Correction to: Combination Glucose-Lowering Therapy Plans in T2DM: Case-Based Considerations.

The article "Combination Glucose-Lowering Therapy Plans in T2DM: Case-Based Considerations".

Combination Glucose-Lowering Therapy Plans in T2DM: Case-Based Considerations.

Type 2 diabetes mellitus (T2DM) is a complex disease, and while lifestyle interventions remain the cornerstone of therapy, most patients will also require pharmacotherapy. Current diabetes treatment guidelines and algorithms recommend an individualized approach to setting glycemic goals and selecting treatment. Although a single antihyperglycemic agent may be appropriate as the initial T2DM pharmacotherapy, the progressive nature of the disease due to declining pancreatic ß-cell function will result in the vast majority of T2DM patients eventually requiring two or more antihyperglycemic agents. The American Association of Clinical Endocrinologists/American College of Clinical Endocrinology T2DM management algorithm recommends initial dual agent combination therapy when a single agent is unlikely to achieve their target glycemia, i.e., for those patients with an HbA1c ≥ 7.5 and an individualized HbA1c target of < 7.5%. The American Diabetes Association Standards of Care recommend combination pharmacotherapy for those patients presenting with very elevated HbA1c levels (e.g., ≥ 9% and < 10%). Metformin (if well tolerated and not contraindicated) is the initial pharmacologic choice for most patients; selection of another antihyperglycemic agent to the regimen will depend on the presence of atherosclerotic cardiovascular disease and other patient-specific factors (e.g., age, known duration of T2DM, history of or risk for hypoglycemia and/or adverse consequences from hypoglycemia, other comorbidities, and available resources), along with drug-specific factors (e.g., risk for hypoglycemia, potential effects on weight, drug adverse event profiles, and cost). Combination therapy may be administered as a multi-pill regimen, a single-pill combination (i.e., fixed-dose combination oral therapy), or as a combination of oral and/or injectable therapies. This paper provides two illustrative case presentations to demonstrate how current treatment recommendations and algorithms can be used to guide the selection of non-insulin-based combination therapy for patients with T2DM in primary care settings and discusses the relative merits of several possible approaches for each patient. FUNDING: Boehringer Ingelheim Pharmaceuticals, Inc.

A p53-Pax2 pathway in kidney development: implications for nephrogenesis.

Congenital reduction in nephron number (renal hypoplasia) is a predisposing factor for chronic kidney disease and hypertension. Despite identification of specific genes and pathways in nephrogenesis, determinants of final nephron endowment are poorly understood. Here, we report that mice with germ-line p53 deletion (p53(-/-)) manifest renal hypoplasia; the phenotype can be recapitulated by conditional deletion of p53 from renal progenitors in the cap mesenchyme (CM(p53-/-)). Mice or humans with germ-line heterozygous mutations in Pax2 exhibit renal hypoplasia. Since both transcription factors are developmentally expressed in the metanephros, we tested the hypothesis that p53 and Pax2 cooperate in nephrogenesis. In this study, we provide evidence for the presence of genetic epistasis between p53 and Pax2: a) p53(-/-) and CM(p53-/-)embryos express lower Pax2 mRNA and protein in nephron progenitors than their wild-type littermates; b) ChIP-Seq identified peaks of p53 occupancy in chromatin regions of the Pax2 promoter and gene in embryonic kidneys; c) p53 binding to Pax2 gene is significantly more enriched in Pax2 -expressing than non-expressing metanephric mesenchyme cells; d) in transient transfection assays, Pax2 promoter activity is stimulated by wild-type p53 and inhibited by a dominant negative mutant p53; e) p53 knockdown in cultured metanephric mesenchyme cells down-regulates endogenous Pax2 expression; f) reduction of p53 gene dosage worsens the renal hypoplasia in Pax2(+/-) mice. Bioinformatics identified a set of developmental renal genes likely to be co-regulated by p53 and Pax2. We propose that the cross-talk between p53 and Pax2 provides a transcriptional platform that promotes nephrogenesis, thus contributing to nephron endowment.

