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.     

Renal Actions of RGS2 Control Blood Pressure

G protein-coupled receptors (GPCRs) have key roles in cardiovascular regulation and are important targets for the treatment of hypertension. GTPase-activating proteins, such as RGS2, modulate downstream signaling by GPCRs. RGS2 displays regulatory selectivity for the Gαq subclass of G proteins, and mice lacking RGS2 develop hypertension through incompletely understood mechanisms. Using total body RGS2-deficient mice, we used a kidney crosstransplantation strategy to examine separately the contributions of RGS2 actions in the kidney from those in extrarenal tissues with regard to BP regulation. Loss of renal RGS2 was sufficient to cause hypertension, whereas the absence of RGS2 from all extrarenal tissues including the peripheral vasculature did not significantly alter BP. Accordingly, these results suggest that RGS2 acts within the kidney to modulate BP and prevent hypertension. These data support a critical role for the renal epithelium and/or vasculature as the final determinants of the intra-arterial pressure in hypertension.The role of G protein-coupled receptors (GPCRs) in hypertension and cardiovascular diseases is well established.¹ Moreover, pharmacologic antagonists of GPCRs, such as β-adrenergic and angiotensin receptors, are cornerstones of therapy in the treatment of hypertension and its complications.² Signaling by GPCRs is triggered by ligand-induced conformational changes in the receptor that promote exchange of guanosine 5′-diphosphate for guanosine 5′-triphosphate on the Gα subunit of the G protein complex,³ followed by dissociation of Gα from the Gβγ dimer. The dissociated subunits can then interact with effector molecules to propagate the signal. The duration and intensity of signaling are further regulated by GTPase-activating proteins.⁴ The regulators of G protein signaling (RGSs) are a family of proteins with GTPase-activating protein activity.⁴ Among these, RGS2 displays regulatory selectivity for the Gαq subclass of G proteins.⁵ Many key cardiovascular hormones such as angiotensin II, endothelin-1, thromboxane A₂, and norepinephrine activate receptors that couple to Gαq.A specific role for RGS2 in maintaining normal vascular tone and BP was established using genetically modified mice.^6,7 RGS2-deficient mice have hypertension^6,7 along with abnormal vascular contraction and relaxation responses.⁷ In addition to its actions to influence the contractile state of vascular smooth muscle, regulated expression of RGS2 has been described in other tissues that are important for BP regulation including the central nervous system⁸ and the kidney.⁹ Here, we use a kidney crosstransplantation strategy to distinguish contributions of RGS2 actions in the kidney from extrarenal tissues to the regulation of BP and the development of hypertension. Our studies indicate that RGS2 effects within the kidney are critical for regulation of BP, suggesting that altered renal epithelial and/or vascular functions are responsible for hypertension in this genetic model.To determine the relative contributions of RGS2 in renal versus extrarenal tissues to the pathogenesis of hypertension, we used a kidney crosstransplantation strategy. By varying the genotype of the transplant donor and recipient, we generated four groups of animals in which renal function was provided entirely by the single transplanted kidney. The wild-type group consisted of wild-type mice transplanted with kidneys from wild-type donors, having normal expression of RGS2 in the kidney transplant and in all systemic tissues. For the systemic knockout (KO) group, RGS2-deficient recipients were transplanted with kidneys from wild-type donors; these animals lack RGS2 in all tissues except the kidney. Kidney KO animals are wild-type recipients of RGS2-deficient kidneys lacking expression of RGS2 only in renal parenchyma and vasculature but with normal expression of receptors in all systemic, nonrenal tissues including peripheral vessels. Finally, the total KO group consists of RGS2-deficient recipients of RGS2-deficient kidneys and therefore completely lacks RGS2 in all tissues.The absence of RGS2 did not significantly affect the normal diurnal variation in BP in any of the groups (Figure 1). Among the transplanted animals, mean systolic BP levels for the period of baseline recording in the wild-type group (123 ± 2 mmHg; n = 7) were in a range similar to previous measurements in nontransplanted, wild-type C57BL/6 mice,¹⁰ supporting our previous observations that the surgical procedure and the presence of only a single transplanted kidney do not significantly alter baseline levels of BP.¹⁰ By contrast (Figure 2), BP levels were significantly increased in the total KO animals completely lacking RGS2 (129 ± 2 mmHg; n = 6) compared with the wild-type controls (P = 0.04). Thus, elimination of RGS2 in all tissues in the total KO group recapitulates the original phenotype of elevated BP described in Rgs2^−/− mice.^6,7,11Open in a separate windowFigure 1.Daytime and nighttime systolic BPs measured by radiotelemetry are elevated in kidney KO and total KO groups. Diurnal variation was preserved in all groups. *P = 0.04.Open in a separate windowFigure 2.Mean systolic BPs are significantly increased in the kidney KO and total KO groups compared with wild-type controls. *P = 0.04. In the systemic KO group, transplantation of a wild-type kidney into a RGS2-deficient mouse generated a normal BP.BP levels in the systemic KO group (120 ± 3 mmHg; n = 6) were not different from wild-type controls. Thus, deletion of RGS2 from all extrarenal tissues including the central nervous system and peripheral vasculature is not sufficient to cause hypertension. On the other hand, BP levels in the kidney KO group (131 ± 3.0 mmHg; n = 7) were significantly increased compared with the wild-type controls (P = 0.046) and comparable with those of the total KO group. This finding is consistent with the view that the kidney is a major determinant of the chronic level of BP and indicates that the absence of signaling pathways linked to RGS2 in the kidney and its vasculature is sufficient to increase BP. The patterns of BP differences between the groups were similar when daytime and nighttime BPs were examined separately (not shown). Furthermore, feeding a high-salt (6% NaCl) diet for 7 days did not significantly affect BP in any of the groups except the total KO group, in which an increase in BP from 131 ± 3 mmHg on the regular (0.4% NaCl) diet to 137 ± 11 mmHg on the high-salt diet was observed, which approached statistical significance (P = 0.0503).At the end of the studies, kidneys and hearts were harvested, and organ weights were determined. As shown in

