Nonselective Cyclooxygenase Inhibition Retards Cyst Progression in a Murine Model of Autosomal Dominant Polycystic Kidney Disease.

Aim: Autosomal dominant polycystic kidney disease is one of the most common genetic renal diseases. Cyclooxygenase plays an important role in epithelial cell proliferation and may contribute to the mechanisms underlying cyst formation. The aim of the present study was to evaluate the role of cyclooxygenase inhibition in the cyst progression in polycystic kidney disease. Method: Pkd2WS25/- mice, a murine model which harbors a compound cis-heterozygous mutation of the Pkd2 gene were used. Cyclooxygenase expression was assessed in both human and murine kidney specimens. Pkd2WS25/- mice were treated with Sulindac (a nonselective cyclooxygenase inhibitor) or vehicle for 8 months starting at three weeks age, and then renal cyst burden was assessed by kidney weight and volume. Results: Cyclooxygenase-2 expression was up-regulated compared to control kidneys as shown by RNase protection in human polycystic kidneys and immunoblot in mouse Pkd2WS25/- kidneys. Cyclooxygenase-2 expression was up-regulated in the renal interstitium as well as focal areas of the cystic epithelium (p<0.05). Basal Cyclooxygenase-1 levels were unchanged in both immunohistochemistry and real-time PCR. Administration of Sulindac to Pkd2WS25/- mice and to control mice for 8 months resulted in reduced kidney weights and volume in cystic mice. Renal function and electrolytes were not significantly different between groups. Conclusion: Thus treatment of a murine model of polycystic kidney disease with Sulindac results in decreased kidney cyst burden. These findings provide additional implications for the use of Cyclooxygenase inhibition as treatment to slow the progression of cyst burden in patients with polycystic kidney disease.

The integrin ß1 subunit regulates paracellular permeability of kidney proximal tubule cells.

Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin ß1 subunit in these cells converts them from a "loose" epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a "tight" epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin ß1 cytoplasmic tail and does not entail ß1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the ß1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin ß1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.

Deleting the TGF-ß receptor attenuates acute proximal tubule injury.

TGF-ß is a profibrotic growth factor in CKD, but its role in modulating the kidney's response to AKI is not well understood. The proximal tubule epithelial cell, which is the main cellular target of AKI, expresses high levels of both TGF-ß and its receptors. To determine how TGF-ß signaling in this tubular segment affects the response to AKI, we selectively deleted the TGF-ß type II receptor in the proximal tubules of mice. This deletion attenuated renal impairment and reduced tubular apoptosis in mercuric chloride-induced injury. In vitro, deficiency of the TGF-ß type II receptor protected proximal tubule epithelial cells from hydrogen peroxide-induced apoptosis, which was mediated in part by Smad-dependent signaling. Taken together, these results suggest that TGF-ß signaling in the proximal tubule has a detrimental effect on the response to AKI as a result of its proapoptotic effects.

Membrane-type 4 matrix metalloproteinase (MT4-MMP) modulates water homeostasis in mice.

MT4-MMP is a membrane-type metalloproteinase (MMP) anchored to the membrane by a glycosyl-phosphatidylinositol (GPI) motif. GPI-type MT-MMPs (MT4- and MT6-MMP) are related to other MT-MMPs, but their physiological substrates and functions in vivo have yet to be identified. In this manuscript we show that MT4-MMP is expressed early in kidney development, as well as in the adult kidney, where the highest levels of expression are found in the papilla. MT4-MMP null mice had minimal renal developmental abnormalities, with a minor branching morphogenesis defect in early embryonic kidney development and slightly dysmorphic collecting ducts in adult mice. Interestingly, MT4-MMP null mice had higher baseline urine osmolarities relative to wild type controls, but these animals were able to concentrate and dilute their urines normally. However, MT4-MMP-null mice had decreased daily water intake and daily urine output, consistent with primary hypodipsia. MT4-MMP was shown to be expressed in areas of the hypothalamus considered important for regulating thirst. Thus, our results show that although MT4-MMP is expressed in the kidney, this metalloproteinase does not play a major role in renal development or function; however it does appear to modify the neural stimuli that modulate thirst.

beta1 integrin is necessary for ureteric bud branching morphogenesis and maintenance of collecting duct structural integrity.

