Transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors.

There has been an increasing interest recently in the possibility of treating renal diseases using gene therapy. The ability to pursue gene therapy for renal diseases has been limited by the availability of an adequate system for gene delivery to the kidney. Adeno-associated virus (AAV) is a defective virus of the parvovirus family that has a number of properties attractive for renal gene delivery: recombinant AAV contains no viral genes; expression of genes delivered by these vectors does not activate cell-mediated immunity; the virus is able to transduce nondividing as well as dividing cells; and both wild-type and recombinant AAV integrate into the host chromosome resulting in long-term gene expression. Studies were performed to determine whether AAV can deliver reporter genes to kidney cells in vitro and in vivo. These studies show that AAV can deliver reporter genes with approximately equal efficiency to human mesangial, proximal tubule, thick ascending limb, collecting tubule, and renal cell carcinoma cells in primary culture. Immortalized mouse mesangial cells are transduced at a much greater efficiency. Transduction can be enhanced by pharmaceutical agents up to sevenfold in primary cells (transducing up to 20% of primary cells per well) and as much as 400-fold in immortalized mesangial cells. AAV delivered in vivo by intraparenchymal injection results in at least 3 mo of reporter gene expression in tubular epithelial, but not glomerular or vascular, cells at the injection site. These data indicate that AAV can deliver genes to renal cells both in vitro and in vivo resulting in prolonged gene expression, and thus AAV can be a useful tool for renal gene delivery.

Adeno-associated virus gene transfer into renal cells: potential for in vivo gene delivery.

The human parvovirus adeno-associated virus (AAV), type 2, has a number of features that make it an attractive choice as a vector for gene delivery to the kidney. AAV vectors permit long-term gene expression in vivo by integration into the host genome, have potential for site-specific integration on chromosome 19, do not express viral genes or generate a cellular immune response, and demonstrate enhancement of gene expression by chemotherapeutic agents that are approved for use in vivo. These properties confer advantages to AAV over other viral and nonviral methods for gene transfer. Preliminary experiments in our laboratory suggest that AAV is able to transfer genes to both renal cells in culture and the kidney in vivo. Thus, AAV has the potential to be an important gene transfer vector for the kidney in vivo.

Identification and characterization of a cell membrane nucleic acid channel.

We have identified a 45-kDa protein purified from rat renal brush border membrane that binds short single-stranded nucleic acid sequences. This activity was purified, reconstituted in proteoliposomes, and then fused with model planar lipid bilayers. In voltage-clamp experiments, the reconstituted 45-kDa protein functioned as a gated channel that allows the passage of nucleic acids. Channel activity was observed immediately after addition of oligonucleotide. Channel activity was not observed in the absence of purified protein or of oligonucleotide or when protein was heat-inactivated prior to forming proteoliposomes. In the presence of symmetrical buffered solution and oligonucleotide, current passed linearly over the range of holding potentials tested. Conductance was 10.4 +/- 0.4 picosiemens (pS) and reversal potential was 0.2 +/- 1.7 mV. There was no difference in channel conductance or reversal potential between phosphodiester and phosphorothioate oligonucleotides. Ion-substitution experiments documented a shift in reversal potential only when a concentration gradient for oligonucleotide was established, indicating that movement of oligonucleotide alone was responsible for current. Movement of oligonucleotide across the bilayer was confirmed by using 32P-labeled oligonucleotides. Channel open probability decreased significantly in the presence of heparan sulfate. These studies provide evidence for a cell surface channel that conducts nucleic acids.

Calcium regulation of a cell surface nucleic acid channel.

We have recently purified a 45 kDa protein from rat renal brush border membrane that functions as a macromolecular nucleic acid channel when reconstituted in artificial lipid bilayers. To explore the role of calcium in the regulation of this channel, purified protein was reconstituted in bilayers and calcium concentration was altered while voltage clamp experiments were performed. Open probability of the channel was less than 1% in 0 calcium and increased an average of 19-fold when calcium concentration was increased from 0 to 0.5 mM and 100-fold when increased to 1.0 mM. Equimolar calcium and EDTA resulted in a fall in open probability to 1%. Increasing calcium concentration had no effect on current/voltage relationship or mean open time of the channel, but decreased the mean closed time significantly. Cooperative channel gating was observed at 1.0 mM. These data demonstrate that open probability of the renal nucleic acid channel is calcium-dependent. The increase in open probability is due to an increase in channel gating activity rather due to an increase in the duration of the open state.

Molecular therapy for renal diseases.

The introduction of molecular therapy through the delivery of nucleic acids either as oligonucleotides or genetic constructs holds enormous promise for the treatment of renal disease. Significant barriers remain, however, before successful organ-specific molecular therapy can be applied to the kidney. These include the development of methods to target the kidney selectively, the definition of vectors that transduce renal tissue, the identification of appropriate molecular targets, the development of constructs that are regulated and expressed for long periods of time, the demonstration of efficacy in vivo, and the demonstration of safety in humans. As the genetic and pathophysiologic basis of renal disease is clarified, obvious targets for therapy will be defined, for example, polycystin in polycystic kidney disease, human immunodeficiency virus (HIV) type 1 in HIV-associated nephropathy, alpha-galactosidase A in Fabry's disease, insulin in diabetic nephropathy, and the "minor" collagen IV chains in Alport's syndrome. In addition, several potential mediators of progressive renal disease may be amenable to molecular therapeutic strategies, such as interleukin-6, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta(TGF-beta). To test the in vivo efficacy of molecular therapy, appropriate animal models for these disease states must be developed, an area that has received too little attention. For the successful delivery of genetic constructs to the kidney, both viral and nonviral vector systems will be required. The kidney has a major advantage over other solid organs since it is accessible by many routes, including intrarenal artery infusion, retrograde delivery through the uroexcretory pathways, and ex vivo during transplantation. To further restrict expression to the kidney, tropic vectors and tissue-specific promoters also must be developed. For the purpose of inhibition of endogenous or exogenous genes, current therapeutic modalities include the delivery of antisense oligodeoxynucleotides or ribozymes. For these approaches to succeed, we must gain a much better understanding of the nature of their transport into the kidney, requirements for specificity, and in vivo mechanisms of action. The danger of a rush to clinical application is that superficial approaches to these issues will likely fail and enthusiasm will be lost for an area that should be one of the most exciting developments in therapeutics in the next decade.

