Peter G. Czarnecki

Blood Pressure in Early Autosomal Dominant Polycystic Kidney Disease

BACKGROUND Hypertension is common in autosomal dominant polycystic kidney disease (ADPKD) and is associated with increased total kidney volume, activation of the renin-angiotensin-aldosterone system, and progression of kidney disease. METHODS In this double-blind, placebo-controlled trial, we randomly assigned 558 hypertensive participants with ADPKD (15 to 49 years of age, with an estimated glomerular filtration rate [GFR] >60 ml per minute per 1.73 m(2) of body-surface area) to either a standard blood-pressure target (120/70 to 130/80 mm Hg) or a low blood-pressure target (95/60 to 110/75 mm Hg) and to either an angiotensin-converting-enzyme inhibitor (lisinopril) plus an angiotensin-receptor blocker (telmisartan) or lisinopril plus placebo. The primary outcome was the annual percentage change in the total kidney volume. RESULTS The annual percentage increase in total kidney volume was significantly lower in the low-blood-pressure group than in the standard-blood-pressure group (5.6% vs. 6.6%, P=0.006), without significant differences between the lisinopril-telmisartan group and the lisinopril-placebo group. The rate of change in estimated GFR was similar in the two medication groups, with a negative slope difference in the short term in the low-blood-pressure group as compared with the standard-blood-pressure group (P<0.001) and a marginally positive slope difference in the long term (P=0.05). The left-ventricular-mass index decreased more in the low-blood-pressure group than in the standard-blood-pressure group (-1.17 vs. -0.57 g per square meter per year, P<0.001); urinary albumin excretion was reduced by 3.77% with the low-pressure target and increased by 2.43% with the standard target (P<0.001). Dizziness and light-headedness were more common in the low-blood-pressure group than in the standard-blood-pressure group (80.7% vs. 69.4%, P=0.002). CONCLUSIONS In early ADPKD, the combination of lisinopril and telmisartan did not significantly alter the rate of increase in total kidney volume. As compared with standard blood-pressure control, rigorous blood-pressure control was associated with a slower increase in total kidney volume, no overall change in the estimated GFR, a greater decline in the left-ventricular-mass index, and greater reduction in urinary albumin excretion. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others; HALT-PKD [Study A] ClinicalTrials.gov number, NCT00283686.).

Defects in the IFT-B Component IFT172 Cause Jeune and Mainzer-Saldino Syndromes in Humans

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A.

Angiotensin Blockade in Late Autosomal Dominant Polycystic Kidney Disease

BACKGROUND Hypertension develops early in patients with autosomal dominant polycystic kidney disease (ADPKD) and is associated with disease progression. The renin-angiotensin-aldosterone system (RAAS) is implicated in the pathogenesis of hypertension in patients with ADPKD. Dual blockade of the RAAS may circumvent compensatory mechanisms that limit the efficacy of monotherapy with an angiotensin-converting-enzyme (ACE) inhibitor or angiotensin II-receptor blocker (ARB). METHODS In this double-blind, placebo-controlled trial, we randomly assigned 486 patients, 18 to 64 years of age, with ADPKD (estimated glomerular filtration rate [GFR], 25 to 60 ml per minute per 1.73 m(2) of body-surface area) to receive an ACE inhibitor (lisinopril) and placebo or lisinopril and an ARB (telmisartan), with the doses adjusted to achieve a blood pressure of 110/70 to 130/80 mm Hg. The composite primary outcome was the time to death, end-stage renal disease, or a 50% reduction from the baseline estimated GFR. Secondary outcomes included the rates of change in urinary aldosterone and albumin excretion, frequency of hospitalizations for any cause and for cardiovascular causes, incidence of pain, frequency of ADPKD-related symptoms, quality of life, and adverse study-medication effects. Patients were followed for 5 to 8 years. RESULTS There was no significant difference between the study groups in the incidence of the composite primary outcome (hazard ratio with lisinopril-telmisartan, 1.08; 95% confidence interval, 0.82 to 1.42). The two treatments controlled blood pressure and lowered urinary aldosterone excretion similarly. The rates of decline in the estimated GFR, urinary albumin excretion, and other secondary outcomes and adverse events, including hyperkalemia and acute kidney injury, were also similar in the two groups. CONCLUSIONS Monotherapy with an ACE inhibitor was associated with blood-pressure control in most patients with ADPKD and stage 3 chronic kidney disease. The addition of an ARB did not alter the decline in the estimated GFR. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others; HALT-PKD [Study B] ClinicalTrials.gov number, NCT01885559.).

