Genetic Variation of DKK3 May Modify Renal Disease Severity in ADPKD

Significant variation in the course of autosomal dominant polycystic kidney disease ( ADPKD) within families suggests the presence of effect modifiers. Recent studies of the variation within families harboring PKD1 mutations indicate that genetic background may account for 32 to 42% of the variance in estimated GFR (eGFR) before ESRD and 43 to 78% of the variance in age at ESRD onset, but the genetic modifiers are unknown. Here, we conducted a high-throughput single-nucleotide polymorphism (SNP) genotyping association study of 173 biological candidate genes in 794 white patients from 227 families with PKD1. We analyzed two primary outcomes: (1) eGFR and (2) time to ESRD (renal survival). For both outcomes, we used multidimensional scaling to correct for population structure and generalized estimating equations to account for the relatedness among individuals within the same family. We found suggestive associations between each of 12 SNPs and at least one of the renal outcomes. We genotyped these SNPs in a second set of 472 white patients from 229 families with PKD1 and performed a joint analysis on both cohorts. Three SNPs continued to show suggestive/significant association with eGFR at the Dickkopf 3 (DKK3) gene locus; no SNPs significantly associated with renal survival. DKK3 antagonizes Wnt/β-catenin signaling, which may modulate renal cyst growth. Pending replication, our study suggests that genetic variation of DKK3 may modify severity of ADPKD resulting from PKD1 mutations.Autosomal dominant polycystic kidney disease ( ADPKD) is the most common monogenic kidney disease worldwide, affecting one in 500 to 1000 births.^1,2 It is characterized by focal development of renal cysts in an age-dependent manner. Typically, only a few renal cysts are clinically detectable during the first three decades of life; however, by the fifth decade, tens of thousands of renal cysts of different sizes can be found in most patients.³ Progressive cyst expansion with age leads to massive enlargement and distortion of the normal architecture of both kidneys and, ultimately, ESRD in most patients. ADPKD is also associated with an increased risk for cardiac valvular defects, colonic diverticulosis, hernias, and intracranial arterial aneurysms. Overall, ADPKD accounts for approximately 5% of ESRD in North America.²Mutations of PKD1 and PKD2 respectively account for approximately 85% and approximately 15% of linkage-characterized European families. Polycystin-1 (PC-1) and PC-2, the proteins encoded by PKD1 and PKD2, respectively, function as a macromolecular complex and regulate multiple signaling pathways to maintain the normal tubular structure and function.¹ Monoclonal expansion of individual epithelial cells that have undergone a somatic “second hit” mutation, resulting in biallelic inactivation of either PKD1 or PKD2, seems to provide a major mechanism for focal cyst initiation,⁴ possibly through the loss of polycystin-mediated mechanosensory function in the primary cilium.⁵ In addition, a large prospective, observational study indicated that renal cysts in ADPKD expand exponentially with increasing age, and patients with large polycystic kidneys are at higher risk for developing kidney failure⁶; however, the key factors that modulate renal disease progression in ADPKD remain incompletely understood.Renal disease severity in ADPKD is highly variable, with the age of onset of ESRD ranging from childhood to old age.^7–11 A strong genetic locus effect has been noted in ADPKD. Adjusted for age and gender, patients with PKD1 have larger kidneys and earlier onset at ESRD than patients with PKD2 (mean age at ESRD 53.4 versus 72.7 years, respectively).^8,9 By contrast, a weak allelic effect (based on the 5′ versus 3′ location of the germline mutations) on renal disease severity may be present for PKD1¹⁰ but not PKD2.¹¹ Marked intrafamilial variability in renal disease is well documented in ADPKD and suggests a strong modifier effect.^10–15 In an extreme example, large polycystic kidneys were present in utero in one of a pair of dizygotic twins affected with the same germline PKD1 mutation, whereas the kidneys of the co-twin remained normal at 5 years of age.¹² Several studies have quantified the role of genetic background in the phenotypic expression of ADPKD. In a comparison of monozygotic twins and siblings, greater variance in the age of onset of ESRD in the siblings supported a role for genetic modifiers.¹³ Two other studies of intrafamilial disease variability in PKD1 have estimated that genetic factors may account for 32 to 42% of the variance of creatinine clearance before ESRD and 43 to 78% of the variance in age at ESRD.^14,15 The magnitude of the modifier gene effect from these studies suggests that mapping such factors is feasible. Here, we report the results of an association study of modifier genes for PKD1 renal disease severity.     

Reduced Ciliary Polycystin-2 in Induced Pluripotent Stem Cells from Polycystic Kidney Disease Patients with PKD1 Mutations

Heterozygous mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause autosomal dominant PKD (ADPKD), whereas mutations in PKHD1, which encodes fibrocystin/polyductin (FPC), cause autosomal recessive PKD (ARPKD). However, the relationship between these proteins and the pathogenesis of PKD remains unclear. To model PKD in human cells, we established induced pluripotent stem (iPS) cell lines from fibroblasts of three ADPKD and two ARPKD patients. Genetic sequencing revealed unique heterozygous mutations in PKD1 of the parental ADPKD fibroblasts but no pathogenic mutations in PKD2. Undifferentiated PKD iPS cells, control iPS cells, and embryonic stem cells elaborated primary cilia and expressed PC1, PC2, and FPC at similar levels, and PKD and control iPS cells exhibited comparable rates of proliferation, apoptosis, and ciliogenesis. However, ADPKD iPS cells as well as somatic epithelial cells and hepatoblasts/biliary precursors differentiated from these cells expressed lower levels of PC2 at the cilium. Additional sequencing confirmed the retention of PKD1 heterozygous mutations in iPS cell lines from two patients but identified possible loss of heterozygosity in iPS cell lines from one patient. Furthermore, ectopic expression of wild-type PC1 in ADPKD iPS-derived hepatoblasts rescued ciliary PC2 protein expression levels, and overexpression of PC1 but not a carboxy-terminal truncation mutant increased ciliary PC2 expression levels in mouse kidney cells. Taken together, these results suggest that PC1 regulates ciliary PC2 protein expression levels and support the use of PKD iPS cells for investigating disease pathophysiology.Polycystic kidney disease (PKD) is associated with defects of primary cilia and replacement of the normal kidney parenchyma with tubular epithelial cysts and fibrosis, leading to progressive deterioration of kidney function. PKD is among the world’s most common life-threatening genetic diseases, affecting approximately 1 in 600 people, and it is a significant contributor to CKD. Autosomal dominant PKD (ADPKD) causes end stage kidney disease by the age of 60 years in approximately 50% of adults with the disease, whereas autosomal recessive PKD (ARPKD) is a more rare form that typically presents earlier in life and causes significant childhood mortality. PKD may be considered a developmental disorder, with renal cysts becoming detectable in utero even in ADPKD.¹ In addition to kidney cysts, hepatic involvement is common, with liver cysts developing in many ADPKD patients and congenital hepatic fibrosis being a hallmark of ARPKD.^1,2ADPKD is inherited as heterozygous mutations in PKD1 or PKD2, whereas ARPKD is caused by biallelic mutations in PKHD1 (polycystic kidney and hepatic disease 1). These three genes encode transmembrane proteins, known as polycystin-1 (PC1), polycystin-2 (PC2), and fibrocystin/polyductin (FPC), respectively. PC1, PC2, and FPC form a receptor channel complex in membrane compartments including the primary cilium,^3,4 a sensory organelle on the apical cell surface, and loss of this localization pattern has been observed in cystic renal epithelia from humans.^5,6 Mutations in more than 50 gene products associated with the cilium cause a spectrum of related diseases known as the ciliopathies, most of which feature cystic kidneys.⁷ Ciliary trafficking signals have recently been identified at the carboxyl terminus of PC1 and the amino terminus of PC2, but the extent to which PC1 is involved in PC2 trafficking is not yet clear.^8–11 The abnormal phenotype in ADPKD has been attributed to loss of epithelial cell heterozygosity as a result of an additional somatic mutation or environmental insult (the two-hit hypothesis), although there is also genetic evidence for a haploinsufficiency model.^12–15There is a need for human disease-specific laboratory models for PKD to better understand disease and develop therapies, because animal models may not fully genocopy or phenocopy the human disease.^16,17 Primary cells taken from nephrectomized ADPKD kidneys have been linked to various epithelial cell phenotypes, but because these cells are derived from kidneys with advanced disease, it remains unclear whether these characteristics represent primary defects central to PKD etiology or secondary consequences of injury or dedifferentiation.^6,18–21 A powerful new technology, induced pluripotent stem (iPS) cells are adult somatic cells which have been reprogrammed into an embryonic pluripotent state.^22,23 The result is a next generation cell culture model that can differentiate into diverse cell types and complex tissues for the purposes of regenerative therapies or investigating disease. As for other hereditary diseases, iPS cells from patients with PKD can be examined for disease-specific abnormalities to better understand the pathophysiology of clinical mutations and screen for potential therapeutics.^7,24 PKD iPS cells derived from unaffected cell types, such as fibroblasts, might be expected to have fewer secondary phenotypes compared with cyst-lining epithelial cells, and they could be used to investigate PKD during development, when PKD disease genes are most highly expressed.^1,16,21,25 Their intrinsic pluripotency, ability to self-renew indefinitely, and immunocompatibility also make PKD iPS cells an attractive potential source for renal replacement tissue. As a first step in this direction, generation of iPS cells from one ADPKD patient was recently reported, although no disease phenotypes were described.²⁶ In our study, we generate iPS cell lines from ADPKD, ARPKD, and healthy control patients and evaluate their ability to ciliate, proliferate, and express PKD disease genes to establish a system in vitro for investigating human PKD. We identify reduced levels of PC2 at the primary cilium in undifferentiated iPS cells, differentiated somatic epithelial cells, and hepatoblasts as a consistent phenotype in three ADPKD patients with PKD1 mutations but not in ARPKD patients. Furthermore, we have found using ADPKD iPS-derived hepatoblasts and cultured kidney cells that wild-type but not mutant PC1 promotes PC2 localization to cilia.     

