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Netrin-1 Overexpression Induces Polycystic Kidney Disease

A Novel Mechanism Contributing to Cystogenesis in Autosomal Dominant Polycystic Kidney Disease
      Despite recent advances in understanding the pathogenesis of polycystic kidney disease (PKD), the underlying molecular mechanisms involved in cystogenesis are not fully understood. This study describes a novel pathway involved in cyst formation. Transgenic mice overexpressing netrin-1 in proximal tubular cells showed increased production and urinary excretion of netrin-1. Although no cysts were detectable immediately after birth, numerous small cysts were evident by the age of 4 weeks, and disease was accelerated along with age. Surprisingly, cyst formation in the kidney was restricted to male mice, with 80% penetrance. However, ovariectomy induced kidney cyst growth in netrin-1–overexpressing female mice. Cyst development in males was associated with albuminuria and polyuria and increased cAMP excretion in netrin-1 transgenic mice. Netrin-1 overexpression significantly increased extracellular signal-regulated kinase and focal adhesion kinase phosphorylation and vimentin expression. Interestingly, p53 expression was increased but in an inactive form. Furthermore, netrin-1 expression was increased in cystic epithelia and urine of various rodent models of PKD. siRNA-mediated suppression of netrin-1 significantly reduced cyst growth and improved kidney function in netrin-1 transgenic mice and in two genetic animal models of PKD. Together, these data demonstrate that netrin-1 up-regulation induced cyst formation in autosomal dominant PKD.
      Autosomal dominant polycystic kidney disease (ADPKD) is a leading cause of end-stage renal disease, which requires chronic dialysis or renal transplantation. It affects approximately 500,000 to 600,000 people in the United States and 4 to 6 million people worldwide.
      • Chapman A.B.
      • Devuyst O.
      • Eckardt K.U.
      • Gansevoort R.T.
      • Harris T.
      • Horie S.
      • Kasiske B.L.
      • Odland D.
      • Pei Y.
      • Perrone R.D.
      • Pirson Y.
      • Schrier R.W.
      • Torra R.
      • Torres V.E.
      • Watnick T.
      • Wheeler D.C.
      Conference Participants
      Autosomal-dominant polycystic kidney disease (ADPKD): executive summary from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference.
      • Grantham J.J.
      Clinical practice: autosomal dominant polycystic kidney disease.
      • Harris P.C.
      • Torres V.E.
      Polycystic kidney disease.
      ADPKD is the primary cause for end-stage renal disease in 5% to 10% of patients.
      • Collins A.J.
      • Foley R.N.
      • Herzog C.
      • Chavers B.
      • Gilbertson D.
      • Ishani A.
      • et al.
      US Renal Data System 2010 Annual Data Report.
      Understanding the pathogenesis of this disease at the cellular and molecular levels is critical for developing effective therapies that prevent the progression to renal failure in ADPKD. Although several targeted treatments for ADPKD have been evaluated during the last two decades, the vasopressin-2 receptor antagonist tolvaptan represents the only targeted treatment with proven efficacy to retard cyst growth.
      • Meijer E.
      • Gansevoort R.T.
      Vasopressin V2 receptor antagonists in autosomal dominant polycystic kidney disease: efficacy, safety, and tolerability.
      However, its efficacy is limited to an approximately 25% to 30% reduction of kidney function decline.
      PKD1 (polycystic kidney disease 1), the gene responsible for 85% of cases, encodes polycystin-1, a large receptor-like integral membrane protein that contains several extracellular motifs indicative of cell-cell and cell-matrix interactions.
      • Harris P.C.
      • Torres V.E.
      Polycystic kidney disease.
      PKD2, accounting for most of the remaining 15% of cases, encodes polycystin-2, a transmembrane protein that acts as a nonspecific calcium-permeable channel. Both polycystin 1 and 2 function together in a nonredundant manner through a common pathway and produce cellular responses that regulate proliferation, migration, differentiation, and kidney morphogenesis.
      • Collins A.J.
      • Foley R.N.
      • Herzog C.
      • Chavers B.
      • Gilbertson D.
      • Ishani A.
      • et al.
      US Renal Data System 2010 Annual Data Report.
      ,
      • Hopp K.
      • Ward C.J.
      • Hommerding C.J.
      • Nasr S.H.
      • Tuan H.F.
      • Gainullin V.G.
      • Rossetti S.
      • Torres V.E.
      • Harris P.C.
      Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity.
      • Mekahli D.
      • Parys J.B.
      • Bultynck G.
      • Missiaen L.
      • De Smedt H.
      Polycystins and cellular Ca2+ signaling.
      • Streets A.J.
      • Newby L.J.
      • O'Hare M.J.
      • Bukanov N.O.
      • Ibraghimov-Beskrovnaya O.
      • Ong A.C.
      Functional analysis of PKD1 transgenic lines reveals a direct role for polycystin-1 in mediating cell-cell adhesion.
      • Ong A.C.
      • Harris P.C.
      Molecular pathogenesis of ADPKD: the polycystin complex gets complex.
      • Yu S.
      • Hackmann K.
      • Gao J.
      • He X.
      • Piontek K.
      • Garcia-Gonzalez M.A.
      • Menezes L.F.
      • Xu H.
      • Germino G.G.
      • Zuo J.
      • Qian F.
      Essential role of cleavage of polycystin-1 at G protein-coupled receptor proteolytic site for kidney tubular structure.
      Single loss-of-function mutations in either gene, most likely in combination with somatic mutations of the remaining normal allele (second hit) in certain cells, result in tubular cells reverting to a less differentiated state or an embryonic dedifferentiated state, which is more prone to proliferation. There is a significant unmet need to develop new and effective therapeutic strategies that target the mechanisms contributing to the pathogenesis of ADPKD. The slow progress in the development of new therapeutics is partially related to an incomplete pathogenetic understanding of PKD. The latter is due to the fact that two genes may be causative as well as the probable need for a somatic mutation of the second allele, which is not adequately copied by most animal models of PKD. Moreover, there is a sex difference in the incidence and severity of kidney disease in ADPKD patients as well as in PKD animal models. On average, female ADPKD patients show a milder disease course and reach end-stage renal disease later, compared with male ADPKD patients.
      • Ishikawa I.
      • Maeda K.
      • Nakai S.
      • Kawaguchi Y.
      Gender difference in the mean age at the induction of hemodialysis in patients with autosomal dominant polycystic kidney disease.
      Netrin-1 is a laminin-related secreted molecule identified as a neuronal guidance cue, predominantly implicated in axonal outgrowth and cell migration orientation in the developing nervous system.
      • Tessier-Lavigne M.
      • Goodman C.S.
      The molecular biology of axon guidance.
      Apart from nervous system, netrin-1 is widely expressed in vascular endothelial cells,
      • Ly N.P.
      • Komatsuzaki K.
      • Fraser I.P.
      • Tseng A.A.
      • Prodhan P.
      • Moore K.J.
      • Kinane T.B.
      Netrin-1 inhibits leukocyte migration in vitro and in vivo.
      kidney tubular epithelial cells, and immune cells.
      • Ranganathan P.
      • Mohamed R.
      • Jayakumar C.
      • Ramesh G.
      Guidance cue netrin-1 and the regulation of inflammation in acute and chronic kidney disease.
      ,
      • Wang W.
      • Reeves W.B.
      • Ramesh G.
      Netrin-1 and kidney injury, I: netrin-1 protects against ischemia-reperfusion injury of the kidney.
      Netrin-1 mediates its biological function by interacting with its receptor deleted in colorectal cancer and the uncoordinated-5 homolog family. Administration or moderate overexpression of netrin-1 induces renal proximal tubular epithelial cell proliferation, migration,
      • Ranganathan P.
      • Mohamed R.
      • Jayakumar C.
      • Ramesh G.
      Guidance cue netrin-1 and the regulation of inflammation in acute and chronic kidney disease.
      ,
      • Wang W.
      • Reeves W.B.
      • Ramesh G.
      Netrin-1 increases proliferation and migration of renal proximal tubular epithelial cells via the UNC5B receptor.
      and angiogenesis. Moreover, netrin-1 promotes cancer cell invasion and tumor growth.
      • Rodrigues S.
      • De Wever O.
      • Bruyneel E.
      • Rooney R.J.
      • Gespach C.
      Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis.
      However, specifics regarding the involvement of netrin-1 in pathophysiology of PKD are sparse. Herein, the study identified a novel mechanism that contributes to cystogenesis in ADPKD. One of the transgenic mice (Tg6) expressing high levels of renal netrin-1 spontaneously developed a cyst in the kidney in a sex-specific manner, which was limited to male mice. This novel model system was further characterized to determine previously unidentified pathways that may regulate cyst development and represent a new target for drug discovery. Furthermore, the study found that expression of netrin-1 is increased in kidney of other rodent models of PKD. These results demonstrate that the overexpression of netrin-1 in kidney is pathogenic, which can contribute to cystogenesis in mice.

