Triptolide is a traditional Chinese medicine-derived inhibitor of polycystic kidney disease

During kidney organogenesis, tubular epithelial cells proliferate until a functional tubule is formed as sensed by cilia bending in response to fluid flow. This flow-induced ciliary mechanosensation opens the calcium (Ca(2+)) channel polycystin-2 (PC2), resulting in a calcium flux-mediated cell cycle arrest. Loss or mutation of either PC2 or its regulatory protein polycystin-1 (PC1) results in autosomal dominant polycystic kidney disease (ADPKD), characterized by cyst formation and growth and often leading to renal failure and death. Here we show that triptolide, the active diterpene in the traditional Chinese medicine Lei Gong Teng, induces Ca(2+) release by a PC2-dependent mechanism. Furthermore, in a murine model of ADPKD, triptolide arrests cellular proliferation and attenuates overall cyst formation by restoring Ca(2+) signaling in these cells. We anticipate that small molecule induction of PC2-dependent calcium release is likely to be a valid therapeutic strategy for ADPKD.     

Kidney-specific inactivation of the KIF3A subunit of kinesin-II inhibits renal ciliogenesis and produces polycystic kidney disease

Polycystic kidney disease (PKD) is the most common genetic cause of renal failure in humans. Several proteins that are encoded by genes associated with PKD have recently been identified in primary cilia in renal tubular epithelia. These findings have suggested that abnormalities in cilia formation and function may play a role in the pathogenesis of PKD. To directly determine whether cilia are essential to maintain tubular integrity, we conditionally inactivated KIF3A, a subunit of kinesin-II that is essential for cilia formation, in renal epithelia. Constitutive inactivation of KIF3A produces abnormalities of left-right axis determination and embryonic lethality. Here we show that tissue-specific inactivation of KIF3A in renal tubular epithelial cells results in viable offspring with normal-appearing kidneys at birth. Cysts begin to develop in the kidney at postnatal day 5 and cause renal failure by postnatal day 21. The cyst epithelial cells lack primary cilia and exhibit increased proliferation and apoptosis, apical mislocalization of the epidermal growth factor receptor, increased expression of beta-catenin and c-Myc, and inhibition of p21(CIP1). These results demonstrate that the absence of renal cilia produces both the clinical and cell biological findings associated with PKD. Most generally, the phenotype of Kif3a mutant mice suggests a role for primary cilia in the maintenance of lumen-forming epithelial differentiation.     

Down-expression of Foxj1 on airway epithelium with impaired cilia architecture in non-cystic fibrosis bronchiectasis implies disease severity

Introduction

The pathogenesis of non-cystic fibrosis bronchiectasis has not been clearly clarified. This study aimed to investigate the expression of ciliary regulating protein forkhead box protein j1 (Foxj1) on airway epithelium in non-cystic fibrosis bronchiectasis and its association with airway cilia structure disorder and disease severity.

Methods

Lung tissue sections excised from 47 patients with non-cystic fibrosis bronchiectasis were included between January 2018 and June 2021. Specimens from 26 subjects who underwent a lobectomy due to lung nodule were chosen as controls. Clinical information was collected, and pathologic analysis was performed to assess the epithelial structure and expression of ciliary regulating Foxj1.

Results

Of the 47 patients with non-cystic fibrosis bronchiectasis, 25 were considered as mild, 12 were moderate whereas the remaining 10 cases were severe according to the bronchiectasis severity index score evaluation. Epithelial hyperplasia, hyperplasia of goblet cells and inflammatory cell infiltration were observed in non-cystic fibrosis bronchiectasis, compared with control subjects. Cilia length in non-cystic fibrosis bronchiectasis patients were shorter than that in the control group, (5.34 ± 0.89) μm versus (7.34 ± 0.71) μm, respectively (P = 0.002). The expression of Foxj1 was (2.69 ± 1.09) × 10⁶ in non-cystic fibrosis bronchiectasis, compared with (6.67 ± 1.15) × 10⁶ in the control group (P = 0.001). Moreover, patients with lower expression of Foxj1 showed shorter airway cilia and worse in disease severity.

Conclusion

Foxj1 declined in the airway epithelium of patients with non-cystic fibrosis bronchiectasis, positively correlated to cilia length and might imply worse disease severity.     