Family History of Renal Disease Severity Predicts the Mutated Gene in ADPKD

Mutations of PKD1 and PKD2 account for 85 and 15% of cases of autosomal dominant polycystic kidney disease (ADPKD), respectively. Clinically, PKD1 is more severe than PKD2, with a median age at ESRD of 53.4 versus 72.7 yr. In this study, we explored whether a family history of renal disease severity predicts the mutated gene in ADPKD. We examined the renal function (estimated GFR and age at ESRD) of 484 affected members from 90 families who had ADPKD and whose underlying genotype was known. We found that the presence of at least one affected family member who developed ESRD at age ≤55 was highly predictive of a PKD1 mutation (positive predictive value 100%; sensitivity 72%). In contrast, the presence of at least one affected family member who continued to have sufficient renal function or developed ESRD at age >70 was highly predictive of a PKD2 mutation (positive predictive value 100%; sensitivity 74%). These data suggest that close attention to the family history of renal disease severity in ADPKD may provide a simple means of predicting the mutated gene, which has prognostic implications.Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disorder, with a prevalence of one in 500 to 1000 in the general population. It is the third most common single cause of ESRD in the United States, accounting for 5% of people with ESRD.^1–5 ADPKD is genetically heterogeneous, with two disease genes (PKD1 on chromosome 16 and PKD2 on chromosome 4) accounting for most of the cases. Mutations of PKD1 and PKD2 are thought to account for 85 and 15% of cases, respectively, in linkage-characterized European populations.^6,7 Although the clinical manifestations overlap completely between two gene types, there is a strong locus effect on renal disease severity. Patients with PKD1 have significantly more severe renal disease than patients with PKD2, with larger kidneys and earlier onset at ESRD (median age 53.4 versus 72.7 yr, respectively).^8,9 By contrast, a weak allelic effect (based on the 5′ versus 3′ location of the germline mutations) on renal disease severity may exist for type 1¹⁰ but not type 2 ADPKD.¹¹ In addition, significant intrafamilial renal disease variability is evident, which is thought to be due to genetic and environmental modifiers.^11,12PKD1 is a large, complex gene containing 46 exons spanning 50 kb, with 33 of these exons at the 5′ end being duplicated elsewhere on chromosome 16. PKD2 is a single-copy gene, consisting of 15 exons spanning a 68-kb genomic region. There is marked allelic heterogeneity for both gene types, with 314 truncating mutations having been described in PKD1 and 91 truncating mutations in PKD2.^1,2 PKD1 encodes polycystin 1 (PC1), a large receptor-like protein, and PKD2 encodes polycystin 2 (PC2), a nonselective cation channel that transports calcium. Both PC1 and PC2 physically interact to form a complex that regulates intracellular levels of calcium and are located in the primary cilia of renal tubular cells. Recent studies suggested that the polycystin complex in the primary cilia of renal tubular cells serves as a mechanosensor for urine flow and that dysfunction of this mechanosensor may lead to cellular proliferation and cystogenesis.^1,2Recent advances in our understanding of the molecular pathobiology of ADPKD have led to the discovery of a number of drugs (e.g., tolvaptan, somatostatin, mammalian target of rapamycin inhibitors) that may target cyst growth and delay renal disease progression.^1,2 Several of these promising drugs are being or will be tested in clinical trials, and disease-modifying treatment may become a reality in the not-too-distant future.¹ In this context, the knowledge of ADPKD gene type may allow for the optimization of the design of such clinical trials, and identification of those affected individuals who are most likely to benefit from these novel therapies should they become available; however, the gene type is seldom known for most families in the clinical setting. Although molecular genetic testing, either by linkage or direct mutation analysis, can elucidate the gene type, such testing has its limitations.¹³ Linkage studies require DNA samples from several affected family members and are of limited utility in small families or de novo cases. Mutation-based screening for ADPKD is expensive and yields a definitive pathogenic mutation in only 42 to 63% of cases because the large and complex structure of PKD1 results in many unclassified missense variants whose pathogenicity often cannot be predicted with complete certainty.^14,15In this study, we explored whether renal disease severity can be used as clinical predictors of underlying gene type in families with ADPKD. To predict PKD1, we explored various cutoffs of early age at ESRD as indicative of severe renal disease. To predict PKD2, we explored various cutoffs of late age with renal sufficiency or at ESRD as indicative of milder renal disease. We then evaluated the performance characteristics of these cutoffs to define the optimal criteria for clinical prediction of ADPKD gene type.     

Rapamycin Ameliorates PKD Resulting from Conditional Inactivation of Pkd1

Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the gradual replacement of normal renal parenchyma by cysts, which culminates in renal failure in approximately 50% of patients.¹ No effective drug treatment is available to slow the progression of ADPKD, which is primarily (85%) caused by mutations in the PKD1 gene encoding polycystin-1 (PC1).² Our previous results suggested that PC1 may regulate the kinase mammalian target of rapamycin (mTOR) via its interaction with tuberin.³ In addition, we have demonstrated that mTOR activity is low in the normal human kidney but strongly upregulated in renal cyst-lining epithelial cells in ADPKD.³ Finally, rapamycin treatment of four nonorthologous rodent PKD models resulted in inhibition of renal cyst growth, regression of kidney size, and preservation of renal function,^3–9 which led to the proposal that mTOR inhibitors, some of which are already in clinical use as immunosuppressants, may be effective in patients with ADPKD.^10–13 Indeed, four clinical trials have been initiated to test the efficacy of mTOR inhibitors in ADPKD.^12–14 Given the immunosuppressive and other adverse effects of mTOR inhibitors, it will be important to establish a compelling rationale for their use in patients with ADPKD.Previous studies used rodent PKD models with mutations in genes that encode proteins (polaris, bicaudal-C, samcystin, and folliculin) with poorly understood function and no known functional link to PC1.^3–9 We hypothesized that the normal function of these and other proteins involved in renal cystic diseases eventually converge on the mTOR pathway,¹³ but it has remained uncertain whether mTOR inhibition would be effective in human ADPKD.To overcome this limitation, we used a mouse model in which the orthologous Pkd1 gene is conditionally inactivated (Pkd1^cond/cond) by Cre-mediated recombination.^15,16 Initially, a Pkd1^cond/cond:MMTV^cre mouse line that resulted in infrequent renal cysts as a result of low renal Cre expression was generated¹⁶. We now report the development of a mouse line, Pkd1^cond/cond:Nestin^cre, in which the nestin promoter drives Cre expression.¹⁷ This results in a mosaic renal expression pattern, mimicking the situation in human ADPKD whereby random somatic, second-hit mutations affect the PKD1 locus,¹ and development of PKD with key features equivalent to the human disease. We report that rapamycin is highly effective in inhibiting all tested aspects of the disease phenotype, resulting in preservation of renal function.     