A Practical Approach to Monitoring Patients on Biological Agents for the Treatment of Psoriasis

Psoriasis is a chronic, systemic, inflammatory skin condition that manifests predominantly as well-demarcated, erythematous, scaly plaques on the elbows, knees, and scalp. While mild cases (minimal body surface) often respond to various topical treatments and light therapy, patients with extensive disease (larger body surface and possibly joint involvement) may require systemic medications for remission. The development of biological agents provides dermatologists valuable ways to help treat psoriatic disease quite efficiently, but literature regarding the monitoring of patients on biological treatments is sparse. Clinical practice varies widely since there is modest strong evidence to recommend or refute most tests currently recommended by the United States Food and Drug Administration. The purpose of this article is to present a practical approach to monitoring patients on biological therapy based on the most up-to-date literature.The use of biological treatments has grown significantly since their introduction and now account for a significant proportion of the systemic therapies used for the treatment of psoriasis. Biological therapies target precise segments of the immune system, offering the advantage of being less immunosuppressive compared to the traditional systemic therapies that broadly cause immunosuppression. Currently, five biological agents (e.g., alefacept, etanercept, infliximab, adalimumab, and ustekinumab) are approved by the United States Food and Drug Administration (FDA) for the treatment of psoriasis, and other newer agents (e.g., ABT-874) are in various stages of development and clinical trials (1–6 The biologicals at present are divided into either tumor necrosis factor alpha (TNF-α) or T-cell lymphocyte inhibitors. Recently, CD4+ T helper (Th) 17 cells and interleukins (IL)-12 and IL-23 have been important in the pathogenesis of T-cell mediated disorders, such as psoriasis, and have influenced the development of medications that specifically target these key immunological players. Both IL-12 and IL-23 stimulate differentiation of naive T-cells into Th1 and Th17 cells, key cells that regulate the production of other pro-inflammatory cytokines significant in the pathogenesis of psoriasis.^7,8 Understanding of these immune cascade complexities has divulged this new class of biological agents that target cytokines (e.g., ustekinumab) important in the pathogenesis of inflammatory skin disease. Each drug class that is used in the treatment of psoriasis works by blocking different steps along the same immune-dysregulation pathway leading to psoriatic disease.