Cystic kidney diseases

Cystic kidney diseases are clinically and genetically heterogeneous. The most important entities are autosomal-dominant and autosomal-recessive polycystic kidney diseases. The proteins encoded by the involved genes are referred to as cystoproteins, which are located predominantly in the primary cilia. Primary cilia play an important role in cyst formation. Inherited polycystic kidney diseases belong to the increasing number of reported ciliopathies, including several syndromic entities. An exact diagnosis is the basis for medical care and genetic counselling; thus, the diagnostic algorithm should include clinical, ultrasonographic and morphological features of the underlying kidney disease, knowledge about further features and family history. Molecular genetic testing may contribute important information towards a definite diagnosis.     

Zystennieren

Cystic kidney diseases are clinically and genetically heterogeneous. The most important entities are autosomal-dominant and autosomal-recessive polycystic kidney diseases. The proteins encoded by the involved genes are referred to as cystoproteins, which are located predominantly in the primary cilia. Primary cilia play an important role in cyst formation. Inherited polycystic kidney diseases belong to the increasing number of reported ciliopathies, including several syndromic entities. An exact diagnosis is the basis for medical care and genetic counselling; thus, the diagnostic algorithm should include clinical, ultrasonographic and morphological features of the underlying kidney disease, knowledge about further features and family history. Molecular genetic testing may contribute important information towards a definite diagnosis.     

Stimulation of ciliary activity by indomethacin in rabbit tracheal epithelial organ culture

Inflammatory mediators influence mucociliary transport function in the lung, which may be dependent on the beating of the underlying cilia. To determine whether a nonsteroidal anti-inflammatory drug affects this function, and, if so, what is the mechanism action, we examined the effect of indomethacin on ciliary beat frequency (CBF) of cultured rabbit tracheal epithelial cells by a photoelectric technique. Addition of indomethacin dose-dependently enhanced CBF without any ciliary discoordination, the maximal increase and Km being 26.5 +/- 4.8% (mean +/- SE, p less than 0.001) and 6.8 X 10(-7) M, respectively. Pretreatment of tissues with nordihydroguaretic acid (10(-5) M), an inhibitor of the lipoxygenase pathway, did not change the baseline CBF, but inhibited the response of CBF to 3 X 10(-6) M indomethacin (p less than 0.01). These results suggest that indomethacin may accelerate mucociliary clearance through the augmentation of ciliary motility and that this ciliostimulation may be mediated by leukotrienes produced by activation of the lipoxygenase pathway of arachidonic acid metabolism.     

The dyskinetic cilia syndrome. Ciliary motility in immotile cilia syndrome

Ciliary motility was studied in three patients with Kartagener syndrome who had previously been found to have absent nasal and pulmonary mucociliary transport and missing dynein arms in nasal cilia. A video system was used to record movement of cilia obtained by nasal brushings for analysis of wave form and beat frequency. Two patterns of abnormal ciliary beat were observed; an oscillating and a rotating type of motion. There was no evidence of planar coordination of metachronicity. This abnormal motion was present in up to 40 percent of cells and the remainder were totally immotile. Thus, in Kartagener syndrome many ciliated cells are motile, but the motion is abnormal. We suggest that "immotile cilia syndrome" is a misnomer, and recommended it be renamed "dyskinetic cilia syndrome."     

Repair of injured proximal tubule does not involve specialized progenitors

Recently we have established that the kidney tubular epithelium is repaired by surviving epithelial cells. It is not known, however, whether a population of intratubular adult progenitor cells are responsible for this epithelial repair after acute kidney injury. In this study, we used an unbiased DNA analog-based approach that does not rely on candidate markers to track multiple rounds of cell division in vivo. In the proximal tubule, robust thymidine analog incorporation was observed postinjury. Cell division was stochastic and enriched among cells that were injured and dedifferentiated. There was no evidence for the presence of a population of specialized progenitors that repeatedly divide in response to injury. Instead, these results indicate that after injury, new epithelial cells arise from self-duplication of surviving cells, most of which are injured. Because the renal papilla contains DNA label-retaining cells and has been proposed as a stem cell niche, we examined the proliferative behavior of these putative progenitors after ischemia-reperfusion injury. Although label-retaining cells in the renal papilla diminished with time after ischemia-reperfusion injury, they neither proliferated nor migrated to the outer medulla or cortex. Thus, nonlethally injured cells repopulate the kidney epithelium after injury in the absence of any specialized progenitor cell population.     

Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis

The basic biochemical mechanisms underlying many heritable human polycystic diseases are unknown despite evidence that most cases are caused by mutations in members of several protein families, the most prominent being the polycystin gene family, whose products are found on the primary cilia, or due to mutations in posttranslational processing and transport. Inherited polycystic kidney disease, the most prevalent polycystic disease, currently affects approximately 500,000 people in the United States. Decreases in proteoglycans (PGs) have been found in tissues and cultured cells from patients who suffer from autosomal dominant polycystic kidney disease, and this PG decrease has been hypothesized to be responsible for cystogenesis. This is possible because alterations in PG concentrations would be predicted to disrupt many homeostatic mechanisms of growth, development, and metabolism. To test this hypothesis, we have generated mice lacking xylosyltransferase 2 (XylT2), an enzyme involved in PG biosynthesis. Here we show that inactivation of XylT2 results in a substantial reduction in PGs and a phenotype characteristic of many aspects of polycystic liver and kidney disease, including biliary epithelial cysts, renal tubule dilation, organ fibrosis, and basement membrane abnormalities. Our findings demonstrate that alterations in PG concentrations can occur due to loss of XylT2, and that reduced PGs can induce cyst development.     

Abnormal sodium pump distribution during renal tubulogenesis in congenital murine polycystic kidney disease.

Congenital polycystic kidney disease is characterized by the formation of large fluid-filled cysts in kidney tubules. It has been postulated that increased epithelial cell proliferation and altered transtubular fluid transport are necessary for cyst formation. To address the latter problem, we have studied the plasma membrane distribution of the alpha 1 and beta 1 subunits of Na+/K(+)-ATPase during progressive stages of proximal and collecting tubular cyst formation in the CPK mouse, a murine model of autosomal recessive polycystic kidney disease. In both control and cystic proximal tubules, Na+/K(+)-ATPase distribution was restricted to the basal-lateral membrane of cells. However, in newborn through day 5 kidney tissue, 16% of control vs. 47% of cystic outer cortical, 6% of control vs. 46% of cystic inner cortical, and 2% of control vs. 63% of cystic medullary collecting tubules demonstrated apical and lateral membrane distribution of Na+/K(+)-ATPase. In all nephrogenic zones, the percentage of control or cystic collecting tubules demonstrating apical membrane distribution of Na+/K(+)-ATPase decreased over time, but the percentage of cystic collecting tubules with apical membrane Na+/K(+)-ATPase remained significantly greater than in developmentally matched controls. No alterations in the normal distributions of other apical or basal-lateral membrane marker proteins were noted at any stage of control or cystic proximal or collecting tubule development. We conclude that apical-lateral membrane Na+/K(+)-ATPase expression is a normal transient feature of early collecting tubule development. However, apical membrane Na+/K(+)-ATPase persists in cystic kidneys, suggesting that such expression may be a manifestation of the relatively undifferentiated phenotype of epithelial cells lining collecting tubule cysts. The persistence of apical membrane Na+/K(+)-ATPase, if the enzyme is functional, may have pathogenic important in abnormal transtubular fluid transport in polycystic kidney disease.     

Polycystic kidney disease--the ciliary connection

CONTEXT: "Cystic degeneration" of the kidneys was first described pathologically in 1841 and "polycystic kidneys" as a clinical syndrome in 1888. The heritable nature in some families was noted in 1899, and autosomal dominant and recessive patterns of inheritance of polycystic kidney disease (PKD) were later recognised. Autosomal dominant PKD is one of the most common human genetic diseases and results from mutations in PKD1 or PKD2. These genes encode two proteins, polycystin-1 and polycystin-2. STARTING POINT: Primary cilia are cellular organelles previously thought by some to be vestigial. New findings from several species, including algae, nematodes, and mice, implicate defects in structure or function of primary cilia as a possible common mechanism central to the development of some forms of recessive PKD. Two recent reports propose a causal link between ciliary dysfunction and autosomal dominant PKD. B Yoder and colleagues (J Am Soc Nephrol 2002; 13:2508-16) show that polycystin-1 and polycystin-2 are localised to primary cilia in cultured renal epithelial cells. S Nauli and colleagues (Nat Genet 2003; 33:129-37) show that polycystin-1 and polycystin-2 function as flow-sensitive mechanosensors in the same signal-transduction pathway. WHERE NEXT? Cystic epithelial cells show many altered cellular properties, including changes in proliferation, apoptosis, adhesion, differentiation, polarity, extracellular matrix synthesis, and fluid transport. The next important steps in PKD research will be to define the physiological roles of primary renal cilia and how defects in ciliary structure and function lead to the development of a cystic phenotype in different forms of PKD.     