Type of PKD1 Mutation Influences Renal Outcome in ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) is heterogeneous with regard to genic and allelic heterogeneity, as well as phenotypic variability. The genotype-phenotype relationship in ADPKD is not completely understood. Here, we studied 741 patients with ADPKD from 519 pedigrees in the Genkyst cohort and confirmed that renal survival associated with PKD2 mutations was approximately 20 years longer than that associated with PKD1 mutations. The median age at onset of ESRD was 58 years for PKD1 carriers and 79 years for PKD2 carriers. Regarding the allelic effect on phenotype, in contrast to previous studies, we found that the type of PKD1 mutation, but not its position, correlated strongly with renal survival. The median age at onset of ESRD was 55 years for carriers of a truncating mutation and 67 years for carriers of a nontruncating mutation. This observation allows the integration of genic and allelic effects into a single scheme, which may have prognostic value.Autosomal dominant polycystic kidney disease (ADPKD) is the most common kidney disorder with a Mendelian inheritance pattern, with a prevalence ranging from 1/400 to 1/1000 worldwide.¹ ADPKD shows both locus and allelic heterogeneity. Two causative genes—PKD1, located at 16p13.3,² and PKD2, located at 4q21³—have been identified, and the ADPKD mutation database (http://pkdb.mayo.edu/) describes >1000 pathogenic mutations (929 in PKD1 and 167 in PKD2 as of June 5, 2012), not including our most recent data.⁴ADPKD also shows high phenotypic variability, as exemplified by the wide variation in the age at onset of ESRD,⁵ which is defined as the requirement of dialysis or transplantation. Genotype-phenotype correlation studies underscore two major issues. First, on average, ESRD occurs 20 years earlier in patients with PKD1 than those with PKD2,^6,7 indicating a genic influence on the ADPKD phenotype. Second, the position of the PKD1 mutation is associated with the age at ESRD onset,⁸ suggesting an allelic influence on ADPKD phenotype. However, these observations were made >10 years ago, when mutational analysis of the PKD1 and PKD2 genes (particularly of the nonunique portion of the PKD1 gene^2,9) was substantially less comprehensive and sophisticated than it is currently,^4,10 the methods for predicting the potential pathogenicity of missense mutations were in their infancy, and the studied patient cohorts were relatively small. To confirm (or refute) these earlier observations, we performed a genotype and phenotype correlation study using the Genkyst cohort, which comprises patients with ADPKD recruited from all private and public nephrology centers in the Brittany region, namely, the western part of France.     

Sirolimus Therapy to Halt the Progression of ADPKD

Activation of mammalian target of rapamycin (mTOR) pathways may contribute to uncontrolled cell proliferation and secondary cyst growth in patients with autosomal dominant polycystic kidney disease (ADPKD). To assess the effects of mTOR inhibition on disease progression, we performed a randomized, crossover study (The SIRENA Study) comparing a 6-month treatment with sirolimus or conventional therapy alone on the growth of kidney volume and its compartments in 21 patients with ADPKD and GFR ≥40 ml/min per 1.73 m². In 10 of the 15 patients who completed the study, aphthous stomatitis complicated sirolimus treatment but was effectively controlled by topical therapy. Compared with pretreatment, posttreatment mean total kidney volume increased less on sirolimus (46 ± 81 ml; P = 0.047) than on conventional therapy (70 ± 72 ml; P = 0.002), but we did not detect a difference between the two treatments (P = 0.45). Cyst volume was stable on sirolimus and increased by 55 ± 75 ml (P = 0.013) on conventional therapy, whereas parenchymal volume increased by 26 ± 30 ml (P = 0.005) on sirolimus and was stable on conventional therapy. Percentage changes in cyst and parenchyma volumes were significantly different between the two treatment periods. Sirolimus had no appreciable effects on intermediate volume and GFR. Albuminuria and proteinuria marginally but significantly increased during sirolimus treatment. In summary, sirolimus halted cyst growth and increased parenchymal volume in patients with ADPKD. Whether these effects translate into improved long-term outcomes requires further investigation.Autosomal dominant polycystic kidney disease (ADPKD) is an inherited systemic disorder with major renal manifestations, which occurs in 1 of 400 to 1000 individuals.¹ ADPKD is genetically heterogeneous. Mutations of the two genes PKD1 (85% of the cases) and PKD 2 (15% of cases), encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively, are implicated in the disease development.² The functions of PC1 and PC2 have not been defined with certainty; however, PC1 is thought to interact with and regulate PC2, which is a member of a subfamily of transient receptor potential channels³ and may act as a cation channel allowing Ca²⁺ entry from the extracellular environment. Consistent with the PC1/PC2 complex having a role in Ca²⁺ regulation, PKD epithelial cells display altered intracellular Ca²⁺ homeostasis,⁴ which alters the response to increased levels of intracellular cAMP.^5–7Another change consistently found in PKD cells is activation of the Ser/Thr kinase mammalian target of rapamycin (mTOR), an enzyme that coordinates cell growth, cell-cycle progression, and proliferation.⁸ mTOR is made up of two distinct complexes: mTORC1 and TORC2. The direct downstream targets of mTORC1, the eukaryotic initiation factor 4E-binding protein and ribosomal protein S6 kinase (p70S6K1),^9,10 tightly regulate the translational initiation machinery to control cell growth and proliferation.⁸ In vitro studies demonstrated that the N-terminal cytoplasmic domain of PC1 co-localizes and interacts with tuberin.¹¹ Activated phospho-mTOR and p70S6K are induced in cyst-lining epithelial cells in cysts from human and mouse kidneys.¹¹ Moreover, p70S6K is increased in Han:SPRD rat kidneys with PKD.¹² These observations led to the hypothesis that defects in PC1 in ADPKD promote disruption of the tuberin-mTOR complex, leading to aberrant mTOR activation and signaling.¹¹ There is also evidence that IGF-1 by binding to its receptor is a major regulator of the mTOR pathway via signaling to phosphatidylinositol-3 kinase, protein kinase B (Akt), and mTOR.⁸ Increase in IGF-1 mRNA levels in the kidneys of the pcy mouse model of PKD¹³ and in IGF-1 protein in Han:SPRD rats¹⁴ has been reported. In addition, the amount of phospho-Akt in cystic Pkd1^−/− mouse kidneys was more than that in wild-type kidneys.¹⁵ Thus, if mTOR is such a converging point in PKD cells, it would be worthwhile as a possible drug target for treatment of renal cystic disorders.Sirolimus (originally referred to as rapamycin) is a macrocyclic lactone that is derived from Streptomyces hygroscopicus and exerts antiproliferative and growth-inhibiting effects as well as antifibrotic effect by inhibition of the mTOR enzyme.^16,17 The drug has been used in kidney transplant recipients as part of maintenance immunosuppressive therapy¹⁸ and more recently as an antitumor agent^19,20 and in drug-eluting stents to prevent coronary artery stenosis.²¹ Short-term treatment with sirolimus markedly reduced kidney size and lowered renal total cyst volume (TCV) density in PKD animal models.^11,12,22 In addition, in renal transplant recipients who had progressed to ESRD because of ADPKD, the size of native kidney and liver cysts decreased while on mTOR inhibitor therapy but did not change appreciably during treatment with other immunosuppressants.^11,23Thus, to assess formally the risk/benefit profile of mTOR inhibitor therapy in PKD, we designed the Sirolimus Treatment in Patients with Autosomal Dominant Polycystic Kidney Disease: Renal Efficacy and Safety (SIRENA; http://clinicaltrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT00491517","term_id":"NCT00491517"}}NCT00491517), a proof-of-concept, randomized clinical trial aimed to compare the changes in total kidney volume (TKV) and in the kidney''s various compartments. This was assessed by serial computed tomography (CT) scan evaluations during 6 months of treatment with sirolimus or conventional therapy alone in 21 patients with ADPKD and normal or moderately decreased kidney function. The study secondarily evaluated whether and to which extent treatment-induced changes in kidney volume and structure translated into concomitant changes in GFR as assessed by standard techniques. The results of these analyses formed the basis of this report.     