Ciliary and centrosomal defects associated with mutation and depletion of the Meckel syndrome genes MKS1 and MKS3

Meckel syndrome (MKS) is a lethal disorder characterized by renal cystic dysplasia, encephalocele, polydactyly and biliary dysgenesis. It is highly genetically heterogeneous with nine different genes implicated in this disorder. MKS is thought to be a ciliopathy because of the range of phenotypes and localization of some of the implicated proteins. However, limited data are available about the phenotypes associated with MKS1 and MKS3, and the published ciliary data are conflicting. Analysis of the wpk rat model of MKS3 revealed functional defects of the connecting cilium in the eye that resulted in lack of formation of the outer segment, whereas infertile wpk males developed spermatids with very short flagella that did not extend beyond the cell body. In wpk renal collecting duct cysts, cilia were generally longer than normal, with additional evidence of cells with multiple primary cilia and centrosome over-duplication. Kidney tissue and cells from MKS1 and MKS3 patients showed defects in centrosome and cilia number, including multi-ciliated respiratory-like epithelia, and longer cilia. Stable shRNA knockdown of Mks1 and Mks3 in IMCD3 cells induced multi-ciliated and multi-centrosomal phenotypes. These studies demonstrate that MKS1 and MKS3 are ciliopathies, with new cilia-related eye and sperm phenotypes defined. MKS1 and MKS3 functions are required for ciliary structure and function, including a role in regulating length and appropriate number through modulating centrosome duplication.

The ciliary transition zone: from morphology and molecules to medicine.

Researchers from various disciplines, including cell and developmental biology, genetics and molecular medicine, have revealed an exceptional diversity of cellular functions that are mediated by cilia-dependent mechanisms. Recent studies have directed our attention to proteins that localize to the ciliary transition zone (TZ), a small evolutionarily conserved subcompartment that is situated between the basal body (BB) and the more distal ciliary axoneme. These reports shed light on the roles of TZ proteins in ciliogenesis, ciliary protein homeostasis and specification of ciliary signaling, and pave the way for understanding their contribution to human ciliopathies. In this review, we describe the interplay of multimeric protein complexes at the TZ, integrating morphological, genetic and proteomic data towards an account of TZ function in ciliary physiology.

B9D1 is revealed as a novel Meckel syndrome (MKS) gene by targeted exon-enriched next-generation sequencing and deletion analysis

Meckel syndrome (MKS) is an embryonic lethal, autosomal recessive disorder characterized by polycystic kidney disease, central nervous system defects, polydactyly and liver fibrosis. This disorder is thought to be associated with defects in primary cilia; therefore, it is classed as a ciliopathy. To date, six genes have been commonly associated with MKS (MKS1, TMEM67, TMEM216, CEP290, CC2D2A and RPGRIP1L). However, mutation screening of these genes revealed two mutated alleles in only just over half of our MKS cohort (46 families), suggesting an even greater level of genetic heterogeneity. To explore the full genetic complexity of MKS, we performed exon-enriched next-generation sequencing of 31 ciliopathy genes in 12 MKS pedigrees using RainDance microdroplet-PCR enrichment and IlluminaGAIIx next-generation sequencing. In family M456, we detected a splice-donor site change in a novel MKS gene, B9D1. The B9D1 protein is structurally similar to MKS1 and has been shown to be of importance for ciliogenesis in Caenorhabditis elegans. Reverse transcriptase-PCR analysis of fetal RNA revealed, hemizygously, a single smaller mRNA product with a frameshifting exclusion of B9D1 exon 4. ArrayCGH showed that the second mutation was a 1.713 Mb de novo deletion completely deleting the B9D1 allele. Immunofluorescence analysis highlighted a significantly lower level of ciliated patient cells compared to controls, confirming a role for B9D1 in ciliogenesis. The fetus inherited an additional likely pathogenic novel missense change to a second MKS gene, CEP290; p.R2210C, suggesting oligogenic inheritance in this disorder.

