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.     

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.     

Polycystic Kidney Disease and Cancer after Renal Transplantation

Autosomal dominant polycystic kidney disease (ADPKD), the most common form of polycystic kidney disease (PKD), is a disorder with characteristics of neoplasia. However, it is not known whether renal transplant recipients with PKD have an increased risk of cancer. Data from the Scientific Registry of Transplant Recipients, which contains information on all solid organ transplant recipients in the United States, were linked to 15 population-based cancer registries in the United States. For PKD recipients, we compared overall cancer risk with that in the general population. We also compared cancer incidence in PKD versus non-PKD renal transplant recipients using Poisson regression, and we determined incidence rate ratios (IRRs) adjusted for age, sex, race/ethnicity, dialysis duration, and time since transplantation. The study included 10,166 kidney recipients with PKD and 107,339 without PKD. Cancer incidence in PKD recipients was 1233.6 per 100,000 person-years, 48% higher than expected in the general population (standardized incidence ratio, 1.48; 95% confidence interval [95% CI], 1.37 to 1.60), whereas cancer incidence in non-PKD recipients was 1119.1 per 100,000 person-years. The unadjusted incidence was higher in PKD than in non-PKD recipients (IRR, 1.10; 95% CI, 1.01 to 1.20). However, PKD recipients were older (median age at transplantation, 51 years versus 45 years for non-PKD recipients), and after multivariable adjustment, cancer incidence was lower in PKD recipients than in others (IRR, 0.84; 95% CI, 0.77 to 0.91). The reason for the lower cancer risk in PKD recipients is not known but may relate to biologic characteristics of ADPKD or to cancer risk behaviors associated with ADPKD.Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of inherited cystic renal disease and the fourth most common cause of ESRD in the United States.^1–3 There are currently>16,000 individuals with polycystic kidney disease (PKD, of which ADPKD is by far the most common type) living with a renal transplant in the United States.⁴ADPKD is a result of mutations in one of two genes: PKD1 and PKD2.^1,5,6 These genes are widely expressed in many tissues, consistent with the multiorgan pathology characterizing ADPKD. A key factor in cyst formation and enlargement in ADPKD is the abnormal proliferation of cyst epithelial cells in a cell-autonomous manner.^7,8 This cyst formation is associated with cellular dedifferentiation and is considered a neoplastic process driven by upregulated proto-oncogenes.^9–13 While published case reports document the occurrence of renal cell carcinomas (RCCs) in ADPKD-affected kidneys,^14,15 these tumors may be partly due to acquired renal cystic disease resulting from long-term dialysis.¹⁶ Because there do not appear to be widespread published reports of other cancers in patients with ADPKD, protective mechanisms might exist in ADPKD to prevent malignant transformation. Indeed, many oncogenes that promote cell proliferation also act as potent growth suppressors (e.g., Ras¹⁷) or inducers of apoptosis (e.g., Myc^18,19). Thus, there is uncertainty whether ADPKD mutations are associated with increased rates of kidney cancer or cancer in general.We therefore designed a study to compare cancer risk in kidney transplant recipients with PKD versus kidney recipients with other causes of ESRD. Organ transplant recipients are at increased risk of cancer, largely because of immunosuppressive therapy.²⁰ An increased risk of cancer in patients with PKD might be detectable in this high-risk cancer population. Alternatively, if there is no increased risk of cancer in ADPKD, the findings would suggest the need for further study to determine whether protective cellular mechanisms may be at work.     

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.     

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.     

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%.     

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.     

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.     

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.     

Albuminuria and Estimated Glomerular Filtration Rate Independently Associate with Acute Kidney Injury