Recurrence of Lupus Nephritis after Kidney Transplantation

The frequency and outcome of recurrent lupus nephritis (RLN) among recipients of a kidney allograft vary among single-center reports. From the United Network for Organ Sharing files, we estimated the period prevalence and predictors of RLN in recipients who received a transplant between 1987 and 2006 and assessed the effects of RLN on allograft failure and recipients'' survival. Among 6850 recipients of a kidney allograft with systemic lupus erythematosus, 167 recipients had RLN, 1770 experienced rejection, and 4913 control subjects did not experience rejection. The period prevalence of RLN was 2.44%. Non-Hispanic black race, female gender, and age <33 years each independently increased the odds of RLN. Graft failure occurred in 156 (93%) of those with RLN, 1517 (86%) of those with rejection, and 923 (19%) of control subjects without rejection. Although recipients with RLN had a fourfold greater risk for graft failure compared with control subjects without rejection, only 7% of graft failure episodes were attributable to RLN compared and 43% to rejection. During follow-up, 867 (13%) recipients died: 27 (16%) in the RLN group, 313 (18%) in the rejection group, and 527 (11%) in the control group. In summary, severe RLN is uncommon in recipients of a kidney allograft, but black recipients, female recipient, and younger recipients are at increased risk. Although RLN significantly increases the risk for graft failure, it contributes far less than rejection to its overall incidence; therefore, these findings should not keep patients with lupus from seeking a kidney transplant.The frequency and clinical impact of recurrent lupus nephritis (RLN) in the kidney allograft of recipients with systemic lupus erythematosus (SLE) varies considerably in both prospective and retrospective studies.^1–25 In 1996, Mojcik and Klippel²⁶ pooled data from a total of 366 allografts transplanted in 338 recipients. In that review, histologic RLN was present in 3.8% of the grafts. Contrasting, in the studies by Goral et al.²⁷ and Nyberg et al.,¹⁰ RLN was reported in a much higher proportion: 30 and 44% of recipients, respectively.The clinical consequences of RLN on patient and allograft survival have ranged from no effect to a significant increase in the risk for graft loss and patient mortality.^24,27–31 In this case-control study, we estimated the period prevalence of RLN in kidney transplant recipients who had ESRD secondary to lupus nephritis and received a transplant between October 1987 and October 2006. We assessed the effects of RLN on graft failure and recipient survival and the risk factors leading to the development of RLN.     

Pkd1 Haploinsufficiency Increases Renal Damage and Induces Microcyst Formation following Ischemia/Reperfusion

Mutations in PKD1 cause the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). Because polycystin 1 modulates cell proliferation, cell differentiation, and apoptosis, its lower biologic activity observed in ADPKD might influence the degree of injury after renal ischemia/reperfusion. We induced renal ischemia/reperfusion in 10- to 12-wk-old male noncystic Pkd1^+/− and wild-type mice. Compared with wild-type mice, heterozygous mice had higher fractional excretions of sodium and potassium and higher serum creatinine after 48 h. In addition, in heterozygous mice, also cortical damage, rates of apoptosis, and inflammatory infiltration into the interstitium at time points out to 14 d after injury all increased, as well as cell proliferation at 48 h and 7 d. The mRNA and protein expression of p21 was lower in heterozygous mice than wild-type mice at 48 h. After 6 wk, we observed dilated tubules, microcysts, and increased renal fibrosis in heterozygotes. The early mortality of heterozygotes was significantly higher than that of wild-type mice when we extended the duration of ischemia from 32 to 35 min. In conclusion, ischemia/reperfusion induces a more severe injury in kidneys of Pkd1-haploinsufficient mice, a process that apparently depends on a relative deficiency of p21 activity, tubular dilation, and microcyst formation. These data suggest the possibility that humans with ADPKD from PKD1 mutations may be at greater risk for damage from renal ischemia/reperfusion injury.Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic renal disease, with a prevalence of 1:400 to 1:1000. Mutations of the PKD1 gene are responsible for approximately 85% of the disease cases, whereas approximately 15% are caused by mutations in PKD2.¹ Only half of patients reach the age of 58 yr without ESRD.² ADPKD is a systemic disorder, however, including extrarenal manifestations typically represented by liver cysts, intracranial aneurysms, and heart valve alterations.The PKD1 gene encodes polycystin 1, a large glycoprotein with an approximately 3000–amino acid extracellular portion that comprises domains that seem to be involved in protein–protein and protein–carbohydrate interactions. A number of studies support the involvement of the primary apical cilium in the pathogenesis of PKD by modulating signal transduction via intracellular Ca²⁺ transients.³ Polycystins 1 and 2 (PC1 and PC2) are thought to participate actively in this process.^3–6 Ciliary mechanosensation has also been associated with STAT6-dependent changes in gene expression.⁷ In addition, the cellular effects of polycystins seem to rely on interaction with the cytoskeleton and mediation of cell–cell adhesion.^8,9PC1 and PC2 activate a number of other pathways. PC1 may function as a G protein–coupled receptor.¹⁰ Its activation, following a process dependent on PC2, may also activate JAK2, leading to phosphorylation and activation of STAT1 and generation of STAT1 homodimers.¹¹ These dimers bind to the p21 promoter in the nucleus, promoting its upregulation, reduction of Cdk2 activity, and cell arrest in G0/G1. It has also been shown that PC1 induces phosphatidylinositol 3 kinase–dependent Akt activation,¹² whereas its C-terminus may interact with tuberin, regulating mammalian target of rapamycin activity.¹³ Moreover, PC1 is subjected to an autoproteolytic process in the G protein-coupled receptor proteolytic site domain, generating an extracellular N-terminal fragment,¹⁴ whereas its C-terminus seems to be cleaved, to be translocated to the nucleus, and to activate AP-1.¹⁵Ischemia/reperfusion (IR) injury is a common cause of acute kidney injury (AKI), including patients with ADPKD. The cellular damage is secondary to a chain of biochemical and biologic abnormalities.^16,17 An abnormal proliferative response of ADPKD cells to cAMP has been reported, apparently associated with defective intracellular Ca²⁺ homeostasis,¹⁸ and animal models of PKD have been associated with dysregulated cell-cycle activity.¹⁹ Piontek et al.,²⁰ however, have shown that the effects of Pkd1 inactivation in mice are determined by a developmental switch on postnatal day 13. Interestingly, in this model, cellular proliferation was not significantly increased.In this scenario, we hypothesized that a lower PC1 biologic activity might amplify the IR injury degree. Although the focal cyst formation in ADPKD is likely dependent on a two-hit mechanism,²¹ the functional effects of PC1 seem to rely on activity thresholds.²² PKD1-haploinsufficient kidney cells, therefore, might be unable to achieve the required PC1 activity level when exposed to IR. Although studies on the ischemia/PC2 relation have brought some potential contributions to this question,^23,24 the relationship between PC1 and IR is basically unknown. By coordinating cell planar polarity in renal tubules, PC1 might exert a protective effect after an ischemic insult. In this study, this potential mechanism was investigated in Pkd1^+/− mice obtained from an inbred mouse line with a Pkd1 null mutation. Our findings of a more severe renal lesion in Pkd1-null heterozygotes suggest an increased risk for renal IR injury in Pkd1-haploinsufficient mice. Development of tubular dilation (TD) and microcysts (MCs) and increased renal fibrosis, in turn, suggest that the IR aggression has a higher long-term negative impact on Pkd1^+/− kidneys.     

