Cilia and polycystic kidney disease.

Polycystic kidney disease (PKD), comprising autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD), is characterized by incessant cyst formation in the kidney and liver. ADPKD and ARPKD represent the leading genetic causes of renal disease in adults and children, respectively. ADPKD is caused by mutations in PKD1 encoding polycystin1 (PC1) and PKD2 encoding polycystin 2 (PC2). PC1/2 are multi-pass transmembrane proteins that form a complex localized in the primary cilium. Predominant ARPKD cases are caused by mutations in polycystic kidney and hepatic disease 1 (PKHD1) gene that encodes the Fibrocystin/Polyductin (FPC) protein, whereas a small subset of cases are caused by mutations in DAZ interacting zinc finger protein 1 like (DZIP1L) gene. FPC is a type I transmembrane protein, localizing to the cilium and basal body, in addition to other compartments, and DZIP1L encodes a transition zone/basal body protein. Apparently, PC1/2 and FPC are signaling molecules, while the mechanism that cilia employ to govern renal tubule morphology and prevent cyst formation is unclear. Nonetheless, recent genetic and biochemical studies offer a glimpse of putative physiological malfunctions and the pathomechanisms underlying both disease entities. In this review, I summarize the results of genetic studies that deduced the function of PC1/2 on cilia and of cilia themselves in cyst formation in ADPKD, and I discuss studies regarding regulation of polycystin biogenesis and cilia trafficking. I also summarize the synergistic genetic interactions between Pkd1 and Pkhd1, and the unique tissue patterning event controlled by FPC, but not PC1. Interestingly, while DZIP1L mutations generate compromised PC1/2 cilia expression, FPC deficiency does not affect PC1/2 biogenesis and ciliary localization, indicating that divergent mechanisms could lead to cyst formation in ARPKD. I conclude by outlining promising areas for future PKD research and highlight rationales for potential therapeutic interventions for PKD treatment.

Renal Reabsorption of Folates: Pharmacological and Toxicological Snapshots.

Folates are water-soluble B9 vitamins that serve as one-carbon donors in the de novo synthesis of thymidylate and purines, and in the conversion of homocysteine to methionine. Due to their key roles in nucleic acid synthesis and in DNA methylation, inhibiting the folate pathway is still one of the most efficient approaches for the treatment of several tumors. Methotrexate and pemetrexed are the most prescribed antifolates and are mainly used in the treatment of acute myeloid leukemia, osteosarcoma, and lung cancers. Normal levels of folates in the blood are maintained not only by proper dietary intake and intestinal absorption, but also by an efficient renal reabsorption that seems to be primarily mediated by the glycosylphosphatidylinositol- (GPI) anchored protein folate receptor α (FRα), which is highly expressed at the brush-border membrane of proximal tubule cells. Folate deficiency due to malnutrition, impaired intestinal absorption or increased urinary elimination is associated with severe hematological and neurological deficits. This review describes the role of the kidneys in folate homeostasis, the molecular basis of folate handling by the kidneys, and the use of high dose folic acid as a model of acute kidney injury. Finally, we provide an overview on the development of folate-based compounds and their possible therapeutic potential and toxicological ramifications.

Polycystin-1, the product of the polycystic kidney disease gene PKD1, is post-translationally modified by palmitoylation.

Multiple distinct mutations in the protein polycystin 1 (PC1) cause autosomal dominant polycystic kidney disease (ADPKD), a common cause of end stage renal disease. Growing evidence supports the theory that the severity and rate of progression of kidney cysts is correlated with the level of functional PC1 expressed in the primary cilia. Factors that regulate trafficking of PC1 to cilia are thus of great interest both as potential causes of ADPKD, but also as possible modifiable factors to treat ADPKD. Cysteine palmitoylation is a common post-translational modification that frequently alters protein trafficking, localization, and expression levels. Here, using multiple complementary approaches, we show that PC1 is palmitoylated, likely at a single cysteine in the carboxyl terminal fragment that is generated by autoproteolysis of PC1. Additional data suggest that protein palmitoylation is important for PC1 localization and expression levels. These data together identify palmitoylation as a novel post-translational modification of PC1 and a possible pharmacologic target to augment PC1 expression in cilia.

Evidence for Bone and Mineral Metabolism Alterations in Children With Autosomal Dominant Polycystic Kidney Disease.

