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All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost close to the time of birth. The generation of new nephron progenitors and their maintenance in culture are central to the success of kidney regenerative strategies. Using a lentiviral screening approach, we previously generated a human induced nephron progenitor-like state in vitro using a pool of six transcription factors. Here, we sought to develop a more efficient approach for direct reprogramming of human cells that could be applied in vivo. PiggyBac transposons are a non-viral integrating gene delivery system that is suitable for in vivo use and allows for simultaneous delivery of multiple genes. Using an inducible piggyBac transposon system, we optimized a protocol for the direct reprogramming of HK2 cells to induced nephron progenitor-like cells with expression of only 3 transcription factors (SNAI2, EYA1, and SIX1). Culture in conditions supportive of the nephron progenitor state further increased the expression of nephron progenitor genes. The refined protocol was then applied to primary human renal epithelial cells, which integrated into developing nephron structures in vitro and in vivo. Such inducible reprogramming to nephron progenitor-like cells could facilitate direct cellular reprogramming for kidney regeneration.
Copyright © 2019 International Society of Nephrology. All rights reserved.
Drug-dose modification in chronic kidney disease (CKD) utilizes glomerular filtration rate (GFR) with the implicit assumption that multiple renal excretory processes decline in parallel as CKD progresses. We compiled published pharmacokinetic data to evaluate if GFR predicts renal clearance changes as a function of CKD severity. For each drug, we calculated ratio of renal clearance to filtration clearance (Rnf). Of 21 drugs with Rnf >0.74 in subjects with GFR >90 mL/min (implying filtration and secretion), 13 displayed significant change in Rnf vs. GFR (slope of linear regression statistically different from zero), which indicates failure of GFR to predict changes in secretory clearance. The dependence was positive (n = 3; group A) or negative (n = 10; group B). Eight drugs showed no correlation (group C). Investigated drugs were small molecules, mostly hydrophilic, and ionizable, with some characterized as renal transporter substrates. In conclusion, dosing adjustments in CKD require refinement; in addition to GFR, biomarkers of tubular function are needed for secreted drugs.
© 2017 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.
Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.
INTRODUCTION - Partial nephrectomy has a 3%-4% incidence of local treatment failure. This study is to present a series of percutaneous cryoablation for locally recurrent renal cell carcinoma after partial nephrectomy.
MATERIALS AND METHODS - Five consecutive patients were referred to our quarternary center's multidisciplinary Small Renal Mass (SRM) Center for assessment after failure of partial nephrectomy. Tumor size and location was noted. CT-guided cryoablation was performed using an argon/helium-based system (Healthtronics, Austin, Texas, USA). Patients were admitted overnight for observation. Patients were followed with serial imaging, laboratory tests and examination at our SRM Center. Tumor size, location, and nephrometry scores were documented for each patient.
RESULTS - Four tumors were endophytic and one was exophytic. The median tumor size was 2.2 cm (1.8 cm-4.0 cm). Nephrometry scores were 8a, 7x, 4p, 6x, 7p, and 6p prior to cryoablation. Median follow up after cryoablation was 32 months (20-39 months). One patient with a 4.0 cm endophytic tumor developed a second recurrence measuring 2.9 cm 13 months following ablation, which was managed successfully with repeat cryoablation with no evidence of disease after an additional 19 months of follow up. Two patients developed self-limited hematuria which was conservatively managed. There were no other complications, and all patients remained at their pretreatment performance status.
CONCLUSIONS - Percutaneous cryoablation appears to be a safe and effective nephron-sparing modality for control of locally recurrent disease following partial nephrectomy. Most recurrent tumors are endophytic. One patient suffered a second local recurrence, which was managed successfully with repeat cryoablation.
BACKGROUND - The Yes-associated-protein-1 (YAP1) is a novel, direct regulator of stem cell genes both in development and cancer. FAT4 is an upstream regulator that induces YAP1 cytosolic sequestering by phosphorylation (p-Ser 127) and therefore inhibits YAP1-dependent cellular proliferation. We hypothesized that loss of FAT4 signaling would result in expansion of the nephron progenitor population in kidney development and that YAP1 subcellular localization would be dysregulated in Wilms tumor (WT), an embryonal malignancy that retains gene expression profiles and histologic features reminiscent of the embryonic kidney.
METHODS - Fetal kidneys from Fat4(-/-) mice were harvested at e18.5 and markers of nephron progenitors were investigated using immunohistochemical analysis. To examine YAP1 subcellular localization in WT, a primary WT cell line (VUWT30) was analyzed by immunofluorescence. Forty WT specimens evenly distributed between favorable and unfavorable histology (n = 20 each), and treatment failure or success (n = 20 each) was analyzed for total and phosphorylated YAP1 using immunohistochemistry and Western blot.
RESULTS - Fat4(-/-) mouse fetal kidneys exhibit nuclear YAP1 with increased proliferation and expansion of nephron progenitor cells. In contrast to kidney development, subcellular localization of YAP1 is dysregulated in WT, with a preponderance of nuclear p-YAP1. By Western blot, median p-YAP1 quantity was 5.2-fold greater in unfavorable histology WT (P = 0.05).