Randomized Clinical Trial of Long-Acting Somatostatin for Autosomal Dominant Polycystic Kidney and Liver Disease

There are no proven, effective therapies for polycystic kidney disease (PKD) or polycystic liver disease (PLD). We enrolled 42 patients with severe PLD resulting from autosomal dominant PKD (ADPKD) or autosomal dominant PLD (ADPLD) in a randomized, double-blind, placebo-controlled trial of octreotide, a long-acting somatostatin analogue. We randomly assigned 42 patients in a 2:1 ratio to octreotide LAR depot (up to 40 mg every 28 ± 5 days) or placebo for 1 year. The primary end point was percent change in liver volume from baseline to 1 year, measured by MRI. Secondary end points were changes in total kidney volume, GFR, quality of life, safety, vital signs, and clinical laboratory tests. Thirty-four patients had ADPKD, and eight had ADPLD. Liver volume decreased by 4.95% ± 6.77% in the octreotide group but remained practically unchanged (+0.92% ± 8.33%) in the placebo group (P = 0.048). Among patients with ADPKD, total kidney volume remained practically unchanged (+0.25% ± 7.53%) in the octreotide group but increased by 8.61% ± 10.07% in the placebo group (P = 0.045). Changes in GFR were similar in both groups. Octreotide was well tolerated; treated individuals reported an improved perception of bodily pain and physical activity. In summary, octreotide slowed the progressive increase in liver volume and total kidney volume, improved health perception among patients with PLD, and had an acceptable side effect profile.Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of renal cysts and a variety of extrarenal manifestations of which polycystic liver disease (PLD) is the most common.¹ It is caused by mutations in one of two genes: PKD1 or PKD2. PKD1 mutations are responsible for approximately 85% of clinically detected cases. Autosomal dominant PLD (ADPLD) also exists as a genetically distinct disease with few or absent renal cysts. Like ADPKD, ADPLD is genetically heterogeneous, with the first two genes identified (PRKCSH and SEC63) accounting for approximately one-third to one-half of isolated ADPLD cases.^2–5Chronic symptoms are frequently associated with massively enlarged PLD, including abdominal distension and pain, dyspnea, gastroesophageal reflux, and early satiety, which may lead to malnutrition, mechanical lower back pain, inferior vena cava, hepatic and portal vein compression (leading to hypotension and inferior vena cava thrombosis, hepatic venous outflow obstruction, and portal hypertension), and biliary obstruction. Surgical approaches may be associated with definitive palliation but are also associated with a risk of morbidity and mortality.⁶Liver cysts arise by excessive proliferation of cholangiocytes and dilation of biliary ductules and peribiliary glands. Alterations in intracellular calcium homeostasis and 3′-5′-cAMP stimulate mitogen-activated protein kinase-mediated cell proliferation and cystic fibrosis transmembrane conductance regulator-driven chloride and fluid secretion.⁷ Cyst growth is enhanced by growth factors and cytokines secreted into the cyst fluid.⁸ Downstream activation of mTOR likely contributes to cystogenesis.⁹ Somatostatin may blunt cyst development by acting at multiple levels: inhibition of secretin release by the pancreas¹⁰; inhibition of secretin-induced cAMP generation and fluid secretion in cholangiocytes^11–13; vasopressin-induced cAMP generation and water permeability in collecting ducts^14–17 by its effects on Gi protein-coupled receptors; and suppression of the expression of IGF-1, vascular endothelial growth factor, and other cystogenic growth factors and of downstream signaling from their receptors.^14–18To determine whether octreotide could be effective in the treatment of PLD, we examined the effects of octreotide in the PCK rat, a recessive model of polycystic liver and kidney disease. We found that octreotide significantly reduced cAMP levels and hepatic cystogenesis in vitro and in vivo.¹⁹ In patients who underwent liver resections for massive PLD, we had observed that administration of octreotide reduced the rate of fluid secretion from unroofed cysts. In one patient with persistent ascites, intramuscular administration of octreotide LAR 40 mg monthly for 8 months was accompanied by a 17.8% reduction in liver volume from 2833 ml to 2330 ml (Figure 1). Two similar instances have been recently reported by van Keimpema et al.²⁰ Finally, a pilot study showed that administration of octreotide LAR significantly inhibited kidney and cyst enlargement in patients with ADPKD.²¹ Encouraged by the results of the preclinical studies, anecdotal clinical experiences, and the pilot study in ADPKD, we initiated a pilot randomized, placebo-controlled, double-blind clinical trial of octreotide LAR in severe PLD.Open in a separate windowFigure 1.Administration of octreotide LAR to a patient with severe PLD resulted in decreased liver and kidney volumes. CT axial sections immediately before (A) and after 8 months of treatment with octreotide (B) are shown. Total liver volume decreased by 18% from baseline, and total kidney volume decreased by 12%.     

Association of 18 Confirmed Susceptibility Loci for Type 2 Diabetes With Indices of Insulin Release, Proinsulin Conversion, and Insulin Sensitivity in 5,327 Nondiabetic Finnish Men

OBJECTIVE

We investigated the effects of 18 confirmed type 2 diabetes risk single nucleotide polymorphisms (SNPs) on insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin.

RESEARCH DESIGN AND METHODS

A total of 5,327 nondiabetic men (age 58 ± 7 years, BMI 27.0 ± 3.8 kg/m²) from a large population-based cohort were included. Oral glucose tolerance tests and genotyping of SNPs in or near PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, LOC387761, CDKN2B, IGF2BP2, CDKAL1, HNF1B, WFS1, JAZF1, CDC123, TSPAN8, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B were performed. HNF1B rs757210 was excluded because of failure to achieve Hardy-Weinberg equilibrium.

RESULTS

Six SNPs (TCF7L2, SLC30A8, HHEX, CDKN2B, CDKAL1, and MTNR1B) were significantly (P < 6.9 × 10⁻⁴) and two SNPs (KCNJ11 and IGF2BP2) were nominally (P < 0.05) associated with early-phase insulin release (InsAUC_0–30/GluAUC_0–30), adjusted for age, BMI, and insulin sensitivity (Matsuda ISI). Combined effects of these eight SNPs reached −32% reduction in InsAUC_0–30/GluAUC_0–30 in carriers of ≥11 vs. ≤3 weighted risk alleles. Four SNPs (SLC30A8, HHEX, CDKAL1, and TCF7L2) were significantly or nominally associated with indexes of proinsulin conversion. Three SNPs (KCNJ11, HHEX, and TSPAN8) were nominally associated with Matsuda ISI (adjusted for age and BMI). The effect of HHEX on Matsuda ISI became significant after additional adjustment for InsAUC_0–30/GluAUC_0–30. Nine SNPs did not show any associations with examined traits.

CONCLUSIONS

Eight type 2 diabetes–related loci were significantly or nominally associated with impaired early-phase insulin release. Effects of SLC30A8, HHEX, CDKAL1, and TCF7L2 on insulin release could be partially explained by impaired proinsulin conversion. HHEX might influence both insulin release and insulin sensitivity.Impaired insulin secretion and insulin resistance, two main pathophysiological mechanisms leading to type 2 diabetes, have a significant genetic component (1). Recent studies have confirmed 20 genetic loci reproducibly associated with type 2 diabetes (2–13). Three were previously known (PPARG, KCNJ11, and TCF7L2), whereas 17 loci were recently discovered either by genome-wide association studies (SLC30A8, HHEX-IDE, LOC387761, CDKN2A/2B, IGF2BP2, CDKAL1, FTO, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B), or candidate gene approach (WFS1 and HNF1B). The mechanisms by which these genes contribute to the development of type 2 diabetes are not fully understood.PPARG is the only gene from the 20 confirmed loci previously associated with insulin sensitivity (14,15). Association with impaired β-cell function has been reported for 14 loci (KCNJ11, SLC30A8, HHEX-IDE, CDKN2A/2B, IGF2BP2, CDKAL1, TCF7L2, WFS1, HNF1B, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, KCNQ1, and MTNR1B) (6,12,13,16–38). Although associations of variants in HHEX (16–22), CDKAL1 (6,21–26), TCF7L2 (22,27–30), and MTNR1B (13,31,32) with impaired insulin secretion seem to be consistent across different studies, information concerning other genes is limited (12,18–25,27,33–38). The mechanisms by which variants in these genes affect insulin secretion are unknown. However, a few recent studies suggested that variants in TCF7L2 (22,39–42), SLC30A8 (22), CDKAL1 (22), and MTNR1B (31) might influence insulin secretion by affecting the conversion of proinsulin to insulin. Variants of FTO have been shown to confer risk for type 2 diabetes through their association with obesity (7,16) and therefore were not included in this study.Large population-based studies can help to elucidate the underlying mechanisms by which single nucleotide polymorphisms (SNPs) of different risk genes predispose to type 2 diabetes. Therefore, we investigated confirmed type 2 diabetes–related loci for their associations with insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin in a population-based sample of 5,327 nondiabetic Finnish men.     