Context: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. Hypophosphatemia was demonstrated in adult patients with preserved renal function, together with high fibroblast growth factor 23 (FGF23) and low soluble Klotho levels. The latter explained the relative FGF23 hyporesponsiveness in this cohort. Objective: Evaluating phosphate and bone mineral metabolism in children with ADPKD compared with what is known in adult ADPKD patients. Design: Observational cross-sectional study. Setting: Multicenter study via ambulatory care in tertiary centers. Participants: Ninety-two children with ADPKD (52 males; mean ± standard deviation age, 10.2 ± 5.0 years) and 22 healthy controls (HCs, 10 males; mean ± standard deviation age, 10.3 ± 4.1 years). Main Outcome Measures: The predictor was early ADPKD stage. Bone mineral metabolism and renal phosphate handling were the main outcome measures. Performed measurements were serum phosphate, tubular maximum phosphorus reabsorption per glomerular filtration rate, FGF23, soluble Klotho, sclerostin, and bone alkaline phosphatase. Results: ADPKD children had significantly lower serum phosphate levels compared with HC. Low tubular maximum phosphorus reabsorption per glomerular filtration rate was observed in 24% of patients, although not significantly different from HC. Serum FGF23 and soluble Klotho levels were comparable between patients and HC. In addition, we showed decreased bone alkaline phosphatase levels in ADPKD children, suggesting suppressed bone formation. Conclusions: This report demonstrates hypophosphatemia and suppressed bone formation in a pediatric ADPKD cohort, with preserved renal function, compared with HC. Although FGF23 levels were not different from controls, they should be considered inappropriate, given the concomitant hypophosphatemia. Further studies are required to elucidate underlying pathophysiology and potential clinical consequences.

Targeted rescue of a polycystic kidney disease mutation by lysosomal inhibition.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal disease. The molecular pathogenesis of ADPKD is not completely known, and there is no approved therapy. To date, there is limited knowledge concerning the molecular consequences of specific disease-causing mutations. Here we show that the ADPKD missense variant TRPP2(D511V) greatly reduces TRPP2 protein stability, and that TRPP2(D511V) function can be rescued in vivo by small molecules targeting the TRPP2 degradation pathway. Expression of the TRPP2(D511V) protein was significantly reduced compared to wild-type TRPP2. Inhibition of lysosomal degradation of TRPP2(D511V) by the US Food and Drug Administration (FDA)-approved drug chloroquine strongly increased TRPP2 protein levels in vitro. The validation of these results in vivo requires appropriate animal models. However, there are currently no mouse models harboring human PKD2 missense mutations, and screening for chemical rescue of patient mutations in rodent models is time-consuming and expensive. Therefore, we developed a Drosophila melanogaster model expressing the ortholog of TRPP2(D511V) to test chemical rescue of mutant TRPP2 in vivo. Notably, chloroquine was sufficient to improve the phenotype of flies expressing mutant TRPP2. Thus, this proof-of-concept study highlights the potential of directed therapeutic approaches for ADPKD, and provides a rapid-throughput experimental model to screen PKD2 patient mutations and small molecules in vivo.

New treatments for autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and results from mutations in PKD1 or PKD2. Cyst initiation and expansion arise from a combination of abnormal cell proliferation, fluid secretion and extracellular matrix defects and results in kidney enlargement and interstitial fibrosis. Since its first description over 200 years ago, ADPKD has been considered an untreatable condition and its management is limited to blood pressure reduction and symptomatic treatment of disease complications. Results of the recently reported TEMPO 3/4 trial thus represent a paradigm shift in demonstrating for the first time that cystic disease and loss of renal function can be slowed in humans. In this paper, we review the major therapeutic strategies currently being explored in ADPKD including a range of novel approaches in preclinical models. It is anticipated that the clinical management of ADPKD will undergo a revolution in the next decade with the translation of new treatments into routine clinical use.