Histone deacetylase (HDAC) activity is critical for embryonic kidney gene expression, growth, and differentiation.

Histone deacetylases (HDACs) regulate fundamental biological processes such as cellular proliferation, differentiation, and survival via genomic and nongenomic effects. This study examined the importance of HDAC activity in the regulation of gene expression and differentiation of the developing mouse kidney. Class I HDAC1-3 and class II HDAC4, -7, and -9 genes are developmentally regulated. Moreover, HDAC1-3 are highly expressed in nephron precursors. Short term treatment of cultured mouse embryonic kidneys with HDAC inhibitors (HDACi) induced global histone H3 and H4 hyperacetylation and H3K4 hypermethylation. However, genome-wide profiling revealed that the HDAC-regulated transcriptome is restricted and encompasses regulators of the cell cycle, Wnt/ß-catenin, TGF-ß/Smad, and PI3K-AKT pathways. Further analysis demonstrated that base-line expression of key developmental renal regulators, including Osr1, Eya1, Pax2/8, WT1, Gdnf, Wnt9b, Sfrp1/2, and Emx2, is dependent on intact HDAC activity. Treatment of cultured embryonic kidney cells with HDACi recapitulated these gene expression changes, and chromatin immunoprecipitation assays revealed that HDACi is associated with histone hyperacetylation of Pax2/Pax8, Gdnf, Sfrp1, and p21. Gene knockdown studies demonstrated that HDAC1 and HDAC2 play a redundant role in regulation of Pax2/8 and Sfrp1 but not Gdnf. Long term treatment of embryonic kidneys with HDACi impairs the ureteric bud branching morphogenesis program and provokes growth arrest and apoptosis. We conclude that HDAC activity is critical for normal embryonic kidney homeostasis, and we implicate class I HDACs in the regulation of early nephron gene expression, differentiation, and survival.

p53 regulates metanephric development.

p53 is best known as a tumor suppressor that regulates cell-cycle, differentiation, and apoptosis pathways, but its potential role in embryonic development and organogenesis remains controversial. Here, p53(-/-) embryos bred on C57Bl6 background exhibited a spectrum of congenital abnormalities of the kidney and urinary tract, including ureteric bud (UB) ectopia, double ureters/collecting systems, delayed primary branching of the UB, and hypoplastic metanephroi. We observed ectopic UB outgrowth from the Wolffian duct (WD) in one third of p53(-/-) embryos. The prevalence of duplex was higher in embryos than in neonates, and ex vivo organ culture suggested that ectopic ureters can regress over time, leaving behind a dysplastic pole ("segmental dysgenesis"). Transgenic expression of dominant negative p53 or conditional inactivation of p53 in the UB but not in the metanephric mesenchyme lineage recapitulated the duplex phenotype. Mechanistically, p53 inactivation in the WD associated with enhanced sensitivity to glial cell line-derived neurotrophic factor (GDNF)-induced ectopic budding and potentiated phosphatidylinositol-3 kinase activation by GDNF in UB cells. Unlike several other models of UB ectopia, hypersensitivity of p53(-/-) WD to GDNF is not accompanied by reduced Sprouty-1 or anterior expansion of the GDNF domain. In summary, our data lend support for a restrictive role for p53 activity in UB outgrowth from the WD.

Ontogeny of bradykinin B1 receptors in the mouse kidney.