Table 1

Currently approved biological medications for the treatment of psoriasis^1–4,6

Kidney Stones Associate with Increased Risk for Myocardial Infarction

Kidney stones are a risk factor for chronic kidney disease (CKD), which, in turn, is a risk factor for myocardial infarction (MI). The objective of this study was to determine whether kidney stones associate with an increased risk for MI. We matched 4564 stone formers (1984 through 2003) on age and gender with 10,860 control subjects among residents in Olmsted County, Minnesota. We identified incident MI by diagnostic codes and validated events by chart review through 2006. We used diagnostic codes to determine incidence of kidney stones and presence of comorbidities (CKD, hypertension, diabetes, obesity, dyslipidemia, gout, alcohol dependence, and tobacco use). During a mean of 9 years of follow-up, stone formers had a 38% (95% confidence interval 7 to 77%) increased risk for MI, which remained at 31% (95% confidence interval 2% to 69%) after adjustment for CKD and other comorbidities. In conclusion, kidney stone formers are at increased risk for MI, and this risk is independent of CKD and other risk factors.Kidney stones have been identified as a risk factor for chronic kidney disease (CKD).¹ Of great concern with the development of CKD is an increased risk for coronary heart disease. Reduction in both GFR and albuminuria is associated with coronary heart disease in the general population.^2,3 It was hypothesized that the increased risk for CKD in individuals with kidney stones would lead to an increased risk for coronary heart disease. Alternatively, biological pathways that cause calcium kidney stones may also contribute to coronary artery calcification, a risk factor for coronary heart disease events. Myocardial infarction (MI) surveillance in Olmsted County, Minnesota, has been in place since 1979.^4–7 We sought to determine whether kidney stone formers in Olmsted County were at an increased risk for MI and whether this risk is related to the development of CKD.A total of 5081 incident stone formers and 14,144 matched control subjects were identified from the Olmsted County general population between 1984 and 2003. The International Classification of Diseases, Ninth Revision (ICD-9) code used to identify stone formers was 592 (calculus of kidney and ureter) in 4467 (88%), 594 (calculus of lower urinary tract) in 613 (12%), and 274.11 (uric acid nephrolithiasis) in one (<1%). Prevalent MIs were similar between stone formers (n = 178 [3.5%]) and control subjects (n = 489 [3.5%]). After exclusion of individuals with prevalent MI and those who lacked clinic visits at least 90 days after the index date, 4564 stone formers and 10,860 control subjects were followed for incident MI. Only 2.1% of these individuals were nonwhite, consistent with the racial distribution of the community (96% white in 1990). As a consequence of the matching, stone formers and control subjects were similar with respect to age (mean 44.6 versus 44.4 year), gender (59 versus 58% male), length of medical record documentation before the index date (mean 18.4 versus 21.9 years), and length of follow-up to last clinic visit or death (mean 8.7 versus 9.0 years). As shown in ComorbidityStone Formers (n = 4564; n [%])Control Subjects (n = 10,860; n [%])PCKD116 (2.5)200 (1.8)0.0051Hypertension848 (18.6)1748 (16.1)0.0002Diabetes420 (9.2)771 (7.1)<0.0001Obesity1017 (22.3)2155 (19.9)0.0007Dyslipidemia860 (18.8)1802 (16.6)0.0007Gout135 (3.0)255 (2.4)0.028Alcohol dependence235 (5.2)777 (7.2)<0.0001Tobacco use619 (13.6)1598 (14.7)0.063Open in a separate windowThere were incident MIs in 96 stone formers and 166 control subjects. Stone formers were at increased risk for MI in analyses that were unadjusted (hazards ratio [HR] 1.43; 95% confidence interval [CI] 1.11 to 1.84; Figure 1), adjusted for age and gender (HR 