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Background: Numerous studies have documented the in vitro differentiation of human pluripotent stem cells (hPSCs) into kidney cells. Fewer studies have followed the fates of such kidney precursor cells (KPCs) inside animals, a more life-like setting. Here, we tested the hypothesis that implanting hPSC-derived KPCs into an in vivo milieu surgically engineered to be highly vascular would enhance their maturation into kidney tissues. Methods: 3D printed chambers containing KPCs were implanted into the thighs of adult immunodeficient mice. In some chambers, an arterial and venous flow-through (AVFT) was surgically fashioned. After 3 weeks and 3 months, implants were studied by histology, using qualitative and quantitative methods. Results: After 3 weeks, chambers containing AVFTs were richer in small vessels than contralateral chambers without AVFTs. Glomeruli with capillary loops and diverse types of tubules were detected in all chambers. At 3 months, chambers contained only rudimentary tubules and glomeruli that appeared avascular. In chambers with AVFTs, prominent areas of muscle-like cells were also detected near tubules and the abnormal tissues immunostained for transforming growth factor β1. These features have similarities to renal dysplasia, a typical histological signature of human congenital kidney malformations. Conclusions: This study urges a note of caution regarding the in vivo fates of hPSC-derived kidney precursors, with pathological differentiation appearing to follow a period of increased vascularity.

Renal tract defects and autism spectrum disorder (ASD) deficits represent the phenotypic core of the 19q12 deletion syndrome caused by the loss of one copy of the TSHZ3 gene. Although a proportion of Tshz3 heterozygous (Tshz3+/lacZ) mice display ureteral defects, no kidney defects have been reported in these mice. The purpose of this study was to characterize the expression of Tshz3 in adult kidney as well as the renal consequences of embryonic haploinsufficiency of Tshz3 by analyzing the morphology and function of Tshz3 heterozygous adult kidney. Here, we described Tshz3 expression in the smooth muscle and stromal cells lining the renal pelvis, the papilla and glomerular endothelial cells (GEnCs) of the adult kidney as well as in the proximal nephron tubules in neonatal mice. Histological analysis showed that Tshz3+/lacZ adult kidney had an average of 29% fewer glomeruli than wild-type kidney. Transmission electron microscopy of Tshz3+/lacZ glomeruli revealed a reduced thickness of the glomerular basement membrane and a larger foot process width. Compared to wild type, Tshz3+/lacZ mice showed lower blood urea, phosphates, magnesium and potassium at 2 months of age. At the molecular level, transcriptome analysis identified differentially expressed genes related to inflammatory processes in Tshz3+/lacZ compare to wild-type (control) adult kidneys. Lastly, analysis of the urinary peptidome revealed 33 peptides associated with Tshz3+/lacZ adult mice. These results provide the first evidence that in the mouse Tshz3 haploinsufficiency leads to cellular, molecular and functional abnormalities in the adult mouse kidney.

Nephrons scar and involute during aging, increasing the risk of chronic kidney disease. Little is known, however, about genetic mechanisms of kidney aging. We sought to define the signatures of age on the renal transcriptome using 563 human kidneys. The initial discovery analysis of 260 kidney transcriptomes from the TRANScriptome of renaL humAn TissuE Study (TRANSLATE) and the Cancer Genome Atlas identified 37 age-associated genes. For 19 of those genes, the association with age was replicated in 303 kidney transcriptomes from the Nephroseq resource. Surveying 42 nonrenal tissues from the Genotype-Tissue Expression project revealed that, for approximately a fifth of the replicated genes, the association with age was kidney-specific. Seventy-three percent of the replicated genes were associated with functional or histological parameters of age-related decline in kidney health, including glomerular filtration rate, glomerulosclerosis, interstitial fibrosis, tubular atrophy, and arterial narrowing. Common genetic variants in four of the age-related genes, namely LYG1, PPP1R3C, LTF and TSPYL5, correlated with the trajectory of age-related changes in their renal expression. Integrative analysis of genomic, epigenomic, and transcriptomic information revealed that the observed age-related decline in renal TSPYL5 expression was determined both genetically and epigenetically. Thus, this study revealed robust molecular signatures of the aging kidney and new regulatory mechanisms of age-related change in the kidney transcriptome.