Kinins are vasoactive peptides that stimulate two G-protein coupled bradykinin receptors (B1R and B2R). B2R-knockout mice are salt sensitive and develop renal dysgenesis and hypertension if salt stressed during embryogenesis. B1R-knockout mice, on the other hand, are protected from inflammation and fibrosis. This study examined the spatiotemporal expression of B1R during renal organogenesis. The segmental nephron identity of B1R immunoreactivity was determined by costaining with markers of the collecting duct (Dolichos biflorus), proximal tubule (Dolichos tetraglonus), and nephron progenitors (Pax2). At E14.5, the B1R was confined to few cells in the metanephric mesenchyme. Abundance of B1R increased progressively during development. On E17.5, B1R was enriched in differentiating proximal tubular cells and by postnatal day 1, B1R was clearly expressed on the luminal aspect of the proximal tubule. Quantitative real-time PCR revealed that the levels of B1R mRNA more than double during renal maturation. We conclude that 1) B1R expression correlates closely with nephron maturation; 2) lack of B1R in nephron progenitors suggests that B1R is unlikely to play a role in early nephrogenesis; and 3) enrichment of B1R in maturing proximal tubule suggests a potential role for this receptor in terminal differentiation of the proximal nephron.

Bradykinin B2 receptor null mice harboring a Ser23-to-Ala substitution in the p53 gene are protected from renal dysgenesis.

A physiological cross talk operates between the tumor suppressor protein p53 and the bradykinin B2 receptor (BdkrB2) during renal organogenesis. Thus, although BdkrB2 is a target for p53-mediated transcriptional activation, BdkrB2 is required to restrict p53 proapoptotic activity. We previously demonstrated that BdkrB2(-/-) embryos exposed to gestational salt stress develop renal dysgenesis as a result of p53-mediated apoptosis of nephron progenitors and repression of the terminal differentiation program. Compared with wild-type kidneys, BdkrB2(-/-) express abnormally high levels of the Checkpoint kinase (Chk1), which activates p53 via Ser23 phosphorylation. To define the functional relevance of p53S23 phosphorylation, we generated a compound strain of BdkrB2(-/-) mice harboring a homozygous Ser23-to-Ala (S23A) mutation in the p53 gene by crossing BdkrB2(-/-) with p53S23A knockin mice. Unlike salt-stressed BdkrB2(-/-) pups, which exhibit renal dysgenesis, homozygous S23A;BdkrB2(-/-) littermates are protected and have normal renal development. Heterozygous S23A;BdkrB2(-/-) mice have an intermediate phenotype. The p53-S23A substitution was associated with amelioration of apoptosis and restored markers of nephrogenesis and tubulogenesis. Real-time quantitative RT-PCR of terminal differentiation genes demonstrated that the S23A substitution restored normal expression patterns of aquaporin-2, Na-Cl cotransporter, Na-K-2Cl cotransporter, Na-bicarbonate cotransporter, and Sglt1. We conclude that p53 phosphorylation on Ser23 is an essential step in the signaling pathway mediating the susceptibility of BdkrB2(-/-) mutants to renal dysgenesis.

The polycystic kidney disease-1 gene is a target for p53-mediated transcriptional repression.

This study provides evidence that the tumor suppressor protein, p53, is a transcriptional repressor of PKD1. Kidneys of p53-null mice expressed higher Pkd1 mRNA levels than wild-type littermates; gamma-irradiation suppressed PKD1 gene expression in p53+/+ but not p53-/- cells; and chromatin immunoprecipitation assays demonstrated the binding of p53 to the PKD1 promoter in vivo. In transient transfection assays, p53 repressed PKD1 promoter activity independently of endogenous p21. Deletion analysis mapped p53-mediated repression to the proximal promoter region of PKD1. Mutations of the DNA binding or C-terminal minimal repression domains of p53 abolished its ability to repress PKD1. Moreover, trichostatin A, an inhibitor of histone deacetylase activity, attenuated p53-induced repression of the PKD1 promoter. These findings, together with previous reports showing that dedifferentiated Pkd1-deficient cells express lower p53 and p21 levels, suggest a model whereby PKD1 signaling activates the p53-p21 differentiation pathway. In turn, p53 cooperates with histone deacetylases to repress PKD1 gene transcription. Loss of a p53-mediated negative feedback loop in PKD1 mutant cells may therefore contribute to deregulated PKD1 expression and cystogenesis.