1.38; 95% CI 1.07 to 1.77), further adjusted for CKD (HR 1.38; 95% CI 1.07 to 1.77), and fully adjusted for all comorbidities (HR 1.31; 95% CI 1.02 to 1.69). The risk for MI remained with adjustment for the number of comorbidities (excluding alcohol dependence), age, and gender (HR 1.35; 95% CI 1.05 to 1.73). By coding subgroup, the risk for MI adjusted for age and gender was statistically significant for ICD-9 code 592 (HR 1.40; 95% CI 1.07 to 1.84) but not for ICD-9 code 594 (HR 1.24; 95% CI 0.70 to 2.21).Open in a separate windowFigure 1.Increased risk for MI in stone formers than in controls among Olmsted County, Minnesota residents.After adjustment for age and gender, most comorbidities were associated with MI: CKD (HR 2.97; 95% CI 1.86 to 4.72), hypertension (HR 1.54; 95% CI 1.18 to 2.01), diabetes (HR 2.18; 95% CI 1.61 to 2.94), obesity (HR 1.77; 95% CI 1.38 to 2.27), dyslipidemia (HR 1.65; 95% CI 1.27 to 2.15), gout (HR 1.40; 95% CI 0.88 to 2.22), alcohol dependence (HR 1.22; 95% CI 0.76 to 1.95), and tobacco use (HR 2.23; 95% CI 1.67 to 2.96). After exclusion of individuals with these comorbidities at baseline, there remained 2366 stone formers with 25 incident MIs and 5818 control subjects with 42 incident MIs, and the risk for MI with kidney stones remained elevated but not statistically significant (HR 1.50; 95% CI 0.92 to 2.47). There was no detectable interaction between these comorbidities and the risk for MI with kidney stones (P ≥ 0.10 for each comorbidity × stone former interaction).We found stone formers to be at a 38% increased risk for MI. A consideration is that this association reflects shared risk factors for both MI and kidney stones, namely, hypertension, diabetes, obesity, and dyslipidemia⁸; however, the risk for MI in stone formers remained elevated with adjustment for these and other known risk factors for MI, including CKD. This finding adds to the literature that kidney stones should be viewed as a metabolic disorder with clinical relevance beyond symptomatic urinary tract obstruction.⁹A Medline (Ovid Technologies) search of English-language human studies on February 2010 with the terms “kidney/renal stone(s)/calculi or nephrolithiasis or urolithiasis” and “MI or coronary heart/artery disease” revealed 67 articles. Among these articles, there were six relevant studies, with most having small samples sizes.^8,10–14 Several studies showed increased risk for MI in stone formers,^8,10,12,14 and other studies showed no association.^11,13 Lack of effective calcification inhibitors may be a common mechanism linking coronary artery calcification to calcium kidney stones (80% of stone formers).¹⁵ High-dosage calcium supplements may overwhelm calcification inhibitors and have been associated with an increased risk for both MI¹⁶ and kidney stones.¹⁷The study strengths include general population cohorts with validated MI end points. Although there was likely some nondifferential misclassification of comorbidities by diagnostic codes, these comorbidities did have the expected associations with kidney stones and with MI. The risk for MI may vary by stone composition just as the risk for CKD may vary by stone composition.¹⁸ Limitations include lack of information on stone burden, stone composition, diet, medications, and laboratory test results. Furthermore, because study participants were mostly non-Hispanic white individuals, a population at increased risk for kidney stones,¹⁹ inferences to other ethnic groups are limited.In conclusion, the increased risk for MI with kidney stones suggests these two diseases share a common pathophysiologic pathway. This could be a target for future intervention strategies. A history of kidney stones may also be a useful addition in risk stratification algorithms for MI. Further studies are needed to assess the relevance of stone composition and stone burden to risk for MI.     