Acute kidney injury (AKI) is increasingly common and a significant contributor to excess death in hospitalized patients. CKD is an established risk factor for AKI; however, the independent graded association of urine albumin excretion with AKI is unknown. We analyzed a prospective cohort of 11,200 participants in the Atherosclerosis Risk in Communities (ARIC) study for the association between baseline urine albumin-to-creatinine ratio and estimated GFR (eGFR) with hospitalizations or death with AKI. The incidence of AKI events was 4.0 per 1000 person-years of follow-up. Using participants with urine albumin-to-creatinine ratios <10 mg/g as a reference, the relative hazards of AKI, adjusted for age, gender, race, cardiovascular risk factors, and categories of eGFR were 1.9 (95% CI, 1.4 to 2.6), 2.2 (95% CI, 1.6 to 3.0), and 4.8 (95% CI, 3.2 to 7.2) for urine albumin-to-creatinine ratio groups of 11 to 29 mg/g, 30 to 299 mg/g, and ≥300 mg/g, respectively. Similarly, the overall adjusted relative hazard of AKI increased with decreasing eGFR. Patterns persisted within subgroups of age, race, and gender. In summary, albuminuria and eGFR have strong, independent associations with incident AKI.It has long been recognized that an episode of acute kidney injury (AKI) can have serious health consequences.^1–4 Even a relatively small degree of renal injury increases a patient''s risk of a prolonged hospital stay, chronic kidney disease (CKD), ESRD, and death.^2,5–10 Over the last 2 decades, the incidence of hospitalized AKI has increased dramatically.^11–14 Precise estimations vary depending on population and method of case identification, but a recent community-based study of AKI estimated the incidence of nondialysis requiring AKI at 522 per 100,000 population per year and dialysis-requiring AKI at 30 per 100,000,¹³ which is well over that of ESRD.¹⁴ This increase in the burden of disease, taken with the associated poor long-term outcomes, has established AKI as a major public health issue.¹⁴Beyond routine supportive care, there exists little established medical therapy for AKI.¹⁵ Many current lines of research are focused on the prevention of AKI. However, few prospective, population-based studies have evaluated the development of AKI.^3,13,16 Hsu et al.,^13,17 along with multiple observational series in various clinical settings, have clearly established older age and CKD as risk factors for AKI.^18–24 Other observed associations with AKI include black race and male gender.^11,18,25 Proteinuria, an established risk factor in the development of cardiovascular disease,^26,27 ESRD,²⁸ and death,²⁹ is less studied in its role in the development of AKI. Hsu and colleagues demonstrated the prospective association of proteinuria with dialysis-requiring AKI; however, the proteinuria classification was binary and based on dipstick measurement.¹⁷ To our knowledge, no study has quantified the independent dose response of albuminuria with AKI hospitalization, including less severe AKI. Our study''s objective was thus to characterize prospectively the association between baseline urine albumin-to-creatinine ratio (UACR) and hospitalizations for AKI, controlling for established and potential risk factors such as CKD, age, and cardiovascular comorbidities.     

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.     

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.     

Activation of p53 Promotes Renal Injury in Acute Aristolochic Acid Nephropathy

Ingestion of aristolochic acid (AA) can cause AA nephropathy (AAN), in which excessive death of tubular epithelial cells (TECs) characterize the acute phase. AA forms adducts with DNA, which may lead to TEC apoptosis via p53-mediated signaling. We tested this hypothesis both by studying p53-deficient mice and by blocking p53 in TECs with its inhibitor pifithrin-α. AA induced acute AAN in wild-type mice, resulting in massive apoptotic and necrotic TEC death and acute renal failure; p53 deficiency or pharmacologic inhibition attenuated this injury. In vitro, AA induced apoptotic and necrotic death of TEC in a time- and dosage-dependent manner, with apoptosis marked by a 10-fold increase in cleaved caspase-3 and terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling–positive/Annexin V-positive propidium iodide–negative TECs (all P < 0.001). AA induced dephosphorylation of STAT3 and the subsequent activation of p53 and TEC apoptosis. In contrast, overexpression of STAT3, p53 inhibition, or p53 knockdown with small interfering RNA all attenuated AA-induced TEC apoptosis. Taken together, these results suggest that AA induces TEC death via apoptosis by dephosphorylation of STAT3 and posttranslational activation of p53, supporting the hypothesis that p53 promotes renal injury in acute AAN.Chinese herb nephropathy was first reported in Belgium in patients with prolonged intake of Chinese herbs during a slimming regimen and is recognized as one of the most severe complications caused by traditional Chinese medicine.^1–3 It is now clear that the major substance that causes Chinese herb nephropathy is the plant nephrotoxin aristolochic acid and its metabolism products.^4–6 Thus, the term aristolochic acid nephropathy (AAN), instead of Chinese herbal nephropathy, is used today.^7,8 AAN has emerged as an important cause of drug-associated renal failure worldwide.⁹Patients with AAN exhibit a rapidly progressive renal deterioration, resulting in acute renal failure that could lead to ESRD.^1–3,10,11 A similar clinical course was observed in experimental animals treated with AA.^12,13 Pathologically, chronic AAN is characterized by extensive interstitial fibrosis with atrophy and loss of renal tubules.^{1–3,10–13} The lesions of chronic AAN are mainly in the cortex involving proximal tubular epithelial cells (TECs)^10–13; glomeruli are relatively spared with minimal inflammation.^9–12 In contrast, progressive TEC death occurs early in the clinical course with an absence of renal fibrosis and inflammation in experimental models and patients with acute AAN.^10,14,15 Although apoptosis is an important pathologic feature in in vivo and in vitro studies of acute AAN,^16–18 the underlying mechanisms remain unclear.In considering the genotoxic effect of AA with the formation of AA-DNA adducts and the importance of the p53 signaling pathway in DNA damage and cell apoptosis,^19–21 we hypothesized that TEC apoptosis in acute AAN is dependent on p53 signaling. We investigated this by inducing acute AAN in p53 knockout (KO) and p53 wild-type (WT) mice and by blocking the p53 activities with a pharmacologic inhibitor. We further studied the toxicity of AA on TEC apoptosis by examining a panel of apoptotic biomarkers. The mechanism that AA induced TEC apoptosis by activating p53 via a STAT3-dependent posttranslational modification was identified.     