Spatiotemporal switch from DeltaNp73 to TAp73 isoforms during nephrogenesis: impact on differentiation gene expression.

p73 is a member of the p53 gene family, which also includes p53 and p63. These proteins share sequence similarity and target genes but also have divergent roles in cancer and development. Unlike p53, transcription of the p73 gene yields multiple full-length (transactivation (TA) domain) and amino terminus-truncated (DeltaN) isoforms. DeltaNp73 acts in a dominant negative fashion to inhibit the actions of TAp73 and p53 on their target genes, promoting cell survival and proliferation and suppressing apoptosis. The balance between TAp73 and its negative regulator, DeltaNp73, may therefore represent an important determinant of developmental cell fate. There is little if anything known regarding the developmental regulation of the p73 gene. In this study, we showed that TAp73 and DeltaNp73 exhibit reciprocal spatiotemporal expression and functions during nephrogenesis. TAp73 was predominantly expressed in the differentiation domain of the renal cortex in an overlapping manner with the vasopressin-sensitive water channel aquaporin-2 (AQP-2). Chromatin immunoprecipitation assays demonstrated that the endogenous AQP-2 promoter was occupied by TAp73 in a developmentally regulated manner. Furthermore TAp73 stimulated AQP-2 promoter-driven reporter expression. TAp73 also activated the bradykinin B2 receptor (B2R) promoter, a developmentally regulated gene involved in regulation of sodium excretion. The transcriptional effects of TAp73 on AQP-2 and B2R were independent of p53. In marked contrast to TAp73, DeltaNp73 isoforms were induced early in development and were preferentially expressed in proliferating nephron precursors. Moreover DeltaNp73 was a potent repressor of B2R gene transcription. We conclude that the p73 gene is developmentally regulated during kidney organogenesis. The spatiotemporal switch from DeltaNp73 to TAp73 may play an important role in the terminal differentiation program of the developing nephron.

Combinatorial control of the bradykinin B2 receptor promoter by p53, CREB, KLF-4, and CBP: implications for terminal nephron differentiation.

Despite a wealth of knowledge regarding the early steps of epithelial differentiation, little is known about the mechanisms responsible for terminal nephron differentiation. The bradykinin B2 receptor (B2R) regulates renal function and integrity, and its expression is induced during terminal nephron differentiation. This study investigates the transcriptional regulation of the B2R during kidney development. The rat B2R 5'-flanking region has a highly conserved cis-acting enhancer in the proximal promoter consisting of contiguous binding sites for the transcription factors cAMP response element binding protein (CREB), p53, and Kruppel-like factor (KLF-4). The B2R enhancer drives reporter gene expression in inner medullary collecting duct-3 cells but is considerably weaker in other cell types. Site-directed mutagenesis and expression of dominant negative mutants demonstrated the requirement of CREB DNA binding and Ser-133 phosphorylation for optimal enhancer function. Moreover, helical phasing experiments showed that disruption of the spatial organization of the enhancer inhibits B2R promoter activity. Several lines of evidence indicate that cooperative interactions among the three transcription factors occur in vivo during terminal nephron differentiation: 1) CREB, p53, and KLF-4 are coexpressed in B2R-positive differentiating cells; 2) the maturational expression of B2R correlates with CREB/p53/KLF-4 DNA-binding activity; 3) assembly of CREB, p53, and KLF-4 on chromatin at the endogenous B2R promoter is developmentally regulated and is accompanied by CBP recruitment and histone hyperacetylation; and 4) CREB and p53 occupancy of the B2R enhancer is cooperative. These results demonstrate that combinatorial interactions among the transcription factors, CREB, p53, and KLF-4, and the coactivator CBP, may be critical for the regulation of B2R gene expression during terminal nephron differentiation.

A novel pathological role of p53 in kidney development revealed by gene-environment interactions.

