Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies.

The differentiation of embryonic or determined stem cell populations into adult liver fates under known conditions yields cells with some adult-specific genes but not others, aberrant regulation of one or more genes, and variations in the results from experiment to experiment. We tested the hypothesis that sets of signals produced by freshly isolated, lineage-dependent mesenchymal cell populations would yield greater efficiency and reproducibility in driving the differentiation of human hepatic stem cells (hHpSCs) into adult liver fates. The subpopulations of liver-derived mesenchymal cells, purified by immunoselection technologies, included (1) angioblasts, (2) mature endothelia, (3) hepatic stellate cell precursors, (4) mature stellate cells (pericytes), and (5) myofibroblasts. Freshly immunoselected cells of each of these subpopulations were established in primary cultures under wholly defined (serum-free) conditions that we developed for short-term cultures and were used as feeders with hHpSCs. Feeders of angioblasts yielded self-replication, stellate cell precursors caused lineage restriction to hepatoblasts, mature endothelia produced differentiation into hepatocytes, and mature stellate cells and/or myofibroblasts resulted in differentiation into cholangiocytes. Paracrine signals produced by the different feeders were identified by biochemical, immunohistochemical, and quantitative reverse-transcription polymerase chain reaction analyses, and then those signals were used to replace the feeders in monolayer and threedimensional cultures to elicit the desired biological responses from hHpSCs. The defined paracrine signals were proved to be able to yield reproducible responses from hHpSCs and to permit differentiation into fully mature and functional parenchymal cells. Conclusion: Paracrine signals from defined mesenchymal cell populations are important for the regulation of stem cell populations into specific adult fates; this finding is important for basic and clinical research as well as industrial investigations. (HEPATOLOGY 2010;52:1443-1454 H uman hepatic stem cells (hHpSCs) are uniquely positioned at the foundation of potential liver regeneration therapies because they are the only parenchymal cell subpopulation identified with both the capacity for self-renewal and the capacity to generate numerous progenitors, such as

Recent reports identified activation of the GABA signaling pathway as a means to induce transdifferentiation of pancreatic α cells into β cells. These reports followed several previous studies that found that α cells were particularly well suited to conversion into β cells in mice, but only after nearly complete β cell loss or forced overexpression of key transcriptional regulators. The possibility of increasing β cell number via reprograming of α cells with a small molecule is enticing, as this could be a potential new pharmacologic therapy for diabetes. Here, we employed rigorous genetic lineage tracing of α cells, using Glucagon-CreER;Rosa-LSL-eYFP mice, to evaluate if activation of GABA signaling caused α-to-β cell reprogramming. In contrast to previous reports, we found that even after long-term treatment of mice with artesunate or GABA, neither α-to-β cell transdifferentiation nor insulin secretion were stimulated, putting into question whether these agents represent a viable path to a novel diabetes therapy.

The effect of acute intravenous infusion of cyclosporine (10 mg/kg) on efferent renal and genitofemoral nerve activity and afferent renal nerve activity was studied in anesthetized rats. AU animals were studied after unilateral renal denervation and extracellular fluid volume expansion. Activity of both efferent sympathetic nerves was increased significantly by cyclosporine infusion (renal, 69%; genitofemoral, 60%). Afferent renal nerve activity was increased 82% after cyclosporine (P < 0.05). Urine flow rate and both absolute and fractional sodium excretion from the innervated kidney were reduced 50% after cyclosporine infusion (P < 0.01). Absolute and fractional sodium excretion from the denervated kidney were significantly increased after cyclosporine. Infusion of vehicle had no significant effect on any measured variable in innervated or denervated kidneys. These studies demonstrate the capacity of cyclosporine to increase efferent sympathetic nerve activity and afferent nerve activity. It is also shown that sodium retention resulting from acute infusion of cyclosporine can be attributed to the increase in efferent renal nerve activity.Cyclosporine (cyclosporin A) is a potent immunosuppressive agent that has significantly improved graft survival in organ and bone marrow transplant recipients. However, toxic side effects are common. Nephrotoxicity and hepatotoxicity have been most often reported as complications of cyclosporine use (1, 2), but clinically important effects such as hypertension, tremors, and tachycardia are also frequently observed (3, 4). These latter indications oftoxicity have been attributed to stimulation of the sympathetic nervous system (5). In view of the known effects of renal efferent nerve activity on renal excretory function (6, 7), we postulated that reports of decreased glomerular filtration rate (GFR) and retention of salt and water associated with an acute infusion of cyclosporine (8,9) were consistent with increased sympathetic nerve activity to the kidneys. Furthermore, the excitatory action of cyclosporine on the sympathetic nervous system may be the result of an activation of peripheral afferent nerves. These hypotheses were tested in rats by measuring the effects of an acute infusion of cyclosporine (total dose 10 mg/kg) on renal function, efferent renal and genitofemoral nerve activity (ERNA and EGNA), and afferent renal nerve activity (ARNA). In the course of preparing renal nerves for activity recording, all nerve bundles from the left kidney were cut and the adventitia associated with the renal pedicle was removed. However, the use of phenol in alcohol to ensure complete destruction of renal nerve fibers was avoided because this would have endangered the dissected nerve bundles intended for activity recordings. METHODS AND MATERIALSNerve Activity Recording. Multifiber activity was recorded from the renal and genitofemoral nerves, both of which contain sympathetic efferent and visceral afferent fibers. In each nerve, activity from afferent or efferent fibers was ...

Orally delivered salt stimulates renal salt excretion more effectively than does iv delivered salt. Although the mechanisms that underlie this "postprandial natriuresis" are poorly understood, the peptide uroguanylin (UGn) is thought to be a key mediator. However, the lack of selective assays for UGn gene products has hindered rigorous testing of this hypothesis. Using peptide-specific assays, we now report surprisingly little UGn in rat intestine or plasma. In contrast, prouroguanylin (proUGn), the presumed-inactive precursor of UGn, is plentiful (at least 40 times more abundant than UGn) in both intestine and plasma. The intestine is the likely source of the circulating proUGn because: 1) the proUGn portal to systemic ratio is approximately two under normal conditions, and 2) systemic proUGn levels decrease rapidly after intestinal resection. Together, these data suggest that proUGn itself is actively involved in enterorenal signaling. This is strongly supported by our observation that iv infusion of proUGn at a physiological concentration produces a long-lasting renal natriuresis, whereas previously reported natriuretic effects of UGn have required supraphysiological concentrations. Thus, our data point to proUGn as an endocrine (i.e. circulating) mediator of postprandial natriuresis, and suggest that the propeptide is secreted intact from the intestine into the circulation and processed to an active form at an extravascular site.

Bladder afferent nerve activity was recorded from the pelvic and hypogastric nerves of rats anesthetized with pentobarbital sodium. Bladder filling with isotonic NaCl at a rate of 250 microl/min excited multiunit afferent activity in the hypogastric nerve by 190 +/- 38% over background at a pressure of 30 mmHg, whereas 150 meq/l KCl at the same filling rate excited hypogastric nerve activity by 498 +/- 103% (P < 0.0001). This difference was localized to a group of chemosensitive fibers that are excited by bladder filling with KCl in a concentration-dependent fashion but are insensitive to bladder filling with NaCl. Bladder filling with 200 meq/l KCl at different filling rates caused a bursting pattern of discharge in these fibers at consistent bladder volumes but with a pressure threshold that increased proportionately with filling rate. Other hypogastric bladder afferent fibers were activated to a similar extent by NaCl and KCl solutions. Chemoreceptive bladder afferent fibers were rare in the pelvic nerve (1 of 15 units), and multiunit preparations showed comparable excitations during bladder filling with NaCl and KCl solutions. The bursting activation of bladder chemoreceptive afferent nerves in hypogastric nerves could signal noxious overdistension and/or inflammation of the bladder.

Understanding the molecular basis of the regenerative response following hepatic injury holds promise for improved treatment of liver diseases. Here, we report an innovative method to profile gene expression specifically in the hepatocytes that regenerate the liver following toxic injury. We used the Fah-/- mouse, a model of hereditary tyrosinemia, which conditionally undergoes severe liver injury unless fumarylacetoacetate hydrolase (FAH) expression is reconstituted ectopically. We used translating ribosome affinity purification followed by high-throughput RNA sequencing (TRAP-seq) to isolate mRNAs specific to repopulating hepatocytes. We uncovered upstream regulators and important signaling pathways that are highly enriched in genes changed in regenerating hepatocytes. Specifically, we found that glutathione metabolism, particularly the gene Slc7a11 encoding the cystine/glutamate antiporter (xCT), is massively upregulated during liver regeneration. Furthermore, we show that Slc7a11 overexpression in hepatocytes enhances, and its suppression inhibits, repopulation following toxic injury. TRAP-seq allows cell type-specific expression profiling in repopulating hepatocytes and identified xCT, a factor that supports antioxidant responses during liver regeneration. xCT has potential as a therapeutic target for enhancing liver regeneration in response to liver injury.

SUMMARY Two major groups of renal chemosensory neural elements have been identified in the rat: one specifically activated by renal ischemia, the previously described "R" chemoreceptors, and the other by backflow of nondiuretic urine into the renal pelvis. The latter group is the object of the present investigation. In anesthetized, male Sprague-Dawley rats, single-unit recordings were obtained by dissection of the centrally cut nerves of the right kidney. The responses of single units to backflow into the renal pelvis of nondiuretic urine, diuretic urine, and solutions containing urea, mannitol, or inorganic ions were compared. The excitatory effect of the backflow of nondiuretic urine was due to its chemical composition rather than to changes in pelvic pressure and pelvic distension. The same units were activated markedly by renal ischemia. The resting discharge rate of the units was very high in nondiuretic conditions, and it declined progressively when diuresis was induced by expansion of the extracellular fluid volume. It is concluded that this group of sensory elements responds to the chemical environment in the renal interstitium as modified by ions crossing the pelvic epithelium, by leakage of ions out of ischemic cells, and by alterations in the excretory function of the kidney and renal blood flow. This group of renal sensory nerve endings has been termed "R2" chemoceptive receptors, to distinguish them from the previously described group of renal "R" chemoreceptors. Circ Res 46: 395-405, 1980LIKE OTHER visceral organs, the kidneys have a profuse sensory innervation. From recordings of impulses conducted along afferent fibers in the renal nerves, several functional classes of renal sensory receptors have been identified so far. In cats, there are receptors sensitive to alterations in ureteral pressure and to venous or arterial perfusion pressure (Beacham and Kunze, 1969;Astrom and Crafoord, 1968); in rabbits, receptors affected by changes in ureteral and arterial perfusion pressure, but not changes in venous pressure (Niijima, 1971); in dogs, units responsive to changes in venous, ureteral, or arterial perfusion pressure (Uchida et al., 1971); in rats, receptors sensitive to increase in venous pressure (Astrom and Crafoord, 1967). In the renal nerves of the rat at least two other populations exist. The fibers of one group are silent under control conditions and are activated only by renal ischemia; those of the other population exhibit a resting discharge and respond markedly to backflow of urine into the renal pelvis (Recordati et al., 1978 characteristics of the first group of receptors which, because of their sensitivity to ischemia and unresponsiveness to mechanical stimuli, were termed renal, "R," chemoreceptors. The second group, on the other hand, was analyzed only superficially. They were considered to be a population of renal mechanoreceptors because previous investigators (Beacham and Kunze, 1969;Astrom and Crafoord, 1968;Niijima, 1971) had thought that changes in pelvic pressure and pelvic d...