TRPV4 Dysfunction Promotes Renal Cystogenesis in Autosomal Recessive Polycystic Kidney Disease

The molecular mechanism of cyst formation and expansion in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood, but impaired mechanosensitivity to tubular flow and dysfunctional calcium signaling are important contributors. The activity of the mechanosensitive Ca²⁺-permeable TRPV4 channel underlies flow-dependent Ca²⁺ signaling in murine collecting duct (CD) cells, suggesting that this channel may contribute to cystogenesis in ARPKD. Here, we developed a method to isolate CD-derived cysts and studied TRPV4 function in these cysts laid open as monolayers and in nondilated split-open CDs in a rat model of ARPKD. In freshly isolated CD-derived cyst monolayers, we observed markedly impaired TRPV4 activity, abnormal subcellular localization of the channel, disrupted TRPV4 glycosylation, decreased basal [Ca²⁺]_i, and loss of flow-mediated [Ca²⁺]_i signaling. In contrast, nondilated CDs of these rats exhibited functional TRPV4 with largely preserved mechanosensitive properties. Long-term systemic augmentation of TRPV4 activity with a selective TRPV4 activator significantly attenuated the renal manifestations of ARPKD in a time-dependent manner. At the cellular level, selective activation of TRPV4 restored mechanosensitive Ca²⁺ signaling as well as the function and subcellular distribution of TRPV4. In conclusion, the functional status of TRPV4, which underlies mechanosensitive Ca²⁺ signaling in CD cells, inversely correlates with renal cystogenesis in ARPKD. Augmenting TRPV4 activity may have therapeutic potential in ARPKD.Polycystic kidney disease (PKD) is a cohort of monogenic disorders that result in development and subsequent growth of renal cysts filled with fluid.^1–5 Cyst enlargement compromises function of surrounding nephrons and progresses to ESRD.^1,6 In the more common form of PKD, autosomal dominant PKD (ADPKD), which is caused by mutations of polycystin 1 (PC1) and polycystin 2 (PC2), renal cysts are formed along the full length of the nephron with prevalence to the collecting duct (CD).^1,7 In the rarer and more severe autosomal recessive PKD (ARPKD), renal cyst formation is virtually restricted to the CD.^1,2,5,8 Mutations of the PKHD1 gene encoding fibrocystin underlie the genetic basis of the disease.^6,8,9 Although the exact function of the protein is unknown, fibrocystin was shown to be expressed in primary cilia where it can interact and form complexes with PC2, possibly participating in mechanotransduction.^10–12It is accepted that the CD cells elevate [Ca²⁺]_i in response to mechanical stress arising from variations in tubular flow or tubular composition.^13–23 Impaired mechanosensitive [Ca²⁺]_i responses, reported for both cultured ADPKD²⁴ and ARPKD^25,26 cells, point to a possible fundamental role of disrupted [Ca²⁺]_i signaling in cystogenesis. The central cilia and cilia-associated PC1 and PC2 were proposed to mediate flow-induced cellular responses.^19,27 However, homomeric PC2 channels are not mechanosensitive and fail to increase [Ca²⁺]_i in response to flow and hypotonicity.^28,29 Furthermore, intercalated cells, which lack primary cilia, respond to flow changes with comparable increases in [Ca²⁺]_i as observed in principal cells, which have primary cilia.^16,30 Therefore, additional mechanisms conferring mechanosensitivity to the CD cells need to be considered.Transient receptor potential (TRP) channels are known to participate in cellular responses to a variety of environmental stimuli, including thermosensation, chemosensation, and mechanical forces (reviewed in Song and Yuan³¹). Several TRP channels, including TRPC3, TRPC6, and TRPV4, can be detected in the native CD cells and CD-originated cultured lines.^19,32–34 Among these channels, TRPV4 has routinely been shown to be activated by mechanical stimuli.^34–38 Indeed, we documented that endogenous TRPV4 in M-1 CD cells is stimulated by increases in flow, a response that is abolished by TRPV4 small interfering RNA knockdown.^34,38 We further demonstrated a lack of flow-mediated [Ca²⁺]_i elevations in CD from TRPV4−/− mice.³⁰ Consistently, flow-mediated Ca²⁺-dependent K⁺ secretion in the CD is disrupted in TRPV4 knockout animals.³⁹ TRPV4 directly interacts with PC2 to form mechanosensitive heteromeric complexes.^28,29 The fact that PC2 interacts with both PC1⁴⁰ and fibrocystin^10–12 suggests that TRPV4 could be an essential part of this mechanotransducing sensory complex.Current PKD management is directed toward pharmacologic interference with abnormal signaling pathways causing exaggerated cell proliferation, dedifferentiation, apoptosis, and cyst growth.⁴¹ Specifically, PKD is associated with elevated circulating vasopressin levels, increased basal cellular cAMP levels, and strong upregulation of cAMP-dependent fluid secretion and proliferation.^42,43 V2 antagonism greatly diminishes disease progression in rodent models of both ADPKD and ARPKD.^42,44 Elevated cAMP levels might be directly related to the reduced [Ca²⁺]_i, possibly due to impaired ability to sense changes in flow.^42,44,45 This raises the possibility that manipulation with the mechanosensitivity in the CD along the TRPV4 axis modulates [Ca²⁺]_i signalization and, in turn, renal cystogenesis.In this study, we developed a new approach to isolate native CD-derived cyst monolayers and nondilated CDs from a rat model of ARPKD to thoroughly investigate how functional TRPV4 status determines the development and growth of renal cysts. We found that the disease leads to disruption of mechanosensitive [Ca²⁺]_i signaling and impaired TRPV4 activity specifically in CD cysts but not in nondilated CDs. Long-term pharmacologic potentiation of TRPV4 activity gradually restores mechanosensitivity in cyst cells and greatly blunts renal ARPKD progression. From a global prospective, this study establishes a temporal link between disruption of TRPV4-based mechanosensitivity in the CD and cystogenesis. This also suggests pharmacologic potential of targeting TRPV4 activity as a treatment strategy in retarding development of ARPKD.     