Gene-environment interactions are implicated in congenital human disorders. Accordingly, there is a pressing need to develop animal models of human disease, which are the product of defined gene-environment interactions. Previously, our laboratory demonstrated that gestational salt stress of bradykinin B(2) receptor (B(2)R)-null mice induces renal dysgenesis and early death of the offspring. In contrast, salt-stressed B(2)R +/+ or +/- littermates have normal development. The present study investigates the mechanisms underlying the susceptibility of B(2)R-null mice to renal dysgenesis. Proteomic and conventional Western blot screens identified E-cadherin among the differentially repressed proteins in B(2)R-/- kidneys, whereas the checkpoint kinase Chk1 and its substrate P-Ser(20) p53 were induced. We tested the hypothesis that p53 mediates repression of E-cadherin gene expression and is causally linked to the renal dysgenesis. Genetic crosses between B(2)R -/- and p53+/- mice revealed that germline reduction of p53 gene dosage rescues B(2)R-/- mice from renal dysgenesis and restores kidney E-cadherin gene expression. Furthermore, gamma-irradiation induces repression of E-cadherin gene expression in p53+/+ but not -/- cells. In transient transfection assays, p53 repressed human E-cadherin promoter-driven reporter activity, whereas a mutant p53, which cannot bind DNA, did not. Functional promoter analysis indicated the presence of a p53-responsive element in exon 1, which partially mediates p53-induced repression. Chromatin immunoprecipitation assays revealed that p53 inhibits histone acetylation of the E-cadherin promoter. Treatment with a histone deacetylase inhibitor reversed both p53-mediated promoter repression and deacetylation. In conclusion, this study demonstrates that gene-environment interactions cooperate to induce congenital defects through p53 activation.

The Bradykinin B2 receptor is required for full expression of renal COX-2 and renin.

Angiotensin converting enzyme (ACE) inhibition leads to increased levels of bradykinin, cyclooxygenase-2 (COX-2), and renin. Since bradykinin stimulates prostaglandin release, renin synthesis may be regulated through a kinin-COX-2 pathway. To test this hypothesis, we examined the impact of bradykinin B2 receptor (B2R) gene disruption in mice on kidney COX-2 and renin gene expression. Kidney COX-2 mRNA and protein levels were significantly lower in B2R-/- mice by 40-50%. On the other hand, renal COX-1 levels were similar in B2R-/- and +/+ mice. Renal renin protein was 61% lower in B2R-/- compared to B2R+/+ mice. This was accompanied by a significant reduction in renin mRNA levels in B2R-/- mice. Likewise, intrarenal angiotensin I levels were significantly lower in B2R-/- mice compared to B2R+/+ mice. In contrast, kidney angiotensin II levels were not different and averaged 261+/-16 and 266+/-15fmol/g in B2R+/+ and B2R-/- mice, respectively. Kidney angiotensinogen, AT1 receptor and ACE activity were not different between B2R+/+ and B2R-/- mice. The results of these studies demonstrate suppression of renal renin synthesis in mice lacking the bradykinin B2R and support the notion that B2R regulation of COX-2 participates in the steady-state control of renin gene expression.

Two functionally divergent p53-responsive elements in the rat bradykinin B2 receptor promoter.

Although p53 is known to have dual functions as a transcriptional activator and repressor, there has not been an example where both p53-activating and -repressing elements reside within one target promoter. Previous work from this laboratory defined two different p53 response elements, termed P1 and P2, located at nucleotide positions -70 and -707, respectively, in the rat bradykinin B2 receptor promoter. In this study, through manipulation of the DNA sequence and context, we demonstrate opposing roles for P1 and P2 as transcriptional activator and repressor, respectively. Deletion of P1 abrogates p53-mediated activation. P1 maintains its role as an activator upon relocation to the P2 site and activates transcription from a heterologous promoter construct. Thus, P1 is a bona fide positive p53-response element. In contrast, deletion of P2 enhances P1-induced activation. P2 represses transcription when substituted for P1 or when relocated midway between P1 and P2. P2-mediated repression is sequence-dependent, because it is reversed to activation when P2 is substituted by the P1 or p53 consensus sequences. Moreover, site-directed mutagenesis that converts P2 to a higher affinity p53-binding site results in transcriptional activation rather than repression. Surprisingly, P2 strongly activates a heterologous promoter. Thus, P2-mediated transcriptional repression is both sequence- and context-dependent. Investigations into the mechanisms of P2-mediated repression indicate that it is trichostatin-insensitive and unaffected by CBP or mutation of the minimal repression C-terminal domain of p53. However, gel shift assays suggest that p53 competes with other transcriptional activators for binding to overlapping binding sequences within the P2 element. In conclusion, this study provides a rare example of a transcription factor having two divergent functional effects that are sequence- and context-dependent. The interplay of P1 and P2 may be important in the regulation of bradykinin B2 receptor gene expression in response to inflammatory stress and during development.