      Materials and Methods

      Transgenic Animals

      Netrin-1–overexpressing transgenic animals were generated, as described before.
      • Mazelin L.
      • Bernet A.
      • Bonod-Bidaud C.
      • Pays L.
      • Arnaud S.
      • Gespach C.
      • Bredesen D.E.
      • Scoazec J.Y.
      • Mehlen P.
      Netrin-1 controls colorectal tumorigenesis by regulating apoptosis.
      Briefly, chicken netrin-1 was cloned upstream of l-fatty acid binding protein promoter and used for microinjection. The l-fatty acid binding protein promoter was thought to be gut specific when the transgenic mice were generated. However, subsequent screening for transgene expression revealed promoter activity and hence transgene expression in other organs or tissues, including the small intestine, colon, spleen, brain, and kidneys.
      • Mazelin L.
      • Bernet A.
      • Bonod-Bidaud C.
      • Pays L.
      • Arnaud S.
      • Gespach C.
      • Bredesen D.E.
      • Scoazec J.Y.
      • Mehlen P.
      Netrin-1 controls colorectal tumorigenesis by regulating apoptosis.
      • Van Raay T.J.
      • Foskett S.M.
      • Connors T.D.
      • Klinger K.W.
      • Landes G.M.
      • Burn T.C.
      The NTN2L gene encoding a novel human netrin maps to the autosomal dominant polycystic kidney disease region on chromosome 16p13.3.
      • Rajasundari A.
      • Pays L.
      • Mehlen P.
      • Ramesh G.
      Netrin-1 overexpression in kidney proximal tubular epithelium ameliorates cisplatin nephrotoxicity.
      Highest expression of the transgene was found in the kidneys. Two strains of transgenic mice were established, Tg3 and Tg6. Tg3 mice were extensively characterized for gut and kidney phenotype, which showed normal kidney histology and function.
      • Wang W.
      • Reeves W.B.
      • Pays L.
      • Mehlen P.
      • Ramesh G.
      Netrin-1 overexpression protects kidney from ischemia reperfusion injury by suppressing apoptosis.
      ,
      • Mohamed R.
      • Jayakumar C.
      • Ranganathan P.V.
      • Ganapathy V.
      • Ramesh G.
      Kidney proximal tubular epithelial-specific overexpression of netrin-1 suppresses inflammation and albuminuria through suppression of COX-2-mediated PGE2 production in streptozotocin-induced diabetic mice.
      However, Tg6 strain was not characterized further because of the development of polycystic kidneys. We obtained this Tg6 strain from European Mouse Mutant Cell Repository and characterized this transgenic strain to determine the role of netrin-1 in polycystic kidney disease. Cyst development was observed only in male mice; further characterization was performed in Tg6 males (n = 6 to 8). All studies were approved by the appropriate institutional animal review board (Institutional Animal Care and Use Committee, Augusta University; approval identifier 07–0044).