The kidney collecting system develops from branching morphogenesis of the ureteric bud (UB). This process requires signaling by growth factors such as glial cell line derived neurotrophic factor (GDNF) and fibroblast growth factors (FGFs) as well as cell extracellular matrix interactions mediated by integrins. The importance of integrin signaling in UB development was investigated by deleting integrin beta1 at initiation (E10.5) and late (E18.5) stages of development. Deletion at E10.5 resulted in a severe branching morphogenesis phenotype. Deletion at E18.5 did not alter renal development but predisposed the collecting system to severe injury following ureteric obstruction. beta1 integrin was required for renal tubular epithelial cells to mediate GDNF- and FGF-dependent signaling despite normal receptor localization and activation in vitro. Aberrations in the same signaling molecules were present in the beta1-null UBs in vivo. Thus beta1 integrins can regulate organ branching morphogenesis during development by mediating growth-factor-dependent signaling in addition to their well-defined role as adhesion receptors.

Apoptosis of the thick ascending limb results in acute kidney injury.

Ischemia- or toxin-induced acute kidney injury is generally thought to affect the cells of the proximal tubule, but it has been difficult to define the involvement of other tubular segments because of the widespread damage caused by ischemia/reperfusion or toxin-induced injury in experimental models. For evaluation of whether thick ascending limb (TAL)-specific epithelial injury results in acute kidney injury, a novel transgenic mouse model that expresses the herpes simplex virus 1 thymidine kinase gene under the direction of the TAL-specific Tamm-Horsfall protein promoter was generated. After administration of gancyclovir, these mice demonstrated apoptosis only in TAL cells, with little evidence of neutrophil infiltration. Compared with control mice, blood urea nitrogen and creatinine levels were at least five-fold higher in the transgenic mice, which also developed oliguria and impaired urinary concentrating ability. These findings suggest that acute injury targeted only to the TAL is sufficient to cause severe acute kidney injury in mice with features similar to those observed in humans.

A WT1 co-regulator controls podocyte phenotype by shuttling between adhesion structures and nucleus.

Glomerular podocyte differentiation state is critical for filtration barrier function and is regulated by WT1, a zinc finger transcription factor. A yeast two-hybrid assay identified a novel, WT1-interacting protein (WTIP) that maps to human chromosome 19q13.1, a region with genes linked to familial focal segmental glomerulosclerosis. The domain structure of WTIP is similar to the zyxin subfamily of cytosolic LIM domain-containing proteins, which contain three carboxyl-terminal LIM protein-protein interaction domains and a proline-rich, pre-LIM region with a nuclear export signal. Other LIM domain-containing proteins (zyxin and mouse muscle LIM protein) did not interact with WT1 in two-hybrid assays, and WTIP did not interact with an unrelated transcription factor, LMX1B. WTIP mRNA was detected in cultured podocytes and was developmentally regulated, with expression peaking in mouse kidney at embryonic day 15-16 (E15-E16) in kidney but persisting into adulthood. In situ hybridization demonstrated WTIP expression in developing E15 glomeruli and in cultured podocytes. The partial WTIP clone, which interacted with WTIP in the two-hybrid assay, co-localized with WT1 in nuclei, co-precipitated with WT1, and inhibited WT1-dependent transcriptional activation of the amphiregulin promoter. In contrast, full-length WTIP was excluded from cell nuclei, but after the addition of leptomycin B, an inhibitor of Crm1-mediated nuclear export, it accumulated in the nucleus and co-precipitated with WT1 in whole cell lysates. Epitope-tagged WTIP co-localized with the adaptor protein CD2AP (CMS) in podocyte actin spots and with Mena at cell-cell junctions. We propose that WTIP monitors slit diaphragm protein assembly as part of a multiple protein complex, linking this specialized adhesion junction to the actin cytoskeleton, and shuttles into the nucleus after podocyte injury, providing a mechanism whereby changes in slit diaphragm structure modulate gene expression.