Haploinsufficiency of the mouse Tshz3 gene leads to kidney defects.

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.

Aberrant Differentiation of Human Pluripotent Stem Cell-Derived Kidney Precursor Cells inside Mouse Vascularized Bioreactors.

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 ß1. 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.

Congenital Disorders of the Human Urinary Tract: Recent Insights From Genetic and Molecular Studies.

The urinary tract comprises the renal pelvis, the ureter, the urinary bladder, and the urethra. The tract acts as a functional unit, first propelling urine from the kidney to the bladder, then storing it at low pressure inside the bladder which intermittently and completely voids urine through the urethra. Congenital diseases of these structures can lead to a range of diseases sometimes associated with fetal losses or kidney failure in childhood and later in life. In some of these disorders, parts of the urinary tract are severely malformed. In other cases, the organs appear grossly intact yet they have functional deficits that compromise health. Human studies are beginning to indicate monogenic causes for some of these diseases. Here, the implicated genes can encode smooth muscle, neural or urothelial molecules, or transcription factors that regulate their expression. Furthermore, certain animal models are informative about how such molecules control the development and functional differentiation of the urinary tract. In future, novel therapies, including those based on gene transfer and stem cell technologies, may be used to treat these diseases to complement conventional pharmacological and surgical clinical therapies.

Uncovering genetic mechanisms of kidney aging through transcriptomics, genomics, and epigenomics.

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.

Generation of Functioning Nephrons by Implanting Human Pluripotent Stem Cell-Derived Kidney Progenitors.

Human pluripotent stem cells (hPSCs) hold great promise for understanding kidney development and disease. We reproducibly differentiated three genetically distinct wild-type hPSC lines to kidney precursors that underwent rudimentary morphogenesis in vitro. They expressed nephron and collecting duct lineage marker genes, several of which are mutated in human kidney disease. Lentiviral-transduced hPSCs expressing reporter genes differentiated similarly to controls in vitro. Kidney progenitors were subcutaneously implanted into immunodeficient mice. By 12 weeks, they formed organ-like masses detectable by bioluminescence imaging. Implants included perfused glomeruli containing human capillaries, podocytes with regions of mature basement membrane, and mesangial cells. After intravenous injection of fluorescent low-molecular-weight dextran, signal was detected in tubules, demonstrating uptake from glomerular filtrate. Thus, we have developed methods to trace hPSC-derived kidney precursors that formed functioning nephrons in vivo. These advances beyond in vitro culture are critical steps toward using hPSCs to model and treat kidney diseases.

Measures of kidney function by minimally invasive techniques correlate with histological glomerular damage in SCID mice with adriamycin-induced nephropathy.

Maximising the use of preclinical murine models of progressive kidney disease as test beds for therapies ideally requires kidney function to be measured repeatedly in a safe, minimally invasive manner. To date, most studies of murine nephropathy depend on unreliable markers of renal physiological function, exemplified by measuring blood levels of creatinine and urea, and on various end points necessitating sacrifice of experimental animals to assess histological damage, thus counteracting the principles of Replacement, Refinement and Reduction. Here, we applied two novel minimally invasive techniques to measure kidney function in SCID mice with adriamycin-induced nephropathy. We employed i) a transcutaneous device that measures the half-life of intravenously administered FITC-sinistrin, a molecule cleared by glomerular filtration; and ii) multispectral optoacoustic tomography, a photoacoustic imaging device that directly visualises the clearance of the near infrared dye, IRDye 800CW carboxylate. Measurements with either technique showed a significant impairment of renal function in experimental animals versus controls, with significant correlations with the proportion of scarred glomeruli five weeks after induction of injury. These technologies provide clinically relevant functional data and should be widely adopted for testing the efficacies of novel therapies. Moreover, their use will also lead to a reduction in experimental animal numbers.

Comparative Proteomics Study of Streptozotocin-induced Diabetic Nephropathy in Rats Kidneys Transfected with Adenovirus-mediated Fibromodulin Gene.

BACKGROUND: Transforming Growth Factor-beta (TGF-ß) 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-ß 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.

Periluminal expression of a secreted transforming growth factor-ß type II receptor inhibits in-stent neointima formation following adenovirus-mediated stent-based intracoronary gene transfer.

Transforming growth factor-ß1 (TGF-ß1) has been shown unequivocally to enhance neointima formation in carotid and ileo-femoral arteries. In our previous studies, however, TGF-ß1 expression in coronary arteries actually reduced neointima formation without affecting luminal loss postangioplasty, while expression of a TGF-ß1 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-ß 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 ß-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-ß antagonism on neointima formation in injured coronary and noncoronary arteries, and suggest that since cellular proliferation is unaffected, TGF-ß1 antagonism might prevent in-stent restenosis without the delayed healing that is associated with drug-eluting stents in current clinical use.

Cytokeratin 15 marks basal epithelia in developing ureters and is upregulated in a subset of urothelial cell carcinomas.

The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells.

Ex vivo modeling of chemical synergy in prenatal kidney cystogenesis.

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.

Muscarinic Acetylcholine Receptor M3 Mutation Causes Urinary Bladder Disease and a Prune-Belly-like Syndrome.

Urinary bladder malformations associated with bladder outlet obstruction are a frequent cause of progressive renal failure in children. We here describe a muscarinic acetylcholine receptor M3 (CHRM3) (1q41-q44) homozygous frameshift mutation in familial congenital bladder malformation associated with a prune-belly-like syndrome, defining an isolated gene defect underlying this sometimes devastating disease. CHRM3 encodes the M3 muscarinic acetylcholine receptor, which we show is present in developing renal epithelia and bladder muscle. These observations may imply that M3 has a role beyond its known contribution to detrusor contractions. This Mendelian disease caused by a muscarinic acetylcholine receptor mutation strikingly phenocopies Chrm3 null mutant mice.

Development of viral vectors for use in cardiovascular gene therapy.

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.

Beta-adrenoceptor blockade markedly attenuates transgene expression from cytomegalovirus promoters within the cardiovascular system.

OBJECTIVE: 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 beta-adrenoceptors might increase transgene expression from MIECMV, and if so, beta-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 beta-galactosidase. beta-galactosidase expression was assayed 72 hours later. Isoprenaline increased transgene expression from MIECMV up to 8-fold (P<0.001), but had no effect on a promoter containing no CRE. The effect of isoprenaline was inhibited by beta-blockade and by specific CRE-decoy oligonucleotides. Beta-blockers did not reduce transgene expression below basal levels. After adenovirus-mediated porcine intracoronary gene transfer, however, beta-blockade reduced beta-galactosidase expression by up to 250-fold compared with non-beta-blocked animals (P<0.01). CONCLUSIONS: Enhancement of promoter activity by endogenous catecholamines is essential for high-level transgene expression from MIECMV within the vasculature. Beta-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.