. Polarity of alpha-galactosidase A uptake by renal tubule cells. Kidney Int Suppl 2002; 61(Suppl 1):S52-5.  Back to cited text no. 16    
17.Clark KR. Recent advances in recombinant adeno-associated virus vector production. Kidney Int 2002;61(Suppl 1):S9-15.  Back to cited text no. 17    
18.Pickles RJ, McCarty D, Matsui H, Hart PJ, Randel SH, Boucher RC. Limited entry of adenovirus vectors into well-differentiated airway epithelium is responsible for inefficient gene transfer. J Virol 1998;72:6014-23.  Back to cited text no. 18    
19.Pilewski JM. Gene therapy for airway diseases: continued progress towards identifying and overcoming barriers to efficiency. Am J Respir Cell Mol Biol 2002;27:117-21.  Back to cited text no. 19    
20.Kone BC. Molecular approaches to renal physiology and therapeutics. Semin Nephrol 1998;18:102-21.  Back to cited text no. 20    
21.Lipkowitz MS, Klotman ME, Bruggeman LA, et al. Molecular therapy for renal diseases. Am J Kidney Dis 1996;28:475-92.  Back to cited text no. 21    
22.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 USA 1996;93:7387-91.  Back to cited text no. 22    
23.No D, Yao TP, Evans RM. Ecdysone­inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 1996;93:3346-51.  Back to cited text no. 23    
24.Yokoo T, Ohashi T, Utsunomiya Y. Inflamed glomeruli-specific gene activation that uses recombinant adenovirus with the Cre/loxP system. J Am Soc Nephrol 2001; 12:2330-7.  Back to cited text no. 24    
25.Kitamura M, Kawachi H. Creation of an in vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular infla­mmation controls transgene activity. J Clin Invest 1997;100:1394-9.  Back to cited text no. 25    
26.Vos JM. Mammalian artificial chromosomes as tools for gene therapy. Curr Opin Genet Dev 1998;8:351-9.  Back to cited text no. 26    
27.Schoneberg T, Sandig V, Wess J, Gudermann T, Schultz G. Reconstitution of mutant V2 vasopressin receptors by adenovirus-mediated gene transfer: Molecular basis and clinical implication. J Clin Invest 1997;100:1547-56.  Back to cited text no. 27    
28.Smith JS, Keller JR, Lohrey NC, McCauslin CS, Ortiz M, Cowan K, Spence SE. Redirected infection of directlly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens. Proc Natl Acad Sci USA 1999;96:8855-60.  Back to cited text no. 28    
29.Lai LW, Chan DM, Erickson RP, Hsu SJ, Lien YH. Correction of renal tubular acidosis in carbonic anhydrase II-deficient mice with gene therapy. J Clin Invest 1998;101:1320-5.  Back to cited text no. 29    
30.Shayakul C, Breton S, Brown D, Alper SL. Gene therapy of inherited renal tubular disease. Am J Kidney Dis 1999;34:374-9.  Back to cited text no. 30    
31.Yang B, Ma T, Dong JY, Verkman A. Partial correction of the urinary concentrating defect in aquaporin-1 null mice by adenovirus-mediated gene delivery. Hum Gene Ther 2000;11:567-75.  Back to cited text no. 31    

Correspondence Address:
Chairat Shayakul
Renal Unit, Department of Medicine, Faculty of Medicine, Siriraj Hospital, 2 Prannok Rd, Bangkoknoi, Bangkok 10700
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PMID: 17657112

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