      Renal Function Tests

      Renal function was assessed by measurement of serum creatinine (catalog number DZ072B; Diazyme Labs, Poway, CA) and blood urea nitrogen (catalog number DIUR-500; Bioassay System, Hayward, CA).

      Netrin-1 Quantification

      For the quantitative determination of netrin-1, we used the commercially available netrin-1 enzyme-linked immunosorbent assay (ELISA) kit for chicken (catalog number CSB-EL016127CH; CEDARLANE Laboratories USA Inc., Burlington, NC), mouse (catalog number MBS733806; MyBioSource, Inc., San Diego, CA), and rat (catalog number MBS728764; MyBiosource, Inc.), as per the manufacturer's instructions.

      Urine Albumin Quantification by ELISA

      Twenty-four–hour urine was collected using a metabolic cage. Urine volume was measured, and urine was centrifuged at 11,000 × g for 10 minutes. The supernatant was aliquoted and stored at −80°C until used. Urine albumin was measured using an ELISA kit (catalog number E90-134; Bethyl Laboratories, Montgomery, TX).

      Quantification of mRNA by Real-Time RT-PCR

      RNA (n = 5 to 6) was isolated using TRIZOL reagent (Life Technologies, Grand Island, NY). Real-time RT-PCR was performed in an Applied Biosystems Inc. 7700 Sequence Detection System (Foster City, CA). A total of 3 μg total RNA was reverse transcribed in a reaction volume of 40 μL using Omniscript RT kit (Qiagen, Germantown, MD) and random primers. The product was diluted to a volume of 150 μL, and 5-μL aliquots were used as templates for amplification using the SYBR Green PCR amplification reagent (Qiagen) and Mouse Transcription Factor PCR Array (catalog number PAMM-075Z; SA Biosciences, Frederick, MD) or gene-specific primers. Data were analyzed using Web analysis tools from SABiosciences.

      Quantification of cAMP in Urine

      cAMP was quantified using ELISA kit (catalog number KGE012B; R&D Systems, Minneapolis, MN), as per the manufacturer's instructions (n = 8).

      Ovariectomy

      Female Tg6–netrin-1 transgenic animals at 5 weeks of age (n = 6 to 8) were ovariectomized or sham operated by the dorsal approach under general anesthesia with pentobarbital. Cyst development was monitored noninvasively every week using an ultrasound machine (Vevo 2100; FUJIFILM VisualSonics Inc., Toronto, ON, Canada). Animals were scarified at 40 weeks, and kidneys and blood were collected. Kidney function was monitored by measuring serum creatinine and blood urea nitrogen.

      Cell Culture and Quantification of Cilium Length

      Mouse proximal tubular epithelial cells were plated in a chamber slide. Cells were cultured with serum or serum-free medium and then treated with 100 ng/mL of netrin-1 or vehicle for a period of 72 hours. Cells were rinsed with phosphate-buffered saline and fixed with methanol. Using rabbit–anti-acetylated tubulin antibody (Sigma-Aldrich, St. Louis, MO), cilia were fluorescently labeled with goat anti-rabbit Cy3 antibody. Nuclei were counterstained with DAPI. Similarly, kidney sections from wild-type (WT) and Tg6–netrin-1 transgenic animals were stained for tubulin using anti-acetylated antibody (Santa Cruz Biotechnology, Inc., Dallas, TX). Images of cilia were oriented parallel to the plane of focus captured from randomly chosen high-power fields (66× objective) in the cortex and outer medulla or in vitro cell culture. Cellsens Standard software (version 1.14; Olympus, Pittsburgh, PA) was used to trace and measure the length of cilia in captured images. Twenty cilia were measured from each of the three mice per group, giving a total of 60 cilia per group.