Differentiated kidney epithelial cells repair injured proximal tubule

Whether kidney proximal tubule harbors a scattered population of epithelial stem cells is a major unsolved question. Lineage-tracing studies, histologic characterization, and ex vivo functional analysis results conflict. To address this controversy, we analyzed the lineage and clonal behavior of fully differentiated proximal tubule epithelial cells after injury. A CreER^T2 cassette was knocked into the sodium-dependent inorganic phosphate transporter SLC34a1 locus, which is expressed only in differentiated proximal tubule. Tamoxifen-dependent recombination was absolutely specific to proximal tubule. Clonal analysis after injury and repair showed that the bulk of labeled cells proliferate after injury with increased clone size after severe compared with mild injury. Injury to labeled proximal tubule epithelia induced expression of CD24, CD133, vimentin, and kidney-injury molecule-1, markers of putative epithelial stem cells in the human kidney. Similar results were observed in cultured proximal tubules, in which labeled clones proliferated and expressed dedifferentiation and injury markers. When mice with completely labeled kidneys were subject to injury and repair there was no dilution of fate marker despite substantial proliferation, indicating that unlabeled progenitors do not contribute to kidney repair. During nephrogenesis and early kidney growth, single proximal tubule clones expanded, suggesting that differentiated cells also contribute to tubule elongation. These findings provide no evidence for an intratubular stem-cell population, but rather indicate that terminally differentiated epithelia reexpress apparent stem-cell markers during injury-induced dedifferentiation and repair.The incidence of acute kidney injury (AKI) is expected to double over the next decade, and outcomes remain disappointing (1). To develop targeted therapies to prevent or treat AKI, a basic prerequisite is a clear understanding of which cells repair injured kidney. Unlike intestine or skin, which possess clearly defined stem-cell populations located in precise niches and responsible for continuous organ homeostasis through regular division, the kidney has a very low rate of cell proliferation during homeostasis. Although damage to intestine or skin elicits a specific repair response from resident stem cells in those organs, the very existence of kidney epithelial stem cells remains a major unresolved question. On one hand the low basal cell proliferation in adult may not require a stem-cell-based mechanism of self-renewal. On the other, it has been known for many years that proximal tubule has a substantial repair capacity after injury (2), and recent reports have highlighted a possible stem-cell source for these proliferating epithelia.We have previously excluded the possibility of an extratubular stem or progenitor population migrating into the tubule using a genetic fate-tracing strategy (3). Our approach left open the possibility that an intratubular stem-cell population might exist and a variety of candidate intratubular progenitors have been described. Lineage analysis has implicated parietal epithelial cells with a CD24+CD133+ phenotype as podocyte precursurs, and these cells possess multilineage potential ex vivo (4–7). Recently LGR5 was shown by lineage analysis to mark a distal tubule progenitor population, lending support to this notion that intratubular progenitors could exist (8). Other characteristics such as side population, label retention, and clonality have also been used to isolate putative intratubular stem cells (9–12).In proximal tubule, the traditional model for epithelial repair after injury has been through a process of dedifferentiation and proliferation of all surviving epithelial cells (2, 13). That proximal tubule cells are poised in G1, ready to reenter the cycle after injury, supports this hypothesis (14). In a lineage analysis of intratubular cells using sequential thymidine analog pulses, we found that cell division at each time point represented a different fraction of the total surviving epithelium, arguing against a common intratubular progenitor that selectively proliferates after injury (15). However, this result has also been interpreted to support a stem-cell-based repair mechanism, because tubular progenitors might preferentially survive and could theoretically represent the dominant population among surviving cells and thus divide only once or twice during repair (16, 17). Lineage tracing of nFatC1+ cells in AKI also support the notion of an intratubular progenitor population (18). Recent reports have revealed individual proximal tubule cells in humans that express vimentin, CD24, and CD133, and these putative progenitor cells are present not only at the urinary pole but also scattered throughout the proximal tubule. These cells can form spheres in vitro, clonally expand in vitro, and ameliorate AKI and contribute to epithelial lineage in experimental models (19–22).It remains unclear whether these cells are committed stem cells or alternately represent transient dedifferentiation and/or injury of a single cell. Smeets et al. have recently showed that kidney injury molecule-1 (KIM-1) is coexpressed with human vimentin+CD24+CD133+ tubule cells. Vimentin and KIM-1 are tubular injury markers (23, 24), and these authors observed no basal expression of either marker in healthy rat kidney but up-regulation of both proteins after injury. Therefore, they argue that vimentin+CD24+CD133+ cells do not reflect a preexisting stem cell population, but rather reflect transient dedifferentiation (25). In the current study we created a mouse with expression of CreERt2 under control of the SLC34a1 locus, which encodes a phosphate transporter that is only expressed in fully differentiated epithelial cells (26). We genetically labeled proximal tubule cells both individually, to perform clonal analysis, and in bulk, to assess for any contribution of an undifferentiated intratubular stem cell to proximal tubule repair. This approach also enabled us to assess whether genetically labeled cells could reactivate expression of apparent stem cell markers that have been used to characterize scattered proximal tubule cells in other studies.     