Colchicine treatment in autosomal dominant polycystic kidney disease: many points in common.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common of the inherited renal cystic diseases and constitutes 10% of the end stage kidney disease population. ADPKD is caused by PKD1 and PKD2 gene mutations in 85% and 15% of the cases respectively. Its high prevalence and negative impact on health outcomes fostered efforts to explain pathophysiologic pathways of cyst formation in kidneys. Among these are increased apoptosis, unopposed proliferation of tubule cells, impaired polarization and planar cell polarity, impaired cAMP pathway, cilier dysfunction, activated mTOR pathway, increased tumor necrosis factor-alpha (TNF-alpha) production. Many drugs have been tried in an attempt to halt cystogenesis in some point. Despite success to some extent in experimental studies, none reached clinical armamentarium yet. Colchicine, originally extracted from Colchicum autunale, is an anti-inflammatory drug that has been in continuous use for more than 3000 years. It has been used successfully to prevent attacks of familial mediterranien fever and amyloidosis, to treat gout and pseudogout attacks for a few decades. Colchicine principally is a microtubule inhibitor, thus prevents cell migration, division, and polarization. It also has anti-apoptotic, anti-proliferative and anti-inflammatory effects and down-regulates (TNF-alpha) receptors. As can easily be seen, many of the effects of colchicine have pathophysiologic counterparts in ADPKD. Thus, we hypothesized that colchicine would be beneficial to prevent or at least delay cyst formation in ADPKD patients. Indirect evidence also support our hypothesis, in which taxol and paclitaxel, other two microtubule inhibitors, were shown to delay cyst formation in experimental models of ADPKD. To our opinion, despite its narrow therapeutic index, widespread experience makes colchicine a suitable candidate for prolonged clinical use, should experimental studies show any benefit in ADPKD.

Role of vasopressin antagonists.

Alterations in intracellular calcium homeostasis and cyclic adenosine 3',5'-phosphate likely underlie the increased cell proliferation and fluid secretion in polycystic kidney disease. Hormone receptors that affect cyclic adenosine 3',5'-phosphate and are preferentially expressed in affected tissues are logical treatment targets. There is a sound rationale for considering the arginine vasopressin V2 receptor as a target. The arginine vasopressin V2 receptor antagonists OPC-31260 and tolvaptan inhibit the development of polycystic kidney disease in cpk mice and in three animal orthologs to human autosomal recessive polycystic kidney disease (PCK rat), autosomal dominant polycystic kidney disease (Pkd2-/WS25 mice), and nephronophthisis(pcy mouse). PCK rats that are homozygous for an arginine vasopressin mutation and lack circulating vasopressin are markedly protected. Administration of V2 receptor agonist 1-deamino-8-D-arginine vasopressin to these animals completely recovers the cystic phenotype. Administration of 1-deamino-8-D-arginine vasopressin to PCK rats with normal arginine vasopressin aggravates the disease. Suppression of arginine vasopressin release by high water intake is protective. V2 receptor antagonists may have additional beneficial effects on hypertension and chronic kidney disease progression. A number of clinical studies in polycystic kidney disease have been performed or are currently active. The results of phase 2 and 2-3 studies indicate that tolvaptan seems to be safe and well tolerated in autosomal dominant polycystic kidney disease. A phase 3,placebo-controlled, double-blind study in 18- to 50-yr-old patients with autosomal dominant polycystic kidney disease and preserved renal function but relatively rapid progression, as indicated by a total kidney volume >750 ml, has been initiated.

Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl channel activity in Xenopus oocytes.

Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl(-) channel activity in Xenopus oocytes. Background. Cyst expansion in autosomal-dominant polycystic kidney disease (ADPKD) is characterized by active Cl(-) secretion in excess of solute reabsorption. However, the connections between elevated epithelial Cl(-) secretion and loss-of-function or dysregulation of either ADPKD gene polycystin-1 (PC1) or polycystin-2 (PC2) remain little understood. Methods. Cl(-) transport in Xenopus oocytes expressing the CD16.7-PKD1 (115-226) fusion protein containing the final 112 amino acid (aa) of the PC1 C-terminal cytoplasmic tail, or in oocytes expressing related PC1 fusion protein mutants, was studied by isotopic flux, two-electrode voltage clamp, and outside-out patch clamp recording. Results. Expression in oocytes of CD16.7-PKD1 (115-226) increased rates of both influx and efflux of (36)Cl(-), whereas CD16.7-PKD1 (1-92) containing the initial 92 aa of the PC1 C-terminal cytoplasmic tail was inactive. The increased Cl(-) transport resembled CD16.7-PKD1 (115-226)-stimulated cation current in its sensitivity to ADPKD-associated missense mutations, to mutations in phosphorylation sites, and to mutations within or encroaching upon the PC1 coiled-coil domain, as well as in its partial suppression by coexpressed PC2. The NS3623- and 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive (36)Cl(-) flux was not blocked by injected ethyleneglycol tetraacetate (EGTA) or by the cation channel inhibitor SKF96365, and was stimulated by the cation channel inhibitor La(3+), suggesting that CD16.7-PKD1 (115-226)-associated cation conductance was not required for (36)CI(-) flux activation. Outside-out patches from oocytes expressing CD16.7-PKD1 (115-226) also exhibited increased NS3623-sensitive Cl(-) current. Conclusion. These data show that CD16.7-PKD1 (115-226) activates Cl(-) channels in the Xenopus oocyte plasma membrane in parallel with, but not secondary to, activation of Ca(2+)-permeable cation channels.