Rituximab: A Review of Dermatological Applications

The treatment of many dermatological disorders, such as autoimmune and immune-mediated diseases, consists of the use of systemic corticosteroids alone or in combination with other steroid-sparing immunosuppressants. Often, these treatment regimens are sufficient to control disease activity with relatively few side effects if monitored by a diligent physician. Some patients, however, may be refractory to treatment or develop intolerable side effects from therapy. For these patients, alternative treatment modalities with less toxicity and greater efficacy are required. Rituximab is a genetically engineered, chimeric monoclonal antibody directed against the B-cell lineage specific CD20 antigen. Originally developed for the treatment of B-cell non-Hodgkin“s lymphoma, rituximab has increasingly been used to treat a variety of autoimmune and immune-mediated disorders, such as rheumatoid arthritis, pemphigus diseases, systemic lupus erythematosus, dermatomyositis, and idiopathic thrombocytopenic purpura to name a few. Since very few randomized, controlled, clinical trials exist regarding the use of rituximab in the treatment of dermatological disorders, guidelines for the off-label use of this medication come from anecdotal case reports and cohort studies. Further clinical studies are needed to validate the safety and efficacy of rituximab therapy in dermatological disorders. Until then, we present a literature review of the emerging use of this B-cell depletion therapy. (J Clin Aesthetic Dermatol. 2009;2(5):29–37.)Rituximab (Rituxan©, Genentech, South San Francisco, California) is a unique, chimeric, murinehuman monoclonal antibody directed against the B-lymphocyte specific antigen CD20 expressed only by pre-B (hematopoietic) and mature (peripheral) B cells.¹ CD20 is suspected to play a significant role in the regulation of cell-cycle initiation and differentiation of the B-cell lineage, evident by a rapid B-cell depletion after treatment, which can be maintained for 6 to 12 months.^2,3 Three mechanisms have been proposed for this finding, including the following: 1) complement-dependent cytotoxicity, 2) antibody-dependent cellular cytotoxicity, and 3) induction of apoptosis.^4–6 Hematopoietic stem cells and plasma cells are spared with rituximab treatment due to their lack of the CD20 antigen; thus, serum immunoglobulin levels typically remain stable.^7–9 Until recently, the primary use of rituximab has been in the induction of B-cell depletion for the treatment of B-lymphocyte malignancies, such as relapsed or refractory, low-grade or follicular, CD20-positive, B-cell non-Hodgkin“s lymphoma (NHL). Rituximab is clinically well tolerated with rare occurrences of serious adverse events, making it an appealing alternative treatment option in patients with refractory autoimmune or immune-mediated conditions (10–12