Transport of phosphorothioate oligonucleotides in kidney: implications for molecular therapy.

The systemic administration of phosphorothioated antisense oligonucleotides has been demonstrated to be an effective strategy for the control of gene expression. Because previous studies have suggested both hepatic and renal accumulation of systemically administered oligonucleotides, we explored whether the kidney might be a site of free DNA transport. [32P]-phosphorothioate oligonucleotides (20 mers) were excreted in urine but cleared at only 30% of glomerular filtration rate. Plasma clearance of the label was very rapid (t1/2 approximately 5 min) but the half life of labeled S-deoxynucleotide excreted in urine was much slower (28 min). Infused oligonucleotide appeared in urine with little degradation. By autoradiography of renal tissue, labeled antisense oligonucleotides appeared within Bowman's capsule and the proximal tubule lumen. DNA was detected in association with brush border membrane and within tubular epithelial cells. Brush border membrane preparations from rat kidney contained oligonucleotide binding proteins as determined by gel mobility shift and UV cross linking assays. Because renal epithelial cells efficiently take up phosphorothioate oligonucleotides without apparent degradation, the kidney appears to be an excellent target for site-directed antisense therapy, but may be a site of antisense toxicity as well.

Alterations in glomerular dynamics in congenital, unilateral hydronephrosis.

We have previously shown that rats with congenital, unilateral hydronephrosis exhibit a reduction in GFR that returns to normal when either the renin angiotensin system or thromboxane A2 (TxA2) is blocked. The current study defines the single nephron defect in congenital, unilateral hydronephrosis and evaluates the roles of angiotensin II (Ang II) and TxA2 in this renal derangement. Renal micropuncture experiments were performed on the right kidney of rats from an inbred colony with unilateral right-sided hydronephrosis (HYDRO), or non-affected litter mates (CONTROL). In addition, four separate groups of hydronephrotic animals were treated with either the TxA2 receptor antagonist SQ-29548 (SQ), one of two Ang II receptor antagonists [saralasin (SAR) or DuP-753 (DUP)]; or combined treatment with DuP-753 and SQ-29,548 (S&D). SNGFR was significantly reduced (P < 0.05) in HYDRO compared to CONTROL (17.6 +/- 2.0 vs. 35.9 +/- 3.7 nl/min, respectively). Treatment with SQ-29,548 normalized SNGFR (29.0 +/- 3.0 nl/min), while saralasin and DuP-753 resulted in only a partial recovery of function (25.6 +/- 1.6 and 27.8 +/- 1.4 nl/min, respectively). Combined SQ-29,548 and DuP-753 treatment resulted in full recovery of SNGFR to 32.9 +/- 4.4 nl/min. The glomerular ultrafiltration coefficient (Kf) was reduced (P < 0.05) approximately 45% in HYDRO compared to CONTROL (1.64 +/- .08 vs. 2.84 +/- .22 nl/min/mm Hg, respectively). Kf returned to control levels in SAR, DUP and SQ, and increased above control in S&D (5.58 +/- 1.6 nl/min/mm Hg). There were no differences (P > 0.05) in hydrostatic or oncotic pressures across the glomerular capillary between any of the groups studied. The observation that Kf increases above CONTROL with combined blockade of TxA2 and Ang II suggests that these regulatory hormones decrease Kf via independent mechanisms. These data indicate that the reduction in SNGFR in congenital, unilateral hydronephrosis is a result of a marked fall in Kf that is mediated by both Ang II and TxA2.

Upregulation of renin-angiotensin system and downregulation of kallikrein in obstructive nephropathy.

The purpose of this study was to delineate the effects of prolonged (1 and 5 wk) unilateral ureteral obstruction (UUO) on the intrarenal renin-angiotensin and kallikrein-kinin systems in the rat. Systolic blood pressure (SBP) and plasma angiotensin (ANG) II levels were significantly higher at 1 and 5 wk of obstruction than in sham-operated groups. Also, plasma renin activity and ANG I levels were elevated at 1 wk (P < 0.05), and plasma angiotensin-converting enzyme (ACE)-kininase II activity was elevated at 5 wk (P < 0.05). Blockade of ANG II receptors with losartan (Dup 753) prevented the rise in SBP after UUO and normalized SBP in chronically hypertensive UUO rats. Renin mRNA levels and ANG II content were elevated in the obstructed kidneys at 1 and 5 wk compared with sham-operated kidneys (P < 0.05). ACE-kininase II activity was elevated in both the obstructed and contralateral kidneys at 5 wk compared with sham-operated kidneys (P < 0.05). In marked contrast to renin, total immunoreactive kallikrein contents and tissue kallikrein mRNA levels in the obstructed kidneys were reduced to 25% of sham-operated kidneys both at 1 and 5 wk (P < 0.001). The results indicate that urinary obstruction activates renin and suppresses kallikrein gene expression. Activation of ACE-kininase II by UUO also serves to enhance intrarenal ANG II generation and kinin degradation. The results implicate ANG II overproduction and kinin deficiency in the pathogenesis of UUO-induced hypertension and intrarenal vasoconstriction.