Matrilysin (MMP-7) expression in renal tubular damage: association with Wnt4.

BACKGROUND: Matrilysin, a secreted matrix metalloproteinase and target gene of Wnt signaling, functions in epithelial repair and host defense, but no role in renal injury has been described. METHODS: Matrilysin expression was assessed in human kidney specimens by immunohistochemistry, and in experimental renal injury in mice by immunohistochemistry, Northern blotting, and RNase protection assays (RPA). A relationship to Wnt4, which is also induced in renal injury, was determined by RPA and in situ hybridization. RESULTS: Matrilysin was not detected in the normal human renal tubular epithelium by immunohistochemistry. However, prominent staining was detected in sections from autosomal-dominant polycystic kidney disease in the cyst lining epithelium, atrophic tubules, and cyst micropolyps, and from hydronephrosis in dilated and atrophic tubules. Matrilysin expression was also induced by acute folic acid nephropathy and unilateral ureteral obstruction (UUO) in the mouse, and expression increased as acute injury progressed to tubulointerstitial fibrosis. Matrilysin staining was primarily localized to epithelium of distal tubule/collecting duct origin in both human and murine renal disease. Wnt signaling can induce matrilysin expression, and we found that the pattern of matrilysin expression during progression of renal fibrosis in the mouse after UUO or folic acid nephropathy, and in the jck model of murine polycystic kidney disease, closely paralleled that of Wnt4. CONCLUSION: These observations suggest that matrilysin may have a role in renal tubular injury and progression of tubulointerstitial fibrosis, and that Wnt4 may regulate matrilysin expression in the kidney.

[Effect of a modified low protein and low fat diet on histologic changes and metabolism in kidneys in an experimental model of polycystic kidney disease]. / Uticaj smanjenog sadrzaja i promenjenog sastava belancevina i masti u ishrani na histoloske promene i metabolizam bubrega u eksperimentalnom modelu policisticne bolesti bubrega.

BACKGROUND: Dietary protein restriction slows progression in numerous animal models of renal diseases. Flax seed has also demonstrated useful anti-inflammatory properties in a number of animal models and human diseases. We undertook several studies to determine if feeding with low protein casein, soy diet and flax seed diet would ameliorate renal injury in Han:SPRD-cy rat model of polycystic kidney disease. METHODS: Male offspring of Han:SPRD-cy heterozygotes received protein modified diet: ad libidum LP 8% casein in test or 20% casein in control group for 8 weeks; 20% heat treated soy protein or 20% casein in control group two separate studies for 8 weeks ad libidum and pair feeding in 6 weeks; and 10% flax seed diet or control rat chow for 8 weeks from weaning. Tissue was harvested for histological assessment and metabolic changes in lipids, citric acid metabolites and osmolytes. Morphometrically after histochemical and immunohistochemical staining cystic changes, renal tubular proliferation and apoptosis, number of interstitial cells/macrophages infiltration and interstitial fibrosis were measured. Gas chromatography was used for lipid analysis in renal and liver tissue. 1-HNMR spectroscopy was used for urine and tissue organic anion and osmolytes content analysis. RESULTS IN PROTEIN MODIFIED DIET: Casein low protein as well as soy protein fed animals demonstrated reduced PKD pathology: significant reduction in cystic changes, interstitial inflammation and fibrosis and also reduction in tubular cells proliferation and apoptosis. Pair feeding protocol in second soy diet study confirmed that significant effect on renal histology was not because of protein deprivation and growth retardation. 1-H NMR spectroscopy revealed that progression of chronic renal failure in Han:SPRD-cy rat PKD is associated with renal depletion of citric acid cycle metabolite and betaine. Amelioration of PKD by soy protein diet is associated with renal retention of citric acid cycle anions, despite increased excretion and preservation of betaine in renal tissue. Soy feeding increased both hepatic and renal content of linoleic acid and increased renal alpha linolenic acid content, while decreased arachidonic hepatic content. RESULTS IN FLAX SEED SUPPLEMENTATION IN DIET: Flax seed fed animals had moderate decrease in cystic size and less interstitial inflammation and fibrosis while there were no differences in epithelial cell apoptosis and proliferation. Lipid analysis revealed significant renal enrichment of 18 and 20 carbon omega 3 polyunsaturated fatty acids. In flax fed animals there was an increased urinary citrate excretion without significant changes in urinary ammonia excretion, so increased citrate excretion was not due to alkaline effect of the diet. Kidney tissue 1H NMR spectroscopy revealed that disease amelioration was associated with tissue retention of succinate and betaine. CONCLUSION: Effect on histology: Low casein and soy feeding ameliorates Han: SPRD-cy rat polycystic kidney disease reducing both tubular remodeling and interstitial inflammation and fibrosis, while flax seed diet effect appears to be through moderation of associated interstitial nephritis. Metabolic effect: Soy diet alters the renal content of polyunsaturated fatty acids and enriched renal betaine content with retention of citric acid cycle metabolites despite increased excretion. Flax seed diet alters renal content of polyunsaturated fatty acids and promotes the formation of less inflammatory classes of renal prostanoides. Flax seed diet also enriched renal content of betaine and succinate. Amelioration of Hans:SPRD-cy rat polycystic kidney disease by diet is associated with alteration in the handling of citric acid cycle metabolites and betaine, and also in content of polyunsaturated fatty acids in kidneys and liver. Metabolic pathways in dietary modified renal pathology have to be established.

