Intensive blood glucose control can decelerate the development of microvascular complications in patients with DN (Gross et al. 2005). In the Kumamoto Study, type 2 diabetic patients receiving multiple insulin therapies for strict glycemic control had slower progression of nephropathy and microvascular complications, including retinopathy (Shichiri et al. 2000). However, the beneficial effects of strict glycemic control are restricted to early-stage DN, prior to the development of albuminuria. Even with strict glycemic control, diabetic patients with developed microalbuminuria do not benefit from a decreased rate of progression to macroabuminuria. (DCCT 1995 and Microalbuminuria Collaborative Study Group 1995).

Renin-angiotensin system (RAS) blockade, with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs), produce positive effects on DN-related albuminuria. Both ACE inhibitors and ARBs are not only effective treatment options for hypertension, but also have an additional renoprotective effect independent of blood pressure reduction. This renoprotective effect is associated with diminished intraglomerular pressure and flow of proteins into the proximal tubule (Thurman and Schrier 2003). This renoprotective effect decreases urine albumin excretion (UAE) and progression to more advanced stages of DN. Various clinical studies have shown that ACE inhibitor and ARB therapies can decrease the rate of progression towards overt DN (Parving et al. 2001) and reduce UAE (Viberti and Wheeldon 2002; Andersen et al. 2003). However, in a recent large-scale clinical study involving 1448 type II diabetic patients, increased adverse effects of combined RAS blockade (ACE inhibitors in combination with ARBs) for treatment of DN have been reported (Fried et al. 2013). The study was terminated early due to safety concerns. Similarly, several other clinical trials with PKC inhibitors, ACE, advanced glycation end products (AGEs), aldose reductase etc. have failed to generate positive effects against DN.

DN is the leading cause of ESRD in the western world (2). For patients with ESRD, the only treatment options available are either dialysis or kidney transplantation (Go et al. 2004). Although dialysis is a lifesaving alternative that can clear out waste and toxins from the blood similar to the kidneys, it is only equivalent to 10 % of healthy kidney function. Besides, long-term dialysis is associated with various complications, such as cardiovascular disease, hernia, peritonitis, etc. (Collins et al. 2009; Diaz-Buxo et al. 2013; Lok and Foley 2013 ; Stuart et al. 2009). The average life expectancy of a patient starting on dialysis is generally 3–5 years (Stokes 2011 and USRDS 2009). The best therapeutic strategy for ESRD patients is kidney transplantation. Kidney transplantation can extend the life expectancy of patients compared to those who stay on dialysis. On average, transplant of a living donor kidney can function for 12–20 years, and a deceased donor kidney from 8 to 12 years (Traynor et al. 2012). Studies have shown that kidney transplantation is especially beneficial for younger patients, but even older adults can favor from an average of 4 or more years of life span compared to patients on dialysis (Briggs 2001; Knoll 2013). However, kidney transplantation is a major surgery with phased recovery. The long-term success of the transplant is limited by the host’s immune response to the foreign kidney graft. Clinical studies have shown that transplants from human leukocyte antigen (HLA)-identical siblings result in highest graft survival (99.17 %, 91.84 %, and 88.96 % at 1, 3, and 5 years, respectively) (Kessaris et al. 2008). To prevent immune rejection, transplanted patients will be required to routinely take immunosuppressant drugs for the rest of their lives. The prolonged immunosuppressant therapies have various adverse effects, including bone disease, hyperglycemia, and most importantly increased susceptibility to opportunistic infections (Alangaden et al. 2006; Marcen 2009). Another major drawback of kidney transplantation, like in any other form of transplantation, is the lack of organ donors. Due to the existing imbalance between demand for kidney transplantation and the number of donor organs, many patients suffering from kidney disease have no option but to remain on dialysis.

The viral vector delivery system is a popular method of transfection due to its ease of production, having a high functional titer and its ability to infect various cell types (Loiler et al. 2003; Work et al. 2009). A gene of interest can be integrated into a viral vector, and exposure of the virus to the targeted cells can lead to transfection. This leads to expression of the transgene in the host cell, resulting in a desired therapeutic effect. There are several pre-clinical studies using viral vectors for targeting DN (Table 17.2). Although this method has been used for clinical studies of cardiovascular disease (Jessup et al. 2011; Grines et al. 2003), there are as yet no clinical studies of viral gene transfection against DN. Despite being approved for clinical trials, the use of viral vectors has several potential drawbacks, including cytotoxicity, expensive production, high immunogenicity and the risk of mutagenesis in the host genome (Thomas et al. 2003).

Fatal accidental deaths have been reported form studies after systemic administration of adenoviral vectors due to over activation of the innate immune response (Marshall 1999). Adeno-associated virus (AAV) has moved to the forefront as the most attractive viral vector. The recombinant form remains almost completely episomal, with only minimal genomic integration in mice (Inagaki et al. 2008) and humans (Kaeppel et al. 2013). While clinical trial experience is growing rapidly with recombinant AAV vectors, immune responses to the capsid and toxicity remain potential concerns. As a result, research into the development of non-viral vectors continues to evolve.