Six-Month Prophylaxis Is Cost Effective in Transplant Patients at High Risk for Cytomegalovirus Infection

The risk of late-onset cytomegalovirus (CMV) infection remains a concern in seronegative kidney and/or pancreas transplant recipients of seropositive organs despite the use of antiviral prophylaxis. The optimal duration of prophylaxis is unknown. We studied the cost effectiveness of 6- versus 3-mo prophylaxis with valganciclovir. A total of 222 seronegative recipients of seropositive kidney and/or pancreas transplants received valganciclovir prophylaxis for either 3 or 6 mo during two consecutive time periods. We assessed the incidence of CMV infection and disease 12 mo after completion of prophylaxis and performed cost-effectiveness analyses. The overall incidence of CMV infection and disease was 26.7% and 24.4% in the 3-mo group and 20.9% and 12.1% in the 6-mo group, respectively. Six-month prophylaxis was associated with a statistically significant reduction in risk for CMV disease (HR, 0.35; 95% CI, 0.17 to 0.72), but not infection (HR, 0.65; 95% CI, 0.37 to 1.14). Cost-effectiveness analyses showed that 6-mo prophylaxis combined with a one-time viremia determination at the end of the prophylaxis period incurred an incremental cost of $34,362 and $16,215 per case of infection and disease avoided, respectively, and $8,304 per one quality adjusted life-year gained. Sensitivity analyses supported the cost effectiveness of 6-mo prophylaxis over a wide range of valganciclovir and hospital costs, as well as variation in the incidence of CMV disease. In summary, 6-mo prophylaxis with valganciclovir combined with a one-time determination of viremia is cost effective in reducing CMV infection and disease in seronegative recipients of seropositive kidney and/or pancreas transplants.Cytomegalovirus (CMV) infection remains one of most common opportunistic infections in solid organ transplant patients despite availability of specific and efficacious anti-viral drugs.^1,2 Solid organ transplant patients who have a negative CMV serology and receive an organ from a positive CMV serologic donor (D+/R−) have the highest incidence of CMV disease with and without prophylaxis.^2–5 Although the risk for CMV disease persists for life, the majority of cases occur shortly after completion of prophylaxis, often within the first year after transplant.⁶ CMV disease causes significant morbidity, increases mortality, and is associated with inferior transplant outcomes, particularly in the case of kidney transplantation.^7–10 Furthermore, the presence of CMV disease is one of the most frequent infectious causes of hospitalization early after transplantation, increasing the total cost of kidney transplantation and reducing its overall effectiveness.^7,11–13Valganciclovir (VGCV) is an effective anti-CMV agent for prophylaxis and treatment of CMV disease that is widely used in transplantation.^2,14–16 Although the recommended dose for CMV prophylaxis is 900 mg daily adjusted for renal function, a recent study showed that VGCV at 450 mg daily provides similar drug exposure compared with oral ganciclovir (GCV) at 1000 mg three times daily in kidney transplant patients, a dose similarly effective for CMV prophylaxis.^2,17 In most studies, VGCV prophylaxis consisted of 100 d after transplant, after which time the risk of CMV infection and disease increased.^2,18,19 Extending the duration of VGCV prophylaxis beyond the early post-transplant period may abrogate this transient increase in the risk of infection and disease.^20,21 In this regard, the optimal duration of prophylaxis for CMV D+/R− patients has not been determined and is the subject of ongoing study.²² Cost, efficacy, and safety are important factors in determining the optimal duration of VGCV prophylaxis. Over the past two decades, various strategies have been used including pre-emptive versus universal prophylaxis and shorter versus longer period of prophylaxis.^20,21,23,24 Although several clinical studies comparing universal prophylaxis versus pre-emptive anti-viral therapy have found similar efficacy and cost in managing CMV infection across various combinations of donor and recipient CMV serologic status, two meta-analyses did find that the use of universal prophylaxis was associated with reduced risk for CMV disease and death.^23–26This study is based on a single center experience comparing two CMV prophylaxis strategies. We report here the clinical outcome and cost-effectiveness analyses of 6- versus 3-mo VGCV prophylaxis in CMV D+/R− de novo kidney and/or pancreas transplant patients.     

Genetics of New-Onset Diabetes after Transplantation

New-onset diabetes after transplantation is a common complication that reduces recipient survival. Research in renal transplant recipients has suggested that pancreatic β-cell dysfunction, as opposed to insulin resistance, may be the key pathologic process. In this study, clinical and genetic factors associated with new-onset diabetes after transplantation were identified in a white population. A joint analysis approach, with an initial genome-wide association study in a subset of cases followed by de novo genotyping in the complete case cohort, was implemented to identify single-nucleotide polymorphisms (SNPs) associated with the development of new-onset diabetes after transplantation. Clinical variables associated with the development of diabetes after renal transplantation included older recipient age, female sex, and percentage weight gain within 12 months of transplantation. The genome-wide association study identified 26 SNPs associated with new-onset diabetes after transplantation; this association was validated for eight SNPs (rs10484821, rs7533125, rs2861484, rs11580170, rs2020902, rs1836882, rs198372, and rs4394754) by de novo genotyping. These associations remained significant after multivariate adjustment for clinical variables. Seven of these SNPs are associated with genes implicated in β-cell apoptosis. These results corroborate recent clinical evidence implicating β-cell dysfunction in the pathophysiology of new-onset diabetes after transplantation and support the pursuit of therapeutic strategies to protect β cells in the post-transplant period.One-year graft survival after renal transplantation is now excellent, exceeding 93% for organs donated after brain death and 96% for those from living donors.^1–3 Technical advancements in surgery, improved understanding of immunology, and innovative developments in pharmacology have altered the landscape of renal transplantation. The goal of preventing early graft loss has largely been achieved and arguably the greatest challenge now is the avoidance of late graft failure. Although there has been a considerable improvement in 1-year renal transplant survival, the rate of graft attrition after the first year remains frustratingly constant.^2,4New-onset diabetes after transplantation (NODAT) is a common and serious disorder that curtails recipient survival.^5–7 NODAT is associated with cardiovascular complications^8–11 and develops in 2%–50%¹² of renal transplant recipients. Approximately 50% of recipients with NODAT require insulin therapy.^{6–8,13–15} A number of clinical variables have been associated with NODAT, including black ethnicity, older recipient age, female sex, obesity, immunosuppression, and viral infections.^{5,6,8,13,16,17}Until recently, the pathophysiology of NODAT was considered to be analogous to type 2 diabetes mellitus. Renal transplant recipients have increased insulin resistance compared with transplant-naïve persons with normal renal function.¹⁸ In a nondiabetic renal transplant population, the main determinants of insulin resistance are obesity and corticosteroid therapy.¹⁹ Insulin resistance improves in renal transplant recipients after successful transplantation^20,21 and recipients have enhanced insulin sensitivity compared with dialysis patients.²² At 1 year, there is no significant difference in insulin resistance between renal transplant recipients with NODAT and those with normal glucose tolerance.^18,23 Furthermore, insulin resistance indices before transplantation and in the early post-transplant period do not predict NODAT development.¹¹Pancreatic β-cell dysfunction may prove to be the main pathologic contributor to NODAT. In glucose clamp studies, a deficit in insulin secretion was common to renal transplant recipients with NODAT.^18,20,21,24 There are a number of possible mechanisms for β-cell toxicity in renal transplantation, including hyperglycemia,²⁵ elevated free fatty acids,²⁶ and the effect of immunosuppressive medication.²⁷ A recent proof-of-concept clinical trial demonstrated that aggressive management of post-transplant hyperglycemia with insulin significantly reduced the 1-year incidence of NODAT.²⁸ This provides further evidence that post-transplant hyperglycemia plays a key role in NODAT development.This study investigates clinical and genetic factors associated with NODAT in a relatively large, white renal transplant population. Clinical variables were identified in a carefully phenotyped, ethnically homogeneous cohort. Initial exploratory analysis was conducted via a genome-wide association study (GWAS) in a subgroup of NODAT cases patients and controls to identify genetic variants associated with NODAT. De novo genotyping was then performed in a larger cohort of NODAT patients and controls to validate the findings.     