Defects in cholangiocyte fibrocystin expression and ciliary structure in the PCK rat

BACKGROUND & AIMS: Recent studies have showed that proteins associated with polycystic kidney disease (PKD) are expressed in cilia, linking this organelle and cyst formation in the kidney, but involvement of cilia in PKD-related biliary cystogenesis has not been shown. We investigated: (1) the expression of fibrocystin (a product of PKHD1, the autosomal-recessive PKD [ARPKD] gene) in cholangiocyte cilia; (2) biliary cyst formation in an orthologous rat model, PCK; and (3) the effect of Pkhd1 mutation on ciliary structure. METHODS: Biliary cystogenesis was assessed by microcomputed tomography. Fibrocystin expression in cholangiocytes of isolated intrahepatic bile ducts (IBDUs) and liver cysts was analyzed by confocal and immunoelectron microscopy, and ciliary structure and length by scanning and transmission electron microscopy. Small interfering RNAs (siRNA) were used to examine the effect of fibrocystin loss on ciliary structure. RESULTS: The biliary tree in the PCK rat was distorted markedly, showing multiple bile duct dilatation and focal budding. In normal IBDUs, each cholangiocyte had a single cilium that expressed fibrocystin. In contrast, cilia in the PCK rat were abnormal with bulbous extensions and diminished length, and were devoid of fibrocystin. In cholangiocytes of normal IBDUs, specific siRNA reduced Pkhd1 messenger RNA by 80%, the length of cilia by 41%, and fibrocystin ciliary expression to an undetectable level. CONCLUSIONS: Our results indicate that fibrocystin is expressed in cholangiocyte cilia and that disruption of Pkhd1 by a germ line mutation in the PCK rat or by siRNA in IBDUs results in abnormalities in ciliary morphology and possibly biliary cystogenesis.     

Regulation of flagellar dynein activity by a central pair kinesin

The motility of cilia and flagella is powered by dynein ATPases associated with outer doublet microtubules. However, a flagellar kinesin-like protein that may function as a motor associates with the central pair complex. We determined that Chlamydomonas reinhardtii central pair kinesin Klp1 is a phosphoprotein and, like conventional kinesins, binds to microtubules in vitro in the presence of adenosine 5'-[beta,gamma-imido]triphosphate, but not ATP. To characterize the function of Klp1, we generated RNA interference expression constructs that reduce in vivo flagellar Klp1 levels. Klp1 knockdown cells have flagella that either beat very slowly or are paralyzed. EM image averages show disruption of two structures associated with the C2 central pair microtubule, C2b and C2c. Greatest density is lost from part of projection C2c, which is in a position to interact with doublet-associated radial spokes. Klp1 therefore retains properties of a motor protein and is essential for normal flagellar motility. We hypothesize that Klp1 acts as a conformational switch to signal spoke-dependent control of dynein activity.