      Histology and Immunostaining

      Kidney tissues were fixed in buffered 10% formalin for 12 hours and then embedded in paraffin. Renal sections (5 μm thick) were rehydrated through a xylene and decreasing ethanol series, washed in phosphate-buffered saline, and incubated with an epitope retrieval solution for 40 minutes in a steam bath (IHC World, Ellicott City, MD). The slides were blocked with 5% bovine serum albumin/phosphate-buffered saline blocking buffer for 1 hour, then incubated with the following antibodies diluted in blocking buffer overnight at 4°C: to determine mouse netrin-1 expression, sections were incubated with goat anti–netrin-1 antibody (Santa Cruz Biotechnology, Inc.); to determine chicken netrin-1 (transgene) expression, sections were incubated with goat anti-chicken netrin-1 antibody (R&D Systems); to determine proximal tubular epithelium localization, section were stained with rabbit anti-megalin antibody (Abcam) and mouse anti–aquaporin-1 (Santa Cruz Biotechnology, Inc.); to determine vimentin expression, sections were stained with rabbit anti-vimentin antibody (Cell Signaling Technology Inc., Danvers, MA); and to determine the cell proliferation, sections were stained with goat anti–Ki-67 antibody (Cell Signaling Technology Inc.). For immunofluorescence, sections were washed with phosphate-buffered saline and then incubated with fluorescence-labeled antibodies diluted in blocking buffer at 37°C for 1 hour (anti-rabbit AlexaFlour488 and anti-goat AlexaFlour594; Thermo Fisher Scientific, Waltham, MA); anti-mouse AlexaFlour488 (Thermo Fisher Scientific); and fluorescence-labeled lotus tetragonolobus lectin (FL-1321; Vector Laboratories, Burlingame, CA). For immunohistochemical analysis, section was washed and incubated with secondary antibody with biotin conjugate. Color was developed after incubation with ABC reagent (Vector Laboratories). For assessment of injury, sections (5 μm thick) were stained with periodic acid–Schiff followed by hematoxylin. Kidney sections were stained with hematoxylin and eosin. A total of 5 to 10 random, nonoverlapping, high-resolution images per mouse at a magnification of ×10 were used for determination of cystic index. Cystic index was calculated as the area percentage of lumen over the total image area, as described before.
      • Happe H.
      • van der Wal A.M.
      • Salvatori D.C.
      • Leonhard W.N.
      • Breuning M.H.
      • de Heer E.
      • Peters D.J.
      Cyst expansion and regression in a mouse model of polycystic kidney disease.
      Fibrosis was determined by staining section with trichrome. Stained sections were imaged using an Olympus inverted microscope with color charge-coupled device camera. Trichrome staining was quantified by tracing the stained area in ×40 field with Cellsens Standard software (Olympus). Five fields for each animal were traced, and percentage-stained area was calculated.

      Western Blot Analysis

      Protein extraction from kidney and Western blot analysis were performed as described before.
      • Mohamed R.
      • Jayakumar C.
      • Ranganathan P.V.
      • Ganapathy V.
      • Ramesh G.
      Kidney proximal tubular epithelial-specific overexpression of netrin-1 suppresses inflammation and albuminuria through suppression of COX-2-mediated PGE2 production in streptozotocin-induced diabetic mice.
      ,
      • Ranganathan P.
      • Jayakumar C.
      • Ramesh G.
      Proximal tubule-specific overexpression of netrin-1 suppresses acute kidney injury-induced interstitial fibrosis and glomerulosclerosis through suppression of IL-6/STAT3 signaling.
      The membrane was probed with goat anti-chicken netrin-1 antibody (R&D Systems), rabbit anti-mouse p53, focal adhesion kinase, Akt and phosphorylated Akt, extracellular signal-regulated kinase (ERK) and phosphorylated ERK, fibronectin, Src-kinase, vimentin and E-cadherin antibodies (Cell Signaling Technology Inc.). Proteins were detected with a horseradish peroxidase–conjugated antibody and enhanced chemiluminescence (Thermo Scientific, Greenville, SC). Intensity of immunoreactivity was measured by densitometry using ImageJ software version 1.51n 1 (NIH, Bethesda, MD; https://imagej.nih.gov/ij, last accessed November 28, 2017). Signals were normalized to glyceraldehyde-3-phosphate dehydrogenase.

      Rodent Models of PKD

      Han:SPRD Rat

      The Han:SPRD rat colony was established in the Animal Facility, University of Zurich, from a litter that was obtained from the Rat Resource and Research Center (Columbia, MO). Nine-week–old heterozygous cystic (Cy/+) and wild-type (+/+) male rats were used in this study. The 24-hour urine samples were collected in metabolic cages at 9 weeks of age. Rats were anesthetized with isoflurane, and kidneys were harvested for histology.

      Pkd1 Conditional Knockout Mouse

      Pkd1 conditional knockout mice (B6.129S4-Pkd1tm2Ggg//J)
      • Piontek K.B.
      • Huso D.L.
      • Grinberg A.
      • Liu L.
      • Bedja D.
      • Zhao H.
      • Gabrielson K.
      • Qian F.
      • Mei C.
      • Westphal H.
      • Germino G.G.
      A functional floxed allele of Pkd1 that can be conditionally inactivated in vivo.
      ,
      • Piontek K.
      • Menezes L.F.
      • Garcia-Gonzalez M.A.
      • Huso D.L.
      • Germino G.G.
      A critical developmental switch defines the kinetics of kidney cyst formation after loss of Pkd1.
      with tamoxifen-inducible Cre [B6.Cg-Tg(CAGcre/Esr1∗)5Amc/J] were supplied by Prof. Gregory G. Germino (National Institutes of Health). We induced Cre recombinase activity by i.p. injection of tamoxifen (1.25 mg/10 g), dissolved in corn oil (Sigma-Aldrich) at day 11 of age into pups. Male and female Pkd1F/F-Cre+ and Pkd1F/F-Cre– were used in this study (equal numbers of males and females in each group). The 24-hour urine samples were collected in metabolic cages at 35 days of age, mice were anesthetized with isoflurane, and kidneys were harvested for histology.