The carrier agents for UMGD are microbubbles; very small, gas-filled bubbles. The gas-filled core is usually made up of a high molecular weight gas, such as decafluorobutane, perfluoropropane or sulfur hexafluoride, while the outer shell is composed of biocompatible lipids or proteins (Klibanov 2006). The low diffusivity and solubility properties of the heavy gas core allows for prolonged stability in circulation. The rheology of microbubbles is comparable to that of red blood cells, with a mean diameter of approximately 2–4 μm; giving them the ability to freely traverse through the microvasculature without being impeded (Lindner et al. 2002). Microbubble contrast agents have been conventionally used as a diagnostic contrast agent for clinical use with ultrasound (Mulvagh et al. 2008; Honos et al. 2007). The ability to modify microbubbles to become a carrier vessel for nucleic acid vectors, such as plasmid DNA, microRNA (miRNA) and silencing-RNA (siRNA) allows them to be used for UMGD.

A schematic diagram of UMGD is depicted in Fig. 17.1. Firstly, microbubble-nucleic acid complexes are generated. This is either by direct incorporation of nucleic acids during microbubble sonication, by conjugation of nucleic acids (plasmid DNA, siRNAs, miRNAs or viral DNA) to the outer surface of microbubbles via electrostatic interactions (charge-coupling), or by simple mixing/co-administration. Secondly, the microbubble-nucleic acid complexes are administered intravenously. Thirdly, the complex circulates through the systemic vascular system during transmission of high power triggered ultrasound over the target tissue/organ. The destructive power of ultrasound causes acoustic disruption of the gas-filled microbubbles and facilitates the nucleic acid uptake by the endothelium and surrounding cells/interstitium. This uptake is via several mechanisms, including formation of microjets, induction of transient pores at the cellular membrane and endocytosis (Christiansen et al. 2003; Kodama et al. 2006; Meijering et al. 2009).

While transfection efficiency of UMGD is relatively modest compared to viral vectors, studies using marker genes, such as luciferase, have demonstrated that transfection can be optimized by (1) using longer pulsing intervals for triggered delivery. This allows replenishment of the microvasculature by microbubble-DNA complexes within the target tissue/organ between destructive delivery pulses of ultrasound (Chen et al. 2003; Song et al. 2002). Conversely, continuous ultrasound transmission results in minimal transfection due to continual destruction of microbubble-DNA complexes as soon as they enter the ultrasound beam, (2) using higher acoustic powers for ultrasound application, with low transmission frequency and a greater mechanical index (Chen et al. 2003). This method must however be balanced against higher adverse bioeffects, especially when applied to the kidney (Williams et al. 2007), (3) the use of intra-arterial injections of carrier microbubbles versus intravenous delivery (Song et al. 2002; Christiansen et al. 2003) also results in greater tissue damage and vessel hemorrhage, and (4) use of repeated deliveries resulting in a more sustained duration of transgene expression (Bekeredjian et al. 2003).

There have been numerous studies of UMGD in multiple organs, delivering gene therapy in many pre-clinical models of human disease. Studies have demonstrated the efficacy of UMGD for therapeutic angiogenesis in a rat model of peripheral arterial disease (PAD). UMGD of a plasmid DNA encoding VEGF and green fluorescent protein (GFP) in a bicistronic vector resulted in increased microvascular perfusion and vessel density (Leong-Poi et al. 2007). Using the same vector and PAD model, UMGD was more effective for therapeutic angiogenesis compared to direct intramuscular (im) injection of the VEGF/GFP plasmid, despite lower transgene expression. The higher efficiency of pro-angiogenic gene therapy by UMGD was likely due to its targeted vascular transfection, as compared to IM injections. While IM injections resulted in local transfection of myocytes and perivascular regions, UMGD resulted in more diffuse transfection of arteriole and capillary endothelium and surrounding myocytes (Kobulnik et al. 2009). UMGD can be effective for targeted cardiac transfection, including delivery of pro-angiogenic genes in pre-clinical models of myocardial infarction (Fujii et al. 2009, 2011) and heart failure (Lee et al. 2013). A major advantage of UMGD is its noninvasive nature, which allows for delivery of multiple genes through repeated gene therapy (Fujii et al. 2011; Smith et al. 2012). Using multiple UMGD deliveries of plasmid DNA encoding for stem cell factor and stromal cell derived factor-1 (SDF-1) in a rat model of myocardial infarction, Fujii et al. showed increased vascular density in the peri-infarct region and greater myocardial perfusion and ventricular function compared with untreated animals (Fujii et al. 2011). The use of UMGD for in-vivo gene transfer has been applied for targeted gene transfection in other organs and tissues that can be imaged by diagnostic ultrasound, including kidney, pancreas (Chen et al. 2006, 2007, 2010) and tumors (Fujii et al. 2013; Carson et al. 2011, 2012).