Loss of Oriented Cell Division Does not Initiate Cyst Formation

Polycystic kidney disease (PKD) can arise from either developmental or postdevelopmental processes. Recessive PKD, caused by mutations in PKHD1, is a developmental defect, whereas dominant PKD, caused by mutations in PKD1 or PKD2, occurs by a cellular recessive mechanism in mature kidneys. Oriented cell division is a feature of planar cell polarity that describes the orientation of the mitotic axes of dividing cells during development with respect to the luminal vector of the elongating nephron. In polycystic mutant mice, the loss of oriented cell division may also contribute to the pathogenesis of PKD. Here, we examined the role of oriented cell division in mouse models based on mutations in Pkd1, Pkd2, and Pkhd1. Precystic tubules after kidney-selective inactivation of either Pkd1 or Pkd2 did not lose oriented division before cystic dilation but lost oriented division after tubular dilation began. In contrast, Pkhd1^del4/del4 mice lost oriented cell division but did not develop kidney cysts. Increased intercalation of cells into the plane of the tubular epithelium maintained the normal tubular morphology in Pkhd1^del4/del4 mice, which had more cells present in transverse tubular profiles. In conclusion, loss of oriented cell division is a feature of Pkhd1 mutation and cyst formation, but it is neither sufficient to produce kidney cysts nor required to initiate cyst formation after mutation in Pkd1 or Pkd2.Defective three-dimensional tissue organization is a phenotypic hallmark of polycystic kidney disease (PKD). Polycystic kidneys are permeated by fluid-filled cysts that grow and deform the organ in a process associated with a decline in glomerular filtration and ESRD. Positional cloning has discovered genes for autosomal dominant (ADPKD; PKD1, PKD2) and autosomal recessive (ARPKD; PKHD1) PKD. The protein products of these PKD genes along with other diseases manifesting with fibrocystic changes in the kidney (e.g., nephronophthisis, Bardet-Biedl syndrome) are expressed in the primary cilia and basal body complex in kidney epithelial cells.¹ In addition, the gene products mutated in spontaneous or induced kidney cystic models in nonprimate vertebrates are associated with cilia.^2–4 The role of cilia in PKD was shown prospectively by the occurrence of cysts after disruption of cilia structure in the kidney by inactivation of Kif3a, a gene not previously known to cause PKD.⁵ In aggregate, these findings have validated the role of cilia in the pathogenesis of fibrocystic diseases in the kidney. There are polycystic disease proteins, including those causing isolated human autosomal dominant polycystic liver disease^6,7 and pronephric cysts in zebrafish,⁸ that do not localize directly to cilia; however, even in these cases, a functional interconnection with cilia has either been shown⁸ or proposed.⁷Whereas many of the mutated genes and the central organelle for the pathogenesis of PKD have been identified, the effecter pathways for cyst formation remain less well defined. Among these, defects in planar cell polarity (PCP), a central determinant of tissue organization (reviewed in reference⁹), have been proposed as fundamental to the pathogenesis of PKD. Discovery that inv, a cystic disease– and cilia-related protein, acts as a switch between canonical and PCP-related noncanonical Wnt signaling¹⁰ led to the hypothesis that cyst growth may be associated with defective polarity within the plane of the tubule epithelium.¹¹ The understanding that orientation of cell division (OCD) is a consequence of planar polarity¹² led Fischer et al.¹³ to test whether defective OCD underlies at least part of the pathogenesis of PKD. Loss of OCD was observed in advance of cyst formation in the pck rat, an orthologous Pkhd1 model, and in cystic disease as a result of mutation of Hnf1β.¹³ More recently, tubules in postnatal Kif3a mutant kidneys showed loss of OCD in the absence of cilia.¹⁴ The converse hypothesis, that loss of PCP proteins can result in PKD, was recently demonstrated with inactivation of PCP-related protocadherin Fat4, resulting in both loss of OCD and PKD.¹⁵ These data support the hypothesis that loss of OCD is associated with mutations that affect cilia function or structure, and mutations in PCP proteins can be associated with kidney cyst formation.In this study, we examined the role of OCD in PKD using orthologous mouse models of human ADPKD (Pkd1, Pkd2) and ARPKD (Pkhd1). We found that after kidney-selective inactivation of either Pkd1 or Pkd2, precystic tubules did not show evidence of loss of OCD in advance of cystic dilation; however, OCD was lost once the tubules began to dilate. By contrast, our Pkhd1^del4 model of recessive PKD, like its rat ortholog,¹³ showed loss of OCD but, unlike the rat model, did not develop kidney cysts.¹⁶ The normal-appearing tubule morphology in Pkhd1^del4/del4 is maintained by increased intercalation of cells into the plane of the epithelium that is associated with a small but significant increase in the number of cells present in transverse tubular profiles. Pkhd1 functions in a PCP pathway to maintain OCD. Loss of OCD is a feature of dilating cysts but is neither a prerequisite for initiation of cyst formation nor sufficient to produce cysts in elongating tubules.     

Pregnancy and Maternal Outcomes Among Kidney Transplant Recipients

Fertility rates, pregnancy, and maternal outcomes are not well described among women with a functioning kidney transplant. Using data from the Australian and New Zealand Dialysis and Transplant Registry, we analyzed 40 yr of pregnancy-related outcomes for transplant recipients. This analysis included 444 live births reported from 577 pregnancies; the absolute but not relative fertility rate fell during these four decades. Of pregnancies achieved, 97% were beyond the first year after transplantation. The mean age at the time of pregnancy was 29 ± 5 yr. Compared with previous decades, the mean age during the last decade increased significantly to 32 yr (P < 0.001). The proportion of live births doubled during the last decade, whereas surgical terminations declined (P < 0.001). The fertility rate (or live-birth rate) for this cohort of women was 0.19 (95% confidence interval 0.17 to 0.21) relative to the Australian background population. We also matched 120 parous with 120 nulliparous women by year of transplantation, duration of transplant, age at transplantation ±5 yr, and predelivery creatinine for parous women or serum creatinine for nulliparous women; a first live birth was not associated with a poorer 20-yr graft or patient survival. Maternal complications included preeclampsia in 27% and gestational diabetes in 1%. Taken together, these data confirm that a live birth in women with a functioning graft does not have an adverse impact on graft and patient survival.One of the many perceived benefits of kidney transplantation has been restoration of pituitary-ovarian function and fertility in women of reproductive age. Prenatal advice for women with a functioning kidney transplant has been primarily based on data derived from observational research,^1–13 and the reported live-birth rates achieved in such women range from 43.2¹⁴ to 82%.¹⁵Although an increased pregnancy event number has been reported for women with a functioning kidney transplant,¹⁶ little is actually known about “pregnancy rate changes” during the past 40 yr. More importantly, long-term graft and maternal survival analyses, referred to when advising women who have undergone transplantation and are considering a pregnancy, have been mostly performed without adequate matching,¹² or, alternatively, matching has been used but outcomes followed up for only brief intervals^14,17,18 and in small cohorts.^19–22 Published graft matching studies to date suggest no adverse impact 10 yr after a live birth.¹⁴In most instances, pregnancies in women with a kidney graft have been encouraged. Historically, renal function,^8,15,17,18 baseline proteinuria,²³ intercurrent hypertension,^1,24 and time from transplantation^{1,3,5,8,14,15,18,24,25} have been used to predict adverse event risks to the mother, kidney, and offspring. To this are added the often unquantifiable inherent risks for genetically transmitted diseases or the problems associated with prematurity.^26,27 More recently, epidemiologic evidence suggests low birth weight may be associated with the development of hypertension,²⁸ cardiovascular disease,²⁹ insulin resistance,³⁰ and end-stage renal failure.³¹ Moreover, low birth weight is associated with an increased risk for hypertension, independent of genetic and shared environmental factors.³²Series published to date have not captured all pregnancy events or their outcomes. Limitations of some of the published studies include short duration of follow-up and studies with no adequate or long-term matching for decade and renal function.We examined fertility rates, pregnancy rates, and pregnancy outcomes over 40 yr in an at-risk population, defined as women who were aged between 15 and 49 and had a functioning kidney transplant, using ANZDATA registry data. In addition, maternal and graft outcomes were analyzed, and, uniquely, a matched cohort analysis of 120 nulliparous and 120 parous women who had undergone transplantation enabled analysis of outcomes at 20 yr.     