Table 1

Therapeutic targets of rituximab

Endothelin-A Receptor Antagonism Modifies Cardiovascular Risk Factors in CKD

Arterial stiffness and impaired nitric oxide (NO) bioavailability contribute to the high risk for cardiovascular disease in CKD. Both asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO production, and endothelin-1 (ET-1) oppose the actions of NO, suggesting that ET-1 receptor antagonists may have a role in cardiovascular protection in CKD. We conducted a randomized, double-blind, three-way crossover study in 27 patients with proteinuric CKD to compare the effects of the ET_A receptor antagonist sitaxentan, nifedipine, and placebo on proteinuria, BP, arterial stiffness, and various cardiovascular biomarkers. After 6 weeks of treatment, placebo and nifedipine did not affect plasma urate, ADMA, or urine ET-1/creatinine, which reflects renal ET-1 production; in contrast, sitaxentan led to statistically significant reductions in all three of these biomarkers. No treatment affected plasma ET-1. Reductions in proteinuria and BP after sitaxentan treatment was associated with increases in urine ET-1/creatinine, whereas reduction in pulse-wave velocity, a measure of arterial stiffness, was associated with a decrease in ADMA. Taken together, these data suggest that ET_A receptor antagonism may modify risk factors for cardiovascular disease in CKD.CKD is common, affecting 6%–11% of the population globally.¹ It is strongly associated with incident cardiovascular disease (CVD).² This increased cardiovascular risk is not adequately explained by conventional (Framingham) risk factors, such as hypertension, hypercholesterolemia, diabetes mellitus, and smoking, all of which are common in patients with CKD. Thus, emerging cardiovascular risk factors have been an area of intense investigation.³ Arterial stiffness⁴ makes an important independent contribution to CVD risk in CKD, and this is promoted by both conventional and emerging cardiovascular risk factors.Hyperuricemia and a shift in the balance of the vasodilator nitric oxide (NO) and vasoconstrictor endothelin (ET) systems have been identified as potential contributors to increased cardiovascular risk in patients with CKD.⁵ These are all common in a typical CKD population.^3,6 Epidemiologic studies report a relationship between serum uric acid and a wide variety of cardiovascular conditions, including hypertension, diabetes mellitus, coronary artery disease, cerebrovascular disease, and CKD.⁷ Indeed, serum uric acid is considered by some to be an independent risk factor for both CVD^8,9 and CKD.¹⁰ Others have noted that an elevated serum uric acid level predicts the development of hypertension and CKD.⁷ Of note, emerging clinical data show that decreasing serum uric acid levels has both cardiovascular and renal benefits.^11–13Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthases. By inhibiting NO formation, ADMA causes endothelial dysfunction, vasoconstriction, elevation of BP, and progression of experimental atherosclerosis.¹⁴ ADMA concentrations are increased in patients with CKD,¹⁴ and clinical data support ADMA as an independent marker of CKD progression, cardiovascular morbidity, and overall mortality.^15–17 Studies have shown a reduction in ADMA after therapy in patients with hypertension and hypercholesterolemia,^18,19 but not in patients with CKD.ET-1 is a potent endogenous vasoconstrictor produced within the vasculature. It is implicated in both the development and progression of CKD.²⁰ Its effects are mediated via two receptors, the ET_A and ET_B receptors; the major pathologic effects are ET_A receptor mediated.²⁰ We have recently shown that long-term selective ET_A receptor antagonist therapy using the orally active drug sitaxentan reduces proteinuria, BP, and arterial stiffness in patients with proteinuric CKD,²¹ effects that are potentially renoprotective. We hypothesized that in this same cohort of patients with CKD, sitaxentan would also reduce levels of serum uric acid, ADMA, and urine ET-1 (as a measure of renal ET-1 production) and so provide broader cardiovascular and renal protection. The current data show the effects of sitaxentan, as well as placebo and an active control agent, nifedipine, on these novel cardiovascular risk factors.As described elsewhere,²¹ after 6 weeks of dosing no significant differences were seen between sitaxentan and nifedipine in the reductions from baseline in BP measures. Despite this, sitaxentan reduced proteinuria to a significantly greater extent than did nifedipine. Pulse-wave velocity (PWV)—a measure of arterial stiffness—decreased to a similar degree with nifedipine as with sitaxentan. Placebo did not affect proteinuria, BP, or PWV (see VariablePlaceboSitaxentanNifedipineBaselineWeek 6BaselineWeek 6BaselineWeek 624-hr proteinuria (g/d)2.06±0.382.00±0.332.07±0.341.46±0.26^a1.95±0.301.99±0.33Protein-to-creatinine ratio (mg/mmol)155±31153±27157±28114±23^b155±27152±29Mean arterial pressure (mmHg)94.6±2.294.3±1.794.4±1.890.7±1.8^c95.5±2.091.7±1.7^cSystolic BP (mmHg)125.4±2.7124.2±1.9124.3±2.2120.7±1.9^c125.7±2.4120.7±1.6^cDiastolic BP (mmHg)77.9±1.577.5±1.277.9±1.374.3±1.3^a78. 9±1.575.7±1.2^aPWV (m/s)7.7±0.38.0±0.48.0±0.37.6±0.3^c7.9±0.37.6±0.3^cCentral augmentation index (%)20±220±220±215±2^a19±217±2Plasma ET-1 (pg/ml)3.6±0.53.7±0.63.6±0.53.7±0.53.5±0.53.5±0.5Urine ET-1 (pg/ml)4.5±0.44.7±0.45.1±0.44.2±2.1^c5.1±0.44.7±0.4Open in a separate windowValues are given as predosing baseline ± SEM.^aP<0.01 for week 6 versus baseline.^bP=0.01 for week 6 versus baseline.^cP<0.05 for week 6 versus baseline.

Table 2.

Renal substudy data from clearance studies performed at baseline and week 6 of each study period

Functional Effector Memory T Cells Enrich the Peritoneal Cavity of Patients Treated with Peritoneal Dialysis