Genetic and physical interaction between the NPHP5 and NPHP6 gene products

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.

Long-term outcome of kidney transplantation in patients with fibrillary glomerulonephritis or monoclonal gammopathy with fibrillary deposits.

To determine the outcome of kidney transplantation in patients with fibrillary glomerulonephritis (FGN), a rare glomerular disease, we followed 12 patients, 5 with FGN and 7 patients with monoclonal gammopathy and fibrillary deposits (MGFD), who underwent 15 kidney transplants since 1988 with a median follow-up of 52 months. Recurrent disease did not arise in any of the patients with FGN but developed in 5 patients with MGFD. Seven allografts failed: 1 in the FGN group and 6 in the MGFD group. Median allograft survival for patients with MGFD was 37 months but had not been reached in FGN patients. One patient with FGN had primary allograft failure secondary to graft thromboembolism. Three patients with MGFD were re-transplanted and one lost the second allograft to recurrent disease, but the other two died from hematological malignancy. Another patient was diagnosed with MPGN type III and did not have detectable fibrillary material 22 months after transplantation. One patient with MGFD had stable allograft function 6 months post-transplant but another, with recurrent disease, underwent peripheral blood stem cell transplantation and regained stable allograft function. Our study shows that kidney transplantation appears safe in patients with FGN with little risk of recurrence. However, patients with MGFD have a significant risk for disease recurrence. Whether the development of hematological malignancies following transplantation in this group is related to their original disease or was coincidental requires further studies.

The Meckel syndrome protein meckelin (TMEM67) is a key regulator of cilia function but is not required for tissue planar polarity.

Meckel syndrome (MKS) is a lethal disorder associated with renal cystic disease, encephalocele, ductal plate malformation and polydactyly. MKS is genetically heterogeneous and part of a growing list of syndromes called ciliopathies, disorders resulting from defective cilia. TMEM67 mutation (MKS3) is a major cause of MKS and the related ciliopathy Joubert syndrome, although the complete etiology of the disease is not well understood. To further investigate MKS3, we analyzed phenotypes in the Tmem67 null mouse (bpck) and in zebrafish tmem67 morphants. Phenotypes similar to those in human MKS and other ciliopathy models were observed, with additional eye, skeletal and inner ear abnormalities characterized in the bpck mouse. The observed disorganized stereociliary bundles in the bpck inner ear and the convergent extension defects in zebrafish morphants are similar to those found in planar cell polarity (PCP) mutants, a pathway suggested to be defective in ciliopathies. However, analysis of classical vertebrate PCP readouts in the bpck mouse and ciliary organization analysis in tmem67 morphants did not support a global loss of planar polarity. Canonical Wnt signaling was upregulated in cyst linings and isolated fibroblasts from the bpck mouse, but was unchanged in the retina and cochlea tissue, suggesting that increased Wnt signaling may only be linked to MKS3 phenotypes associated with elevated proliferation. Together, these data suggest that defective cilia loading, but not a global loss of ciliogenesis, basal body docking or PCP signaling leads to dysfunctional cilia in MKS3 tissues.

ANKS6 is the critical activator of NEK8 kinase in embryonic situs determination and organ patterning

The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6Streaker and Nek8Roc. Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6Strkr mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8Roc mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.