Renal and blood pressure phenotype in 18-mo-old bradykinin B2R(-/-)CRD mice.

Aberrant gene-environment interactions are implicated in the pathogenesis of congenital renal dysgenesis (CRD), a leading cause of renal failure in infants and children. We have recently developed an animal model of CRD that is caused by gestational salt stress (5% NaCl diet; HS) of bradykinin B2R null mice [B2R(-/-)CRD; El-Dahr SS, Harrison-Bernard LM, Dipp S, Yosipiv IV, and Meleg-Smith S. Physiol Genomics 3: 121-131, 2000.]. Developing B2R(-/-)CRD mice exhibit tubular and glomerular cysts, stromal expansion, and loss of corticomedullary differentiation. In addition, B2R(-/-)CRD mice exhibit transient hypertension from 2 to 4 mo of age. The present study was designed to determine the long-term consequences of CRD on renal morphology and salt sensitivity of blood pressure in B2R(-/-)CRD mice. One-year- and 18-mo-old B2R(-/-)CRD mice exhibited stunted renal growth, glomerular cystic abnormalities, and collecting duct ectasia. Moreover, tumors of mesenchymal cell origin emerged in the dysplastic kidneys of 90% of 1-yr-old and 100% of 18-mo-old B2R(-/-)CRD mice but not in age-matched B2R(-/-) or wild-type mice. When challenged with an HS diet, 18-mo-old B2R(-/-)CRD exhibited a significant rise in systolic and diastolic blood pressures and more pronounced natriuresis and diuresis compared with salt-loaded 18-mo-old wild-type mice. Kidney aquaporin-2 expression was decreased by 50%, whereas renin, ANG type 1 receptor, and Na+-K+-ATPase levels were not different in B2R(-/-)CRD mice compared with controls. In conclusion, this study demonstrates that B2R(-/-)CRD mice exhibit permanent phenotypic and functional abnormalities in renal growth and differentiation. This novel model of human disease links gene-environment interactions with renal development and blood pressure homeostasis.

A role for p53 in terminal epithelial cell differentiation.

Terminal epithelial cell differentiation is a crucial step in development. In the kidney, failure of terminal differentiation causes dysplasia, cystogenesis, and cancer. The present study provides multiple lines of evidence implicating the tumor suppressor protein p53 in terminal differentiation of the renal epithelium. In the developing kidney, p53 is highly enriched in epithelial cells expressing renal function genes (RFGs), such as receptors for vasoactive hormones, the sodium pump, and epithelial sodium and water channels. In comparison, proliferating renal progenitors express little if any p53 or RFGs. p53 binds to and transactivates the promoters of RFGs. In contrast, expression of a dominant negative mutant form of p53 inhibits endogenous RFG expression. Moreover, binding of endogenous p53 to the promoters of RFGs coincides with the differentiation process and is attenuated once renal epithelial cells are fully differentiated. Finally, p53-null pups exhibit a previously unrecognized aberrant renal phenotype and spatial disorganization of RFGs. Interestingly, the p53-related protein p73 is unable to functionally compensate for the loss of p53 and fails to efficiently activate RFG transcription. We conclude that p53 promotes the biochemical and morphological differentiation of the renal epithelium. Aberrations in p53-mediated terminal differentiation may therefore play a role in the pathogenesis of nephron dysgenesis and dysfunction.