The frequency and severity of episodes of peritonitis adversely affect the structure and function of the peritoneal membrane in patients treated with peritoneal dialysis (PD), but the underlying mechanisms are not well understood. Alterations in the phenotype and function of resident peritoneal cells may contribute. Because effector memory T cells play a pivotal role in maintaining peripheral tissue immunity, we hypothesized that these cells may initiate or perpetuate the peritoneal inflammatory response. Here, we characterized the phenotype and effector function of peritoneal memory T cells. We found that functional effector memory T cells capable of mounting long-term recall responses enrich the peritoneal cavity of PD patients. Peritoneal T cells were able to mount a Th1-polarized response to recall antigens, and these responses were greater in peritoneal T cells compared with T cells in the peripheral blood. We also observed that the peritoneal T cells had altered telomeres; some cells had ultrashort telomeres, suggesting a highly differentiated local population. In summary, we describe a resident population of memory T cells in the peritoneum of PD patients and speculate that these cells form part of the first line of defense against invading pathogens.Despite advances in treatment, peritoneal infection remains one of the main causes of technique failure in peritoneal dialysis (PD) patients. There is a strong association between peritonitis (frequency and severity) and the loss of membrane function.^1–3 In view of this, there has been considerable interest in understanding the basic processes that regulate peritoneal early responses to infection. Most of these studies have focused on the contribution of peritoneal macrophages or mesothelial cells to these processes.^4–9 Despite representing up to 25% of the resident peritoneal leukocyte population and forming a significant proportion of the leukocyte population present in resolving peritonitis, the function and phenotype of human peritoneal T cells is poorly defined.^10–12 Consequently we understand very little about the adaptive arm of the peritoneal immune responseRecent developments in the field of immunology have greatly enhanced our understanding of T cell phenotype, activation status, differentiation, and tissue homing capacity. Many studies have highlighted the important role of T cells in providing long-term immunological memory.^13–19 According to current definitions, memory T cells are distinguished from naïve T cells (which are yet to encounter their cognate antigen) by the expression of CD45RO (rather than CD45RA).²⁰ Within the memory T cell population, distinct functional subsets have been characterized based on the expression the lymph node homing signal CCR7.^13–19 These subsets differ in both their tissue homing capability and in their response to antigenic stimulation.^15,17,18 Central memory (T_CM) cells (CD45RO/CCR7⁺) are thought to migrate through lymph tissue, whereas effector memory (T_EM) cells (CD45RO /CCR7⁻), which lack lymph node homing signals, are thought to reside primarily in peripheral tissue.^16,17 The T_EM subset rapidly produces effector cytokines such as IFN-γ and IL-4 and are thought to form a first line of defense in vulnerable peripheral tissues. In contrast, T_CM cells lack immediate effector function but retain proliferative capacity and are capable of generating a secondary wave of antigen-specific effector T cells.^17–19 The memory subsets also differ in their replicative history and degree of differentiation. Within the T cell population, telomere lengths decrease from naïve through T_CM through T_EM cells, suggesting that the latter have undergone more cell divisions and are a more highly differentiated population.^17,20Most of our current understanding of T cell memory is derived from murine data or from experiments performed on peripheral blood T cells. Because of the logistical difficulties involved in sample collection, there are very little data available regarding the phenotype and function of memory T cells in human peripheral tissue. The aim of our study was to characterize the phenotype, replicative history, and effector function of the peritoneal memory T cell population during steady-state (non-infected) PD.Our results demonstrate that as compared with peripheral blood, the peritoneal cavity is enriched in cells displaying a T_EM phenotype, with very few intraperitoneal naïve T cells (Figure 1, A and B) (we found no significant difference in the proportion of T_CM cells between blood and peritoneum). Subgroup analysis shows that neither time on PD nor recurrent peritonitis have a significant effect on the proportion of T_EM within the peritoneal cavity, suggesting that T_EM enrichment is a characteristic of the quiescent peritoneal cavity (Supplementary Figure 1). Further phenotypic analysis demonstrated increased expression of the proinflammatory chemokine receptor CCR5 on the T_EM subset, but only low-level expression of the lymph node homing signal CD62L (Figure 1C).Open in a separate windowFigure 1.Paired samples of peripheral blood mononuclear cells (PBMCs) and peritoneal leukocytes were collected from PD patients. Cells were labeled with fluorescently conjugated monoclonal antibody directed against CD4/CD8/CCR7 and CD45RO. A combined gate was set on peritoneal T cells on the basis of CD4 or CD8 expression and their FSC:SSC profiles. (A) Representative example of CD4 data. (B) Summary of data obtained from paired blood and peritoneal T cells obtained from 20 patients. Differences between groups were tested using the Wilcoxon signed ranks test (*P < 0.05). (C) Peritoneal leukocytes were collected from ten separate PD patients. Cells were labeled with fluorescently conjugated monoclonal antibody directed against CD4/CCR7/CD45RO and CD62L or CCR5. A combined gate was set on peritoneal T cells on the basis of CD4 expression and their FSC:SSC profiles. Further gate was plotted on the T_EM subset and percentage CD62L⁺/CCR5⁺ within this calculated subset. See ​and22 for patient demographics.

Table 1.