      Human PKD1 Knock-In Mouse

      PKD1 knock-in mice, PKD1RC/RC, were provided by Dr. Peter Harris (Mayo Clinic College of Medicine, Rochester, MN). In these mice, the endogenous mouse PKD1 gene as a human PKD1 mutation, p.R3277C, was introduced.
      • Hopp K.
      • Ward C.J.
      • Hommerding C.J.
      • Nasr S.H.
      • Tuan H.F.
      • Gainullin V.G.
      • Rossetti S.
      • Torres V.E.
      • Harris P.C.
      Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity.
      Homozygous PKD1RC/RC mice start developing cysts at 10 weeks after birth and progress to severe cystic kidney disease as they age.
      • Hopp K.
      • Ward C.J.
      • Hommerding C.J.
      • Nasr S.H.
      • Tuan H.F.
      • Gainullin V.G.
      • Rossetti S.
      • Torres V.E.
      • Harris P.C.
      Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity.

      In Vivo siRNA Infusion and Suppression of Netrin-1 Expression

      To determine the direct role of transgene netrin-1 on cyst development, specific siRNA to chicken netrin-1 (sense, 5′-AGAGUCAACAUGAAGAAGUAUU-3′; antisense, 5′-UACUUCUUCAUGUUGAUCUUU-3′) or scrambled siRNA was synthesized (Thermo Scientific) and annealed as double-stranded RNA before injection. Four-week–old Tg6 male mice were then administered with chicken netrin-1–specific siRNA or scrambled siRNA (50 μg/mouse, intravenously, twice a week for another 4 weeks; n = 6 to 8 mice in each group). To confirm the role of netrin-1 on cyst development in established PKD animal models, Pkd1 knockout and knock-in (PKD1RC/RC) mice were treated with specific siRNA to mouse netrin-1 (On-Targetplus mouse netrin-1 siRNA; catalog number L-048013-01-0050; Dharmacon, Lafayette, CO) or scrambled siRNA (50 μg/animal, intravenously, twice a week) at 10 weeks of age for next 4 weeks (n = 6 mice in each group). At the end of the treatment, animals were sacrificed and kidney tissue was harvested. Kidneys were weighed, imaged, and processed for histopathologic analysis. Urine chicken netrin-1 and mouse netrin-1 were quantified to confirm suppression of transgene expression by ELISA and Western blot analysis, respectively.

      Statistical Analysis

      All assays were performed in triplicate, and the data are shown as means ± SEM. Statistical significance was assessed by an unpaired, two-tailed t-test for single comparison or analysis of variance for multiple comparisons using GraphPad Prism version 7.0 software (GraphPad Software, La Jolla, CA). P < 0.05 was considered significant.

      Results

      Netrin-1 Overexpression Induces Sex-Specific Cystic Kidney Disease

      As described in Materials and Methods, two netrin-1 transgenic lines (expressing chicken netrin-1) were established.
      • Mazelin L.
      • Bernet A.
      • Bonod-Bidaud C.
      • Pays L.
      • Arnaud S.
      • Gespach C.
      • Bredesen D.E.
      • Scoazec J.Y.
      • Mehlen P.
      Netrin-1 controls colorectal tumorigenesis by regulating apoptosis.
      The Tg3 strain, which showed moderate levels of transgene overexpression in proximal tubular epithelium and exhibited normal kidney histology and function,
      • Mohamed R.
      • Jayakumar C.
      • Ranganathan P.V.
      • Ganapathy V.
      • Ramesh G.
      Kidney proximal tubular epithelial-specific overexpression of netrin-1 suppresses inflammation and albuminuria through suppression of COX-2-mediated PGE2 production in streptozotocin-induced diabetic mice.
      protected against different forms of acute
      • Rajasundari A.
      • Pays L.
      • Mehlen P.
      • Ramesh G.
      Netrin-1 overexpression in kidney proximal tubular epithelium ameliorates cisplatin nephrotoxicity.
      ,
      • Wang W.
      • Reeves W.B.
      • Pays L.
      • Mehlen P.
      • Ramesh G.
      Netrin-1 overexpression protects kidney from ischemia reperfusion injury by suppressing apoptosis.
      and chronic kidney injury
      • Mohamed R.
      • Jayakumar C.
      • Ranganathan P.V.
      • Ganapathy V.
      • Ramesh G.
      Kidney proximal tubular epithelial-specific overexpression of netrin-1 suppresses inflammation and albuminuria through suppression of COX-2-mediated PGE2 production in streptozotocin-induced diabetic mice.
      and accelerated the recovery after acute kidney injury.
      • Ranganathan P.
      • Jayakumar C.
      • Ramesh G.
      Proximal tubule-specific overexpression of netrin-1 suppresses acute kidney injury-induced interstitial fibrosis and glomerulosclerosis through suppression of IL-6/STAT3 signaling.
      These Tg3 mice do not develop any cysts in the kidneys.
      • Mohamed R.
      • Jayakumar C.
      • Ranganathan P.V.
      • Ganapathy V.
      • Ramesh G.
      Kidney proximal tubular epithelial-specific overexpression of netrin-1 suppresses inflammation and albuminuria through suppression of COX-2-mediated PGE2 production in streptozotocin-induced diabetic mice.
      However, the Tg6 strain of netrin-1 transgenic mice developed multiple cysts in the kidneys with accelerated growth after 4 weeks of age (Figure 1, A and B). Exponential growth of the cystic kidneys is apparent by the kidney weight measurements shown in Figure 1C. By 10 weeks of age, the average kidney weight/percentage body weight reached >4.5 g; and by 22 weeks of age, the average percentage kidney/body weight was about 6.5 g. No differences in cystic index were observed in Tg6 mice at 1 week of age. However, cystic index was significantly increased along with age in Tg6 mice (Figure 1D). Consistent with progressive cyst growth, renal function deteriorated, as reflected by increased blood urea nitrogen and serum creatinine levels with age in transgenic mice compared with WT mice (Figure 1E and Supplemental Figure S1A). Surprisingly, cyst formation is sex specific and disease penetrance occurred only in the male animals, 64% bilateral and 17% unilateral cyst formation. However, there is no cyst in female mice (Table 1). Because the netrin-1 transgene is expressed in proximal tubular epithelium of the nephron,
      • Mohamed R.
      • Jayakumar C.
      • Ranganathan P.V.
      • Ganapathy V.
      • Ramesh G.
      Kidney proximal tubular epithelial-specific overexpression of netrin-1 suppresses inflammation and albuminuria through suppression of COX-2-mediated PGE2 production in streptozotocin-induced diabetic mice.
      ,
      • Ranganathan P.
      • Jayakumar C.
      • Ramesh G.
      Proximal tubule-specific overexpression of netrin-1 suppresses acute kidney injury-induced interstitial fibrosis and glomerulosclerosis through suppression of IL-6/STAT3 signaling.
      the cyst origin was determined by staining with a proximal tubular epithelial cell marker. As shown in Figure 1F, all cyst linings were positive for megalin, suggesting the cysts originated from proximal tubular epithelium.
      Figure thumbnail gr1
      Figure 1Netrin-1 overexpression induces development of cyst in kidney. Netrin-1 transgenic mice (Tg6) and their wild-type (WT) littermates were sacrificed at 1, 4, 10, 22, and 35 weeks of age. Kidneys were harvested and imaged. A: Representative images of kidneys from Tg6 male mice showing an age-dependent increase in cyst size, bilateral (week 4 to 22) and unilateral (week 35), compared with WT littermates. B: Hematoxylin and eosin (H&E)–stained kidney sections showing histologic overview of WT and Tg6 kidney with age. C: Kidney weight at different ages from WT and Tg6 netrin-1 transgenic animals, expressed as percentage kidney weight/body weight. D: Cystic index was calculated as area percentage of lumen over the total image area using H&E as stained kidney sections. E: Kidney function was assessed by measuring blood urea nitrogen (BUN) at 10 weeks. F: WT and Tg6 mouse kidneys were stained with the proximal tubular epithelium marker megalin (red) and counterstained with nuclear stain DAPI (blue). All cyst linings were positive for megalin, suggesting that cysts (#) originated from proximal tubular epithelium. Data are expressed as means ± SEM (CE). n = 6 to 8 (CE).∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 versus WT littermates. Scale bars = 100 μm (B and F).
      Table 1Total Numbers of Transgenic Male and Female Animals that Were Analyzed for the Presence of Cysts in the Kidney and Expressed in Percentage
      SexTotal Tg6 miceCyst in both kidneyCyst in one kidneyNo cystMice with cyst, %
      Male17113382
      Female1600160