Renal concentration of alpha-tocopherol: dependence on gender and lack of effect on polycystic kidney disease in Han:SPRD rats.

A gender-associated dimorphism, with males being more severely affected than females, has been observed in autosomal dominant polycystic kidney disease, acquired renal cystic disease, and the renal cystic disease of the Han:SPRD rat. A recent study has suggested that gonadal hormones may be responsible for this dimorphism. Because gonadal hormones have an effect on the concentration of alpha-tocopherol in the liver and adrenal glands and because recent studies indicate that oxidative stress may be important in the pathogenesis of polycystic kidney disease, we wanted to determine whether the renal concentration of alpha-tocopherol is higher in female than in male rats and whether this difference accounts for the gender dimorphism of polycystic kidney disease in Han:SPRD rats. At 3 weeks of age, male and female heterozygous cystic (cy/+) rats were divided into three groups fed a vitamin E-deficient diet or the same diet supplemented with either 65 IU or 10,000 IU alpha-tocopherol/kg laboratory chow. At 8 weeks of age, blood samples and kidneys were obtained for determinations of plasma creatinine and urea, renal concentration of alpha-tocopherol and glutathione, kidney weights, and histomorphometric analysis. Female rats had higher renal concentrations of alpha-tocopherol and less severe renal cystic disease, as reflected by plasma creatinine and urea values, kidney weight corrected by body weight, and histomorphometric analysis, than male rats. The difference in renal alpha-tocopherol concentration, however, could not account for the different severity of the renal cystic disease, because depletion or enrichment of vitamin E in the diet had marked effects on the renal concentration of alpha-tocopherol without affecting the severity of the renal cystic disease. Cy/+ rats had higher renal concentrations of alpha-tocopherol than +/+ animals, possibly reflecting a disturbance of redox metabolism associated with polycystic kidney disease. Renal concentrations of glutathione were unaffected by the vitamin E content of the diet. Although these results do not support the use of vitamin E in the treatment of polycystic kidney disease, observations in the Han:SPRD rat may or may not be relevant to human polycystic kidney disease.

The effect of dietary flaxseed supplementation on organic anion and osmolyte content and excretion in rat polycystic kidney disease.

Progression of chronic renal failure in the Han:SPRD-cy rat polycystic kidney disease is associated with renal depletion of citric acid cycle metabolites and betaine. Amelioration of this disease by a soy protein diet is associated with retention of citric acid cycle anions, despite increased excretion, and preservation of tissue levels of betaine. As we have recently found that modest dietary supplementation with flaxseed preserves renal function and reduces histologic injury in the Han:SPRD-cy rat, we undertook a high-resolution 1H NMR spectroscopic study of urine and renal tissue extracts from Han:SPRD-cy rats to explore the renal biochemical consequences of a flaxseed diet. There was no significant dietary effect upon organic anion, methylamine, or osmolyte excretion in healthy animals. There was increased citrate excretion in Han:SPRD-cy rats fed flaxseed. Urinary ammonium excretion did not differ, suggesting that the observed increase in citrate excretion was not due to an alkaline effect of diet. Tissue extract studies revealed that disease amelioration was associated with tissue retention of succinate and betaine. Amelioration of Han:SPRD-cy rat polycystic kidney disease by diet is associated with alteration in the handling of citric acid cycle metabolites. Betaine may have a metabolic role in the reduction of chronic renal injury.