Intraflagellar Transport Proteins Are Essential for Cilia Formation and for Planar Cell Polarity

The highly conserved intraflagellar transport (IFT) proteins are essential for cilia formation in multiple organisms, but surprisingly, cilia form in multiple zebrafish ift mutants. Here, we detected maternal deposition of ift gene products in zebrafish and found that ciliary assembly occurs only during early developmental stages, supporting the idea that maternal contribution of ift gene products masks the function of IFT proteins during initial development. In addition, the basal bodies in multiciliated cells of the pronephric duct in ift mutants were disorganized, with a pattern suggestive of defective planar cell polarity (PCP). Depletion of pk1, a core PCP component, similarly led to kidney cyst formation and basal body disorganization. Furthermore, we found that multiple ift genes genetically interact with pk1. Taken together, these data suggest that IFT proteins play a conserved role in cilia formation and planar cell polarity in zebrafish.The cilium is a cell surface organelle that is almost ubiquitously present on vertebrate cells. Protruding from the cell into its environment, the cilium is involved in multiple signaling pathways, including the Sonic hedgehog (Shh) pathway, the Wnt pathways, and the target of rapamycin (TOR) pathway.^1–5 Not surprisingly, defects in the cilium have been linked to a growing list of human diseases, coined “ciliopathies,” ranging from laterality defects, retinal degeneration, polycystic kidney disease (PKD), and other hepatorenal fibrocystic disorders to obesity and diabetes (for a review, see reference ⁶).Many studies have demonstrated that the formation and maintenance of the cilium depends on intraflagellar transport (IFT) particles, which are composed of at least 17 polypeptides.^7,8 These IFT particles move along microtubules in cilia and are thought to act as vehicles for transporting cargos needed for the assembly, maintenance, and function of cilia. Homologs of IFT proteins have been found in a wide spectrum of organisms including Caenorhabditis elegans, Drosophila, and mammals and have also been shown to be required for cilia formation.^3,9–12In zebrafish, mutants of ift57, ift81, ift88, and ift172 have numerous defects commonly associated with ciliary abnormalities.^13,14 However, cilia in these mutants are able to form initially but degenerate over time, giving rise to the hypothesis that IFT is essential for cilia maintenance rather than cilia assembly in zebrafish.¹⁴ Interestingly, products of many genes in zebrafish are deposited maternally. One hypothesis for the initial formation of cilia in zebrafish ift mutants is that maternal contribution of ift gene products masks the function of ift genes during early embryonic development. Accordingly, cilia formation is severely impaired in a maternal-zygotic mutant of ift88.¹⁵ In this study, we demonstrate that products of ift57 and ift172 are indeed maternally deposited. We further show that although cilia destined to form early in development show only maintenance defects in ift57^hi3417 and ift172^hi2211 mutants, cilia programmed to assemble later in development fail to form, providing further support for a conserved role of IFT in cilia formation in zebrafish.One of the most extensively studied phenotypes associated with ciliary defects is the formation of kidney cysts. Both the canonical and the noncanonical Wnt pathway, or planar cell polarity (PCP) pathway, have been implicated in kidney cyst formation.^4,16–21 However, in contrast to the well-established role of cilia in the hedgehog pathway,^2,3,22 the role of cilia in the Wnt pathways is unclear.^4,15,23,24 In this study, we demonstrate that, in the kidney duct of ift57^hi3417 and ift172^hi2211 mutants, the organization of basal bodies is impaired, a phenotype consistent with compromised PCP signaling. We further show that knockdown of prickle 1 (pk1), a core PCP player, leads to disorganization of basal bodies and kidney cyst formation. Finally, we provide evidence that ift57, ift88, and ift172 genetically interact with pk1 in convergence-extension (CE) movements during gastrulation, a process regulated by the PCP pathway. Together, these data support an intricate relationship between IFT and the PCP pathway.     

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

RESEARCH DESIGN AND METHODS

We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r^−/− and Gcgr^−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG.

RESULTS

Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR.

CONCLUSIONS

Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.Obesity is an important risk factor for type 2 diabetes, and ∼90% of patients with type 2 diabetes are overweight or obese (1). Among new therapies for type 2 diabetes, peptidyl mimetics of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) stimulate insulin biosynthesis and secretion in a glucose-dependent manner (2,3) and cause modest weight loss in type 2 diabetic patients. The glucose-lowering and antiobesity effects of incretin-based therapies for type 2 diabetes have prompted evaluation of the therapeutic potential of other glucagon-family peptides, in particular oxyntomodulin (OXM). The OXM peptide is generated by post-translational processing of preproglucagon in the gut and is secreted postprandially from l-cells of the jejuno-ileum together with other preproglucagon-derived peptides including GLP-1 (4,5). In rodents, OXM reduces food intake and body weight, increases energy expenditure, and improves glucose metabolism (6–8). A 4-week clinical study in obese subjects demonstrated that repeated subcutaneous administration of OXM was well tolerated and caused significant weight loss with a concomitant reduction in food intake (9). An increase in activity-related energy expenditure was also noted in a separate study involving short-term treatment with the peptide (10).OXM activates both, the GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in vitro, albeit with 10- to 100-fold reduced potency compared with the cognate ligands GLP-1 and glucagon, respectively (11–13). It has been proposed that OXM modulates glucose and energy homeostasis solely by GLP1R agonism, because its acute metabolic effects in rodents are abolished by coadministration of the GLP1R antagonist exendin(9–39) and are not observed in Glp1r^−/− mice (7,8,14,15). Other aspects of OXM pharmacology, however, such as protective effects on murine islets and inhibition of gastric acid secretion appear to be independent of GLP1R signaling (14). In addition, pharmacological activation of GCGR by glucagon, a master regulator of fasting metabolism (16), decreases food intake in rodents and humans (17–19), suggesting a potential role for GCGR signaling in the pharmacology of OXM. Because both OXM and GLP-1 are labile in vivo (T_1/2 ∼12 min and 2–3 min, respectively) (20,21) and are substrates for the cell surface protease dipeptidyl peptidase 4 (DPP-4) (22), we developed two long-acting DPP-4–resistant OXM analogs as pharmacological agents to better investigate the differential pharmacology and therapeutic potential of dual GLP1R/GCGR agonism versus GLP1R-selective agonism. Peptide DualAG exhibits in vitro GLP1R and GCGR agonist potency comparable to that of native OXM and is conjugated to cholesterol via a Cys sidechain at the C-terminus for improved pharmacokinetics. Peptide GLPAG differs from DualAG by only one residue (Gln³→Glu) and is an equipotent GLP1R agonist, but has no significant GCGR agonist or antagonist activity in vitro. The objective of this study was to leverage the matched GLP1R agonist potencies and pharmacokinetics of peptides DualAG and GLPAG in comparing the metabolic effects and therapeutic potential of a dual GLP1R/GCGR agonist with a GLP1R-selective agonist in a mouse model of obesity.