Patient demographics

Adolescent Scalp Psoriasis: Update on Topical Combination Therapy

Plaque psoriasis can begin early in life and negatively affect quality of life. Topical agents are generally recommended as first-line therapy for plaque psoriasis. The synergy of a vitamin D analog and a steroid in a topical fixed-combination formulation provides more favorable effectiveness and tolerability as compared with either agent alone. The safety and effectiveness of a once-daily calcipotriene/betamethasone dipropionate topical suspension have been established in children 12 to 17 years of age with scalp plaque psoriasis. Combination topical formulations and once-daily dosing decrease regimen complexity and may increase adherence. Accommodation of vehicle preference may also improve adherence and real-life effectiveness.Psoriasis, a common dermatologic disorder affecting individuals of all ages, can begin early in life. Up to 35 percent of cases begin before 18 years of age, affecting 0.7 percent of children.1-3 This incidence doubled between 1970 and 1999 and is currently 40.8 cases per 100,000.4 The median age at diagnosis (10.6 years) has remained stable in the United States.4It is important for clinicians to appreciate the impact of psoriasis on children and teenagers. Even mild forms of psoriasis can affect childhood psychosocial functioning and quality of life (QoL).2,5-7 The scalp plaques and possible associated alopecia can be particularly troublesome during adolescence and detrimental to the individual’s sense of self during the transition to adulthood.8,9Topical therapy can be safe and effective in juvenile psoriasis. Topical treatments are a first-line option in adults, and some have been approved for use in adolescents (10-19 This article reviews the distinguishing features of psoriasis in younger patients and the considerations that enter into the choice of treatment.

TABLE 1

Approved topical corticosteroid formulations for adolescents10-14, 16-19

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.     

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.     

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.     

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.     

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%.     

Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V₂ receptor (V₂R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V₂R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus.The anti-diuretic hormone arginine-vasopressin (AVP) is the primary regulator of water reabsorption in the renal collecting duct (CD) and is critically involved in the regulation of water balance and maintenance of plasma osmolality.¹ AVP acts on the CD through the G_s protein–coupled vasopressin V₂ receptor (V₂R) to stimulate adenylyl cyclase (AC) and thus the synthesis of cAMP.² cAMP activates protein kinase A (PKA), which phosphorylates the water channel aquaporin-2 (AQP2) in its COOH-terminal tail on serine residue 256 (S256), thereby resulting in apical plasma membrane accumulation of AQP2.^3–6 In addition, cAMP-independent activation of AQP2 has been reported, which may involve V₂R action of phosphoinositide-specific phospholipase C.⁷ Other non–PKA-targeted AQP2 phosphorylation sites include S261, S264, and S269.⁸ Phosphorylation of AQP2 at S264 and S269 requires prior phosphorylation of S256.⁹ Immunohistochemistry showed that pS269-AQP2 localizes exclusively in the apical plasma membrane of the connecting tubule and CD.^8,10 AVP-stimulated AQP2 phosphorylation thus induces plasma membrane accumulation and retention of the channel.^8,10,11 Genetic defects in the V₂R or AQP2 impair AVP-induced increases in tubular water permeability and cause nephrogenic diabetes insipidus (NDI).^12–14 In comparison, less is known about the role of genetic variation of the proteins involved in signaling from V₂R to AQP2.Generation of cAMP involves the activation of ACs, of which nine different membrane-bound isoforms have been identified (AC1 to 9).¹⁵ Studies on AC isoform expression in the kidney have been almost exclusively confined to the rat, in which mRNA analyses have shown that all membrane-bound isoforms, except for AC1 and 8, are expressed.¹⁶ The same pattern was found for mRNA expression of ACs in inner medullary CD (IMCD) suspensions of rats.¹⁷Based on in situ hybridization studies and the relative expression of AC6 mRNA in CD principal cells of rats, as well as studies using small interfering RNA designed to knock down AC6 in primary cultured mouse IMCD, it has been proposed that this AC isoform may contribute to AVP-stimulated cAMP formation,^18,19 although studies in rat IMCD have indicated that AVP-promoted Ca²⁺/calmodulin-dependent cAMP accumulation involves AC3 activity.¹⁷ In these experiments, we examined mice that lack AC6 (AC6^−/−) to gain insights regarding the role of AC6 in the formation of cAMP, phosphorylation of AQP2 at S256 and S269, and urinary concentration in vivo. The results indicate an important contribution of AC6 to AVP action and urinary concentration in vivo and that AC6^−/− mice have NDI.