Transforming growth factor-β1 (TGF-β1) has been shown unequivocally to enhance neointima formation in carotid and ileo-femoral arteries. In our previous studies, however, TGF-β1 expression in coronary arteries actually reduced neointima formation without affecting luminal loss postangioplasty, while expression of a TGF-β1 antagonist (RIIs) in balloon-injured coronary arteries reduced luminal loss without affecting neointima formation. These observed effects may be a consequence of the mode of coronary artery gene transfer employed, but they may also represent differences in the modes of healing of coronary, carotid, and ileo-femoral arteries after endoluminal injury. To help clarify whether a gene therapy strategy to antagonize TGF-β might have application within the coronary vasculature, we have investigated the effect of high-level periluminal expression of RIIs using stent-based adenovirus-mediated intracoronary gene transfer. Porcine coronary arteries were randomized to receive a custom-made CoverStent preloaded with saline only, or with 1×10(9) infectious units of adenovirus expressing RIIs or β-galactosidase (lacZ). Vessels were analyzed 28 days poststenting, at which time angiographic in-stent diameter was significantly greater in RIIs-treated arteries, and in-stent luminal loss significantly reduced. Computerized morphometric minimum in-stent lumen area was ~300% greater in RIIs-exposed vessels than in lacZ or saline-only groups. This was because of significantly reduced neointima formation in the RIIs group. RIIs had no demonstrable effect on cellular proliferation or apoptosis, but greater normalized neointimal/medial collagen content was observed in RIIs-exposed arteries. These data highlight the qualitatively similar effect of TGF-β antagonism on neointima formation in injured coronary and noncoronary arteries, and suggest that since cellular proliferation is unaffected, TGF-β1 antagonism might prevent in-stent restenosis without the delayed healing that is associated with drug-eluting stents in current clinical use.

Cardiovascular disease represents the most common cause of mortality in the developed world but, despite two decades of promising pre-clinical research and numerous clinical trials, cardiovascular gene transfer has so far failed to demonstrate convincing benefits in the clinical setting. In this review we discuss the various targets which may be suitable for cardiovascular gene therapy and the viral vectors which have to date shown the most potential for clinical use. We conclude with a summary of the current state of clinical cardiovascular gene therapy and the key trials which are ongoing.

Background Transforming Growth Factor-beta (TGF-β) activation appears to be crucial for tissue injury in Diabetic Nephropathy (DN). Fibromodulin, the small leucine-rich proteoglycan, has been proposed to be the potent TGF-β modulator. In this study, the therapeutic effects of fibromodulin in the kidneys of streptozotocin (STZ)-induced diabetic rats were investigated. Methods Diabetic rats received intraperitoneal (IP) injections of recombinant adenovirus expression vectors (RAd5) containing fibromodulin (RAd-FMOD) and were killed after 10 weeks. Proteins were isolated from the rat kidney and separated using two-dimensional gel electrophoresis. The differentially expressed proteins were analyzed using Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Results Ten spots were identified using MALDI-TOF-MS. The identified proteins were primarily responsible for cell metabolism, cytoskeleton formation, and oxidative stress. RAd-FMOD treatment markedly attenuated the albuminuria in diabetic rats. Conclusion Taken together, these results provide a valuable clue in exploring the mechanism underlying the therapeutic effects of fibromodulin in diabetic nephropathy suggesting that it can be a potential agent in the treatment of this disease.