Racial Composition of Residential Areas Associates with Access to Pre-ESRD Nephrology Care

Referral to a nephrologist before initiation of chronic dialysis occurs less frequently for blacks than whites, but the reasons for this disparity are incompletely understood. Here, we examined the contribution of racial composition by zip code on access and quality of nephrology care before initiation of renal replacement therapy (RRT). We retrospectively studied a cohort study of 92,000 white and black adults who initiated RRT in the United States between June 1, 2005, and October 5, 2006. The percentage of patients without pre-ESRD nephrology care ranged from 30% among those who lived in zip codes with <5% black residents to 41% among those who lived in areas with >50% black residents. In adjusted analyses, as the percentage of blacks in residential areas increased, the likelihood of not receiving pre-ESRD nephrology care increased. Among patients who received nephrology care, the quality of care (timing of care and proportion of patients who received a pre-emptive renal transplant, who initiated therapy with peritoneal dialysis, or who had a permanent hemodialysis access) did not differ by the racial composition of their residential area. In conclusion, racial composition of residential areas associates with access to nephrology care but not with quality of the nephrology care received.Clinical practice guidelines for chronic kidney disease emphasize the importance of timely referral to a nephrologist for patients expected to require renal replacement therapy.^1–3 Nevertheless, approximately 33% of end-stage renal disease (ESRD) patients in the United States do not see a nephrologist before initiation of chronic dialysis.^4,5 Lack of timely access to nephrology care is associated with several adverse outcomes after initiation of dialysis including higher mortality rates,^6–9 higher rates of hospitalization,¹⁰ lower rates of renal transplantation,^11,12 delayed creation of arteriovenous fistulae,¹³ lower rates of achievement of dialysis treatment targets,^14,15 and a lower likelihood of receiving home-based dialysis therapies such as peritoneal dialysis and home hemodialysis.^3,16–18In the United States, black dialysis patients are less likely than white patients to have received nephrology care before onset of ESRD.^9,19–21 They are also less likely to receive a pre-emptive kidney transplant,^22,23 select peritoneal dialysis over hemodialysis,²⁴ and have a vascular access in place at onset of hemodialysis.^25,26 Several factors may contribute to these disparities including differences in insurance status, level of education, physician knowledge or biases, and patient preferences.^9,27–29 In addition, geographic factors such as proximity to dialysis facilities and degree of urbanization also affect access to nephrology care.^30–33A substantial proportion of black patients are also more likely to live in areas where most other residents are black. A recent study demonstrated that both black and white dialysis patients living in predominantly black zip codes were less likely to receive a kidney transplant than patients living in other areas.³⁴ Although levels of income, wealth and education tended to be lower among residents of predominantly black zip codes, lower transplant rates among dialysis patients living in these areas were not completely explained by these measures. Patients who live in predominantly black zip codes may face unique barriers to care that are not explained by measured socioeconomic characteristics of those zip codes. We therefore hypothesized that dialysis patients living in zip codes with a greater proportion of black residents would be less likely to have received pre-ESRD care and less likely to have received high-quality nephrology care than patients living in other zip codes. We also hypothesized that these relationships would not be completely explained by differences in zip code socioeconomic status or patient race.     