CONCLUSIONS - Fetal kidneys in Fat4(-/-) mice exhibit a phenotype reminiscent of nephrogenic rests, a WT precursor lesion. In WT, YAP1 subcellular localization is dysregulated and p-YAP1 accumulation is a novel biomarker of unfavorable histology.
© 2013 Wiley Periodicals, Inc.
BACKGROUND - Regression is an important process in the normal development of many organs. In this study, we investigated whether glomerular regression occurs after normal glomerulogenesis and determined the time course for this process.
METHODS - Glomerular number was analyzed in normal mouse kidneys at postnatal day (P)7, P10, P14, P18, P21, P25, and P28 by the gold standard fractionator/dissector method, which involves exhausting the kidney tissue. Vascular regression markers, angiopoietin 2 (ANGPT2), and thrombospondin 1 (THBS1), were examined by immunohistochemistry.
RESULTS - The maximum glomerular number was reached at P7 with 14,051 glomeruli per kidney (95% confidence interval: 12,084-16,018). This peak was followed by a progressive reduction, with a nadir of 11,060 (10,393-11,727) occurring at P18 (P < 0.05 as compared with P7). Thereafter, glomerular number remained constant. Complementary immunohistochemical examination of vascular regression markers showed peak expression of glomerular ANGPT2 and THBS1 at P14.
CONCLUSION - Our study reveals that the tissue- and time-saving Weibel-Gomez method commonly used to assess glomerular number is valid only after P18. The data indicate that regulation of glomerular number by regression occurs in normally maturing mouse kidneys. These findings suggest that the process of glomerular regression could be therapeutically targeted to prevent oligonephronia, which otherwise predisposes to chronic kidney disease.
PURPOSE - SIX2 and CITED1 are transcriptional regulators that specify self-renewing nephronic progenitor cells of the embryonic kidney. We hypothesized that SIX2, which promotes and maintains this stem cell population, and CITED1 remain active in Wilms' tumor (WT).
METHODS - To evaluate expression domains and the pathogenic significance of SIX2 and CITED1 across WT, the Children's Oncology Group provided 40 WT specimens of stages I to IV (n = 10 per stage), which were enriched for unfavorable histology (n = 20) and treatment failure (relapse or death, n = 20). SIX2 and CITED1 protein expression was evaluated qualitatively (immunohistochemistry) and quantitatively (Western blot, or WB). Gene transcription was estimated using quantitative real-time polymerase chain reaction (qRT-PCR).
RESULTS - SIX2 was visualized by immunohistochemistry in 36 (94.7%) of 38 specimens. Protein and messenger RNA expression of SIX2 were quantitatively similar across all stages of disease (P = .48 WB; P = 0.38 qPCR), in favorable or unfavorable histology (P = 0.51 WB; P = 0.58 qPCR), and in treatment failure or success (P = 0.86 WB; P = 0.49 qPCR). Although CITED1 expression paralleled SIX2 qualitatively, no quantitative correlation between SIX2 and CITED1 expression was observed (Spearman correlation coefficient, 0.28; P = 0.08). As in the fetal kidney, overlapping, but also distinct, WT cellular expression domains were observed between SIX2 and CITED1.
CONCLUSION - SIX2 and CITED1 remain active across all disease characteristics of WT. Activity of these genes in WT potentially identifies a population of self-renewing cancer cells that exhibit an embryonic, stemlike phenotype. Taken together, these transcriptional regulators may be fundamental to WT cellular self-renewal and may represent targets for novel therapies that promote terminal differentiation.
Copyright © 2012 Elsevier Inc. All rights reserved.
The identity of niche signals necessary to maintain embryonic nephron progenitors is unclear. Here we provide evidence that Fgf20 and Fgf9, expressed in the niche, and Fgf9, secreted from the adjacent ureteric bud, are necessary and sufficient to maintain progenitor stemness. Reduction in the level of these redundant ligands in the mouse led to premature progenitor differentiation within the niche. Loss of FGF20 in humans, or of both ligands in mice, resulted in kidney agenesis. Sufficiency was shown in vitro where Fgf20 or Fgf9 (alone or together with Bmp7) maintained isolated metanephric mesenchyme or sorted nephron progenitors that remained competent to differentiate in response to Wnt signals after 5 or 2 days in culture, respectively. These findings identify a long-sought-after critical component of the nephron stem cell niche and hold promise for long-term culture and utilization of these progenitors in vitro.
Copyright © 2012 Elsevier Inc. All rights reserved.
Studies of the complex responses of the kidney to acute injury have yielded important insights into mechanisms of tissue injury and repair. A variety of injury models have contributed to this impressive body of knowledge, but the ischemia-reperfusion (IR) model has perhaps been the most widely used. This chapter contains a detailed method description for IR injury in the mouse together with notes on blood sampling and tissue harvesting. The aim of the chapter is to provide the novice with a step-by-step guide to establishing this procedure in their research program.
Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment, distinguished by expression of CITED1, possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood, and their elucidation will provide key insights into normal and dysregulated nephrogenesis, as well as into regenerative processes that follow kidney injury. In this study, we found that the mouse CITED1(+) progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo, RAS inactivation by expression of sprouty 1 (Spry1) in CITED1(+) nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation, infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS.