There remains a need for robust mouse models of diabetic nephropathy (DN) that mimic key features of advanced human DN. The recently developed mouse strain BTBR with the ob/ob leptin-deficiency mutation develops severe type 2 diabetes, hypercholesterolemia, elevated triglycerides, and insulin resistance, but the renal phenotype has not been characterized. Here, we show that these obese, diabetic mice rapidly develop morphologic renal lesions characteristic of both early and advanced human DN. BTBR ob/ob mice developed progressive proteinuria beginning at 4 weeks. Glomerular hypertrophy and accumulation of mesangial matrix, characteristic of early DN, were present by 8 weeks, and glomerular lesions similar to those of advanced human DN were present by 20 weeks. By 22 weeks, we observed an approximately 20% increase in basement membrane thickness and a Ͼ50% increase in mesangial matrix. Diffuse mesangial sclerosis (focally approaching nodular glomerulosclerosis), focal arteriolar hyalinosis, mesangiolysis, and focal mild interstitial fibrosis were present. Loss of podocytes was present early and persisted. In summary, BTBR ob/ob mice develop a constellation of abnormalities that closely resemble advanced human DN more rapidly than most other murine models, making this strain particularly attractive for testing therapeutic interventions. Diabetic nephropathy (DN) is the largest single cause of ESRD in the United States, accounting for nearly half of the patients who enter the dialysis patient population each year and currently accounting for 45% of prevalent kidney failure in the United States. 1-4 Although both type 1 and type 2 diabetes lead to DN, the current epidemic of DN is due to type 2 diabetes; however, understanding the mechanisms that produce the constellation of clinical and pathologic alterations that define DN in humans remains very incomplete, in part because clinical DN is a slowly progressive disease, and relevant animal models that produce this constellation of pathologic and clinical abnormalities have important limitations. Mice rendered hyperglycemic by administration of streptozotocin (STZ) or through genetic predisposition such as the db/db mouse can develop some features of DN, most notably glomerular mesangial expansion, but do so only over prolonged periods and do not progress to ESRD. [5][6][7][8][9] Most murine models to date have failed to develop reliably marked mesangial expansion or the

Rationale: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) exhibit either a "working" chamber or a nodal-like phenotype. To generate optimal hESC-CM preparations for eventual clinical application in cell-based therapies, we will need to control their differentiation into these specialized cardiac subtypes.Objective: To demonstrate intact neuregulin (NRG)-1␤/ErbB signaling in hESC-CMs and test the hypothesis that this signaling pathway regulates cardiac subtype abundance in hESC-CM cultures. Methods and Results:All experiments used hESC-CM cultures generated using our recently reported directed differentiation protocol. To support subsequent action potential phenotyping approaches and provide a higher-throughput method of determining cardiac subtype, we first developed and validated a novel genetic label that identifies nodal-type hESC-CMs. Next, control hESC-CM preparations were compared to those differentiated in the presence of exogenous NRG-1␤, an anti-NRG-1␤ neutralizing antibody, or the ErbB antagonist AG1478. We used 3 independent approaches to determine the ratio of cardiac subtypes in the resultant populations: direct action potential phenotyping under current-clamp, activation of the aforementioned genetic label, and subtype-specific marker expression by RT-PCR. Using all 3 end points, we found that inhibition of NRG-1␤/ErbB signaling greatly enhanced the proportion of cells showing the nodal phenotype. Conclusions: NRG-1␤/ErbB signaling regulates the ratio of nodal-to working-type cells in differentiatinghESC-CM cultures and presumably functions similarly during early human heart development. We speculate that, by manipulating NRG-1␤/ErbB signaling, it will be possible to generate preparations of enriched working-type myocytes for infarct repair, or, conversely, nodal cells for potential use in a biological pacemaker. (Circ Res. 2010;107:776-786.) Key Words: embryonic stem cell Ⅲ electrophysiology Ⅲ pacemaker Ⅲ neuregulin A number of groups, including our own, have reported methods for generating large quantities of highly purified cardiomyocytes from human embryonic stem cells (hESCs) and shown that transplantation of these cardiomyocytes can partially remuscularize infarcted rodent hearts and help preserve contractile function. [1][2][3] However, concerns remain that currently available hESC-derived cardiomyocyte (hESC-CM) preparations include myocytes with both nodal and "working" (ie, atrial and ventricular chamber) type action potential (AP) properties. 4 -6 This electrophysiological diversity, also reported for cardiomyocytes derived from murine ESCs (mESCs), 7-9 induced pluripotent stem cells, 10 and resident cardiac stem cells, 11 represents both an opportunity and a challenge to the development of stem cell-based therapies. An enriched preparation of nodal cells would be of potential use in the formation of a biological pacemaker. On the other hand, we may want to exclude nodal cells from cardiomyocyte preparations for infarct repair, as their sustained pacemaking activity and un...

The reversibility of diabetic nephropathy remains controversial. Here, we tested whether replacing leptin could reverse the advanced diabetic nephropathy modeled by the leptin-deficient BTBR ob/ob mouse. Leptin replacement, but not inhibition of the renin-angiotensin-aldosterone system (RAAS), resulted in near-complete reversal of both structural (mesangial matrix expansion, mesangiolysis, basement membrane thickening, podocyte loss) and functional (proteinuria, accumulation of reactive oxygen species) measures of advanced diabetic nephropathy. Immunohistochemical labeling with the podocyte markers Wilms tumor 1 and p57 identified parietal epithelial cells as a possible source of regenerating podocytes. Thus, the leptin-deficient BTBR ob/ob mouse provides a model of advanced but reversible diabetic nephropathy for further study. These results also suggest that restoration of lost podocytes is possible but is not induced by RAAS inhibition, possibly explaining the limited efficacy of RAAS inhibitors in promoting repair of diabetic nephropathy. Diabetic nephropathy (DN) is now the major cause of CKD and ESRD throughout the world and is the largest single cause of ESRD in the United States, accounting for nearly half of the patients entering dialysis each year. [1][2][3][4][5] The mainstays of current therapy for DN are control of hyperglycemia and BP and inhibition of the renin-angiotensin-aldosterone system (RAAS). 6,7 These therapies can be effective in slowing progression but have not been effective in reversing established complications, such as DN. The recently reported Renin-Angiotensin System Study, a prospective 5-year clinical trial in which early and sustained therapy with inhibitors of the RAAS in diabetic patients did not prevent development of DN, was particularly disappointing in this regard. 8 Two of the major obstacles to progress in the treatment of DN are the lack of relevant animal models in which reversal of advanced DN can be tested and uncertainty about whether podocytes, a cell type that has long thought to be nonreplicating and nonrenewable and to be lost during development of DN, can be replaced and hence permit reconstitution of a normal glomerulus. 9 In this study, we show that both of these obstacles can be overcome. We have recently characterized a new murine model of type 2 DN, the BTBR ob/ob leptin-deficient mouse, which better mirrors human DN than do most previous murine models. 10,11 We have extended our previous characterization of this model by administering leptin to mice with advanced DN and demonstrating, uniquely among both experimental models and human DN, that DN can be reversed with pharmacologic therapy.

SUMMARY Recent evidence indicates that mouse and human embryonic stem (ES) cells are fixed at different developmental stages, with the former positioned earlier. We show that a narrow concentration of the naturally occurring short chain fatty acid, sodium butyrate, supports the extensive self-renewal of mouse and human ES cells, while promoting their convergence toward an intermediate stem cell state. In response to butyrate human ES cells regress to an earlier developmental stage characterized by a gene expression profile resembling that of mouse ES cells, preventing precocious Xist expression, while retaining the ability to form complex teratomas in vivo. Other histone deacetylase inhibitors (HDACi) also support human ES cell self-renewal. Our results indicate that HDACi can promote ES cell self-renewal across species, and demonstrate that ES cells can toggle between alternative states in response to environmental factors.

Heme oxygenase is a mammalian enzyme that converts heme to biliverdin and carbon monoxide. Carbon monoxide activates soluble guanylate cyclase and relaxes vascular smooth muscle, and it has been implicated as a potential neuromessenger. The regulatory functions of endogenous carbon monoxide on hemodynamics are not known. Zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) inhibits heme oxygenase in rats and thus permits assessment of the hemodynamic response to inhibition of endogenous carbon monoxide synthesis. In chronically instrumented, awake male Sprague-Dawley rats, ZnDPBG (45 mumol/kg IP) increased mean arterial pressure (19 +/- 2%, P < .05) and total peripheral resistance (47 +/- 4%, P < .05), decreased cardiac output (-16 +/- 2%, P < .05), but did not affect heart rate. Another heme oxygenase inhibitor, zinc protoporphyrin IX (45 mumol/kg IP), also increased arterial pressure (17 +/- 5%, P < .05), with no effect on heart rate. In contrast, neither the nonmetallic deuteroporphyrin 2,4-bis glycol (45 mumol/kg IP) nor bilverdin (45 mumol/kg IP) had any effect on blood pressure or heart rate. These findings suggest that ZnDPBG and zinc protoporphyrin IX increase arterial pressure by inhibiting heme oxygenase activity. After pretreatment with chlorisondamine (5 mg/kg IP) or prazosin (5 mg/kg IP) to inhibit autonomic ganglionic or alpha 1-adrenoceptor functions, respectively, ZnDPBG did not affect arterial pressure or heart rate. This suggests that ZnDPBG-induced increases in blood pressure rely on autonomic nervous function. We conclude that the pressor response to heme oxygenase inhibitors results from withdrawal of the inhibitory influence of endogenous carbon monoxide on a pressor mechanism mediated by the autonomic nervous system.