Metabolite Profiling Identifies Markers of Uremia

ESRD is a state of small-molecule disarray. We applied liquid chromatography/tandem mass spectrometry-based metabolite profiling to survey >350 small molecules in 44 fasting subjects with ESRD, before and after hemodialysis, and in 10 age-matched, at-risk fasting control subjects. At baseline, increased levels of polar analytes and decreased levels of lipid analytes characterized uremic plasma. In addition to confirming the elevation of numerous previously identified uremic toxins, we identified several additional markers of ESRD, including dicarboxylic acids (adipate, malonate, methylmalonate, and maleate), biogenic amines, nucleotide derivatives, phenols, and sphingomyelins. The pattern of lipids was notable for a universal decrease in lower-molecular-weight triacylglycerols, and an increase in several intermediate-molecular-weight triacylglycerols in ESRD compared with controls; standard measurement of total triglycerides obscured this heterogeneity. These observations suggest disturbed triglyceride catabolism and/or β-oxidation in ESRD. As expected, the hemodialysis procedure was associated with significant decreases in most polar analytes. Unexpected increases in several metabolites, however, indicated activation of a broad catabolic program, including glycolysis, lipolysis, ketosis, and nucleotide breakdown. In summary, this study demonstrates the application of metabolite profiling to identify markers of ESRD, provide perspective on uremic dyslipidemia, and broaden our understanding of the biochemical effects of hemodialysis.Emerging technologies have enhanced the feasibility of acquiring high-throughput “snapshots” of a whole organism''s metabolic status (metabolite profiling, or metabolomics).^1,2 These techniques, which view substrates and products in the context of fundamental biochemical pathways, are particularly relevant for characterizing metabolic disease states. They have been applied to identify markers of obesity and insulin resistance^3,4 and the response to acute perturbations such as glucose ingestion.^5,6 Furthermore, a growing body of data assigns circulating small molecules with unanticipated hormone-like functions.^7–10 Hence, plasma small molecules have the potential to serve as biomarkers and effectors of complex metabolic conditions.ESRD is a state of disturbed metabolism, although important features such as dyslipidemia^11,12 and catabolism^13–15 remain incompletely understood. Because these factors are believed to contribute to mortality in ESRD,^16–18 new insights into their pathogenesis are needed. Small molecules that accumulate with impaired renal function are also believed to contribute to mortality in ESRD, and decades of research have identified numerous small molecules as potential uremic toxins.¹⁹ However, the discovery of these proposed toxins antedates current systematic approaches that place these small molecules in the context of whole-body metabolism. Such an integrated view could illuminate the intersection between ESRD and metabolic disturbances and highlight select pathways as therapeutic targets. Furthermore, understanding the metabolic response to hemodialysis could lead to salutary modifications to the procedure.We have previously utilized targeted liquid chromatography (LC)/tandem mass spectrometry (MS)-based metabolite profiling to study human plasma during glucose ingestion,⁵ myocardial ischemia,²⁰ and planned myocardial infarction.²¹ We have since expanded this platform from a survey of polar metabolites to include an analysis of plasma lipids. We applied this platform to the study of subjects with ESRD with the goal of identifying novel metabolic markers of ESRD and to examine the immediate effects of hemodialysis.