      Tg6 Netrin-1 Transgenic Mouse Kidneys Show Extremely High Levels of Netrin-1 Expression, Which Is Mainly Localized in Proximal Tubular Epithelial Cells

      To determine the expression levels of the transgene and endogenous mouse netrin-1 in WT, Tg3, and Tg6 transgenic mouse kidneys, expression in kidney and excretion were quantified by Western blot analysis, RT-PCR, and ELISA, respectively. Although cyst size increases with age (Figure 1C), transgene protein level in the kidney did not change with age (Figure 2A). Previous studies with the Tg3 strain of transgenic animals showed that transgene expression was localized in the proximal tubular epithelial cells.
      • Wang W.
      • Reeves W.B.
      • Pays L.
      • Mehlen P.
      • Ramesh G.
      Netrin-1 overexpression protects kidney from ischemia reperfusion injury by suppressing apoptosis.
      ,
      • Mohamed R.
      • Jayakumar C.
      • Ranganathan P.V.
      • Ganapathy V.
      • Ramesh G.
      Kidney proximal tubular epithelial-specific overexpression of netrin-1 suppresses inflammation and albuminuria through suppression of COX-2-mediated PGE2 production in streptozotocin-induced diabetic mice.
      To determine whether the Tg6 transgenic strain also showed a similar or different transgene expression pattern, immunolocalization was performed with proximal tubular cell marker aquaporin-1, lotus tetragonolobus lectin, and peanut agglutinin. As shown in Figure 2B, no staining for chicken netrin was seen in WT mouse kidneys, but transgene netrin-1 expression was intense and colocalized in the proximal tubular epithelial cells in the Tg6 strain compared with WT mice. Endogenous netrin-1 excretion in the urine is approximately 0.8 ng/mg of creatinine in WT mice, which is not changed in Tg3 netrin-1 transgenic animals (Figure 2C). However, in Tg6 netrin-1 transgenic mice, the excretion of endogenous mouse netrin-1 is significantly increased compared with WT (Figure 2C). The excretion of transgene chicken netrin-1 in Tg3 mice was >10-fold, whereas the excretion of chicken netrin-1 in Tg6 mice was about 100-fold compared with WT mice (Figure 2, D and E). In addition, the transgene mRNA expression in the kidney is also threefold higher in Tg6 mice compared with Tg3 mice (Figure 2F). Moreover, Tg6 mice show polyuria (Supplemental Figure S1B) and albuminuria (Supplemental Figure S1C).
      Figure thumbnail gr2
      Figure 2Biochemical parameters in wild type (WT) and two different strains of netrin-1 transgenic mice at 10 weeks of age. A: Western blot analysis of renal expression of transgene chicken netrin-1 at different ages. B: Transgene chicken netrin-1 expression localizes in proximal tubular epithelial cells, as determined by colocalization of chicken netrin-1 (red) with aquaporin-1 (AQP-1; green), lotus tetragonolobus lectin (LTL)–fluorescein isothiocyanate (FITC; green), and peanut agglutinin (PNA)–FITC (green) in WT and Tg6 mice. C: Transgenic overexpression of chicken netrin-1 up-regulated endogenous netrin-1 expression. Mouse netrin-1 was measured in urine by enzyme-linked immunosorbent assay (ELISA). D: Transgene chicken netrin-1 in urine was measured by ELISA, expressed as ng/mg of creatinine. E: Chicken netrin-1 excretion in transgenic animals expressed as fold over WT endogenous netrin-1. Netrin-1 is produced in nanogram quantities in transgenic animals, whereas only in picogram quantities in WT animals. Urine netrin-1 excretion was >100-fold in Tg6 animal compared with WT animal urine. F: Chicken netrin-1 mRNA expression in two transgenic strains. Tg6 kidney mRNA levels of chicken netrin-1 are significantly higher than in Tg3 animal kidneys. Data are expressed as means ± SEM (CF). n = 6 to 8 mice (CF). ∗P < 0.05 versus WT; ∗∗P < 0.01 versus WT; ∗∗∗P < 0.001 versus WT or Tg3; P < 0.05, †††P < 0.001 versus Tg3. Scale bars = 100 μm (B).

      Netrin-1 Overexpression Increases Urinary cAMP Excretion and Expression of Early Response Genes Known to Be Associated with PKD Development

      PKD kidney epithelial cells show increased levels of cAMP production, which is associated with cyst growth.
      • Hanaoka K.
      • Guggino W.B.
      cAMP regulates cell proliferation and cyst formation in autosomal polycystic kidney disease cells.
      Because netrin-1 increases cAMP production,
      • Ming G.L.
      • Song H.J.
      • Berninger B.
      • Holt C.E.
      • Tessier-Lavigne M.
      • Poo M.M.
      cAMP-dependent growth cone guidance by netrin-1.
      ,
      • Corset V.
      • Nguyen-Ba-Charvet K.T.
      • Forcet C.
      • Moyse E.
      • Chedotal A.
      • Mehlen P.
      Netrin-1-mediated axon outgrowth and cAMP production requires interaction with adenosine A2b receptor.
      the study addressed whether increased expression of netrin-1 was associated with increased expression of cAMP regulated genes in the kidney and increased excretion of cAMP in urine. As shown in Figure 3A, Tg6 animals showed significantly increased levels of cAMP excretion compared with WT and Tg3 animals. There was no significant increase in the expression of netrin-1 receptor or polycystin-1 and polycystin-2 (Figure 3B) or glucosidase II alpha subunit (GANAB) (Supplemental Figure S2A) mRNA in kidney. However, endogenous mouse netrin-1 expression was increased (Figure 3B) and additional PKD-related genes, such as PKHD1 and HNF1B, were down-regulated in Tg6 transgenic mice (Supplemental Figure S2A). Interestingly, several early response genes and transcription factors known to be associated with cAMP pathways and cell proliferation were significantly up-regulated. These include Atf3, Cebpb, Nfatc4, PPARγ, and Fos (Supplemental Figure S2B). Atf3 and Fos up-regulation has been previously associated with ADPKD in humans.
      • Le N.H.
      • van der Wal A.
      • van der Bent P.
      • Lantinga-van Leeuwen I.S.
      • Breuning M.H.
      • van Dam H.
      • de Heer E.
      • Peters D.J.
      Increased activity of activator protein-1 transcription factor components ATF2, c-Jun, and c-Fos in human and mouse autosomal dominant polycystic kidney disease.
      In addition, several transcription factors, like Foxa2, Foxg1, Gata1, Id1, Kcnh8, Myt5, Myod1, Nanos2, and Pax6, were down-regulated (Supplemental Figure S2C). However, the role of these transcription factors in cyst formation is not clear, which needs further investigation.
      Figure thumbnail gr3
      Figure 3cAMP excretion and cell proliferation and dedifferentiation marker expression at 10 weeks of age in wild-type (WT) and netrin-1 transgenic animal kidneys at 10 weeks of age. A: cAMP excretion is significantly increased in Tg6 animals compared with WT and Tg3 animals. B: Mouse Netrin-1 (mNetrin-1) and its receptors [uncoordinated-5A-D homolog (UNC5A-D) and deleted in colorectal cancer (DCC)], and polycystin 1 (Pkd1) and polycystin-2 (Pkd2) gene expression in WT and netrin-1 transgenic mouse kidneys. No significant alteration in the expression of these genes is seen, except for endogenous mouse netrin-1 mRNA, which is up-regulated moderately in transgenic animal kidneys. C: Cell proliferation determined by staining with Ki-67 antibodies (red) with peanut agglutinin (PNA)–fluorescein isothiocyanate (green). Yellow arrows indicate Ki-67–positive cells. Tg6 animal kidneys showing larger numbers of Ki-67–positive cells only in PNA-positive proximal tubular epithelium. Dedifferentiation of epithelial cell was determined by checking the expression of vimentin. Vimentin localizes mainly in the glomerulus and inner medulla of WT mouse kidney but is highly expressed in the interstitium of Tg6 animal kidney. Fibrosis was determined by trichrome staining. Tg6 animal kidney showing extensive fibrosis compared with WT mice kidney. D: Quantification of Ki-67–positive nuclei in PNA-positive proximal tubular epithelium and PNA-negative tubular epithelium in WT and Tg6 mouse kidney. E: Quantification of fibrosis in the kidney, as explained in . F: Western blot analysis of different kinases, pro-apoptotic protein p53 in WT, and netrin-1 transgenic mouse kidneys. GI: Densitometric quantification of Western blot analyses. Levels expressed as fold over WT after normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Total p53, but not phosphorylated p53 (p-p53) or phosphorylated focal adhesion kinase (p-FAK), is significantly increased in Tg6 mouse kidney compared with WT and Tg3 mouse kidney. Phosphorylated extracellular signal-regulated kinase (p-ERK) increases equally in both Tg6 and Tg3 compared with WT animal kidney. Data are expressed as means ± SEM (A, B, D, E, and GI). n = 6 mice (A, B, D, E, and GI).∗P < 0.05, ∗∗∗P < 0.001 versus WT; P < 0.05 versus Tg3 mice. Scale bars = 100 μm (C).

      Tubular Epithelial Cell Proliferation Increases with Fibrosis in Tg6 Netrin-1 Transgenic Mice

      Because cyst formation is usually associated with increased epithelial cell proliferation, cell proliferation was determined with Ki-67 staining. As shown in Figure 3C, not many cells were positive for Ki-67 in WT kidney, whereas a large number of Ki-67–positive epithelial cells was seen in Tg6 animal kidneys. Proliferating cells were localized in the proximal tubular epithelium, as shown by colocalization with proximal tubule–specific lectin peanut agglutinin (Figure 3C), and quantitative data are shown in Figure 3D. It has been suggested that proliferating cystic epithelial cells may dedifferentiate to acquire an embryonic phenotype.
      • Humphreys B.D.
      • Valerius M.T.
      • Kobayashi A.
      • Mugford J.W.
      • Soeung S.
      • Duffield J.S.
      • McMahon A.P.
      • Bonventre J.V.
      Intrinsic epithelial cells repair the kidney after injury.
      • Humphreys B.D.
      • Czerniak S.
      • DiRocco D.P.
      • Hasnain W.
      • Cheema R.
      • Bonventre J.V.
      Repair of injured proximal tubule does not involve specialized progenitors.
      • Kusaba T.
      • Lalli M.
      • Kramann R.
      • Kobayashi A.
      • Humphreys B.D.
      Differentiated kidney epithelial cells repair injured proximal tubule.
      Therefore, the study determined whether netrin-1 affects epithelial cell dedifferentiation using vimentin expression. As shown in Figure 3C, vimentin expression was confined to the glomerulus in WT kidneys, whereas intense expression was seen in the interstitium of Tg6 mouse kidneys. Because most of the vimentin staining was seen in the interstitium but not in epithelial cells, this suggested that vimentin may be synthesized from myofibroblasts. PKD is also associated with increased fibrosis.
      • Harris P.C.
      • Torres V.E.
      Polycystic kidney disease.
      ,
      • Smith L.A.
      • Bukanov N.O.
      • Husson H.
      • Russo R.J.
      • Barry T.C.
      • Taylor A.L.
      • Beier D.R.
      • Ibraghimov-Beskrovnaya O.
      Development of polycystic kidney disease in juvenile cystic kidney mice: insights into pathogenesis, ciliary abnormalities, and common features with human disease.
      ,
      • Thivierge C.
      • Kurbegovic A.
      • Couillard M.
      • Guillaume R.
      • Cote O.
      • Trudel M.
      Overexpression of PKD1 causes polycystic kidney disease.
      Therefore, fibrosis was determined using trichrome staining and real-time PCR. Although WT kidneys did not stain for trichrome, Tg6 mouse kidneys showed extensive fibrosis in the interstitial space (Figure 3, C and E) and associated with increased expression of profibrotic genes (Supplemental Figure S2D).

      Netrin-1 Overexpression Inactivates p53 but Increases the Activation of ERK and Focal Adhesion Kinase and Expression of Vimentin

      p53 expression plays a critical role in the transition from undifferentiated embryonic phenotype to fully differentiated polarized epithelial cells in renal tubules.
      • Saifudeen Z.
      • Dipp S.
      • El-Dahr S.S.
      A role for p53 in terminal epithelial cell differentiation.
      In addition, a p53 knockout mouse developed PKD.
      • Li H.
      • Zhang Y.
      • Dan J.
      • Zhou R.
      • Li C.
      • Li R.
      • Wu X.
      • Kumar Singh S.
      • Chang J.T.
      • Yang J.
      • Luo Y.
      p53 Mutation regulates PKD genes and results in co-occurrence of PKD and tumorigenesis.
      Because netrin-1 suppresses the activation of p53, thereby promoting cancer cell growth,
      • Tanikawa C.
      • Matsuda K.
      • Fukuda S.
      • Nakamura Y.
      • Arakawa H.
      p53RDL1 regulates p53-dependent apoptosis.
      p53 activity was determined in Tg6 mice. Surprisingly, the expression of p53 increased severalfold in Tg6 kidney compared with WT and Tg3 kidney. However, all p53 was in an inactive form, and no phosphorylated p53 was detected in the Tg6 mice (Figure 3, F and G). In addition, Tg6 kidneys showed significantly increased activation of ERK compared with WT kidneys and focal adhesion kinase compared with WT and Tg3 kidneys (Figure 3, F, H, and I). The expression of vimentin also increased significantly (Supplemental Figure