Cyclic adenosine monophosphate (cAMP) drives genetic polycystic kidney disease (PKD) cystogenesis. Yet within certain PKD families, striking differences in disease severity exist between affected individuals, and genomic and/or environmental modifying factors have been evoked to explain these observations. We hypothesized that PKD cystogenesis is accentuated by an aberrant fetal milieu, specifically by glucocorticoids. The extent and nature of cystogenesis was assessed in explanted wild-type mouse embryonic metanephroi, using 8-Br-cAMP as a chemical to mimic genetic PKD and the glucocorticoid dexamethasone as the environmental modulator. Cysts and glomeruli were quantified by an observer blinded to culture conditions, and tubules were phenotyped using specific markers. Dexamethasone or 8-Br-cAMP applied on their own produced cysts predominantly arising in proximal tubules and descending limbs of loops of Henle. When applied together, however, dexamethasone over a wide concentration range synergized with 8-Br-cAMP to generate a more severe, glomerulocystic, phenotype; we note that prominent glomerular cysts have been reported in autosomal dominant PKD fetal kidneys. Our data support the idea that an adverse antenatal environment exacerbates renal cystogenesis. © 2013 Anders et al.

Poor long-term graft patency remains a major limitation of coronary artery bypass grafting using saphenous vein aortocoronary grafts. Neointimal hyperplasia (NIH) represents the principal mechanism of graft failure; a substantial body of evidence implicates transforming growth factor-beta1 (TGF-beta1) in the pathogenesis of NIH. The small leucine-rich proteoglycans decorin and fibromodulin possess TGF-beta-antagonist activity to differing extents and with differing avidities for the isoforms of TGF-beta. We compared their ability to inhibit NIH in an ex vivo model of human saphenous vein organ culture following adenovirus-mediated gene transfer. Surgically prepared human saphenous vein segments received adenovirus expressing fibromodulin (Ad5-Fmod), decorin (Ad5-Dcn), beta-galactosidase (Ad5-lacZ) or vehicle-only. Computerized morphometry 14 days after infection revealed significantly reduced neointimal area, neointimal thickness and intima/media ratio in Ad5-Fmod- and Ad5-Dcn-infected veins. Each parameter was significantly smaller in Ad5-Fmod- than in Ad5-Dcn-exposed segments. Fibrillar collagen content and levels of biologically active TGF-beta were lower in vessels receiving Ad5-Fmod or Ad5-Dcn than in those receiving Ad5-lacZ or vehicle-only. Fibromodulin is a more potent inhibitor of NIH in cultured human saphenous vein than decorin and offers potential therapeutic benefits in saphenous vein graft failure (and possibly in other forms of accelerated atherosclerosis) by reduction of associated neointima formation.

Objectives— The major immediate–early cytomegalovirus enhancer/promoter (MIECMV), widely used in cardiovascular gene therapy, contains several positively regulatory cAMP response elements (CRE). Catecholamine signaling via β-adrenoceptors might increase transgene expression from MIECMV, and if so, β-blockers may have a detrimental effect on the efficacy of clinical cardiovascular gene therapy strategies. Methods and Results— Cultured smooth muscle cells were exposed to isoprenaline, atenolol, or propranolol, alone and in combination before infection with adenoviruses expressing β-galactosidase. β-galactosidase expression was assayed 72 hours later. Isoprenaline increased transgene expression from MIECMV up to 8-fold ( P P Conclusions— Enhancement of promoter activity by endogenous catecholamines is essential for high-level transgene expression from MIECMV within the vasculature. β-blocker-mediated suppression of transgene expression from MIECMV in vascular tissues has a significant bearing on clinical studies of cardiovascular gene transfer. This is the first described interaction to our knowledge between widely prescribed pharmaceuticals and a commonly used promoter of clinical transgene expression.

Introduction: The success of coronary artery bypass grafting (CABG) is limited by poor long-term graft patency. Saphenous vein (SV) is the most commonly used conduit for CABG with a high failure rate due to intimal thickening and superimposed atherosclerosis. In the absence of any effective pharmacological therapies, late vein graft failure has been identified as a putative target for gene therapy. Two small leucine-rich proteoglycans, decorin and fibromodulin, possess TGF-|[beta]|1-antagonist activity in vitro and in vivo. Local overexpression of decorin reduces neointima formation in response to arterial injury in animal models. However fibromodulin is a more effective competitor for TGF-|[beta]| binding than decorin. The purpose of this study was to evaluate the ability of adenoviral-mediated gene transfer of bovine fibromodulin to inhibit neointima formation in an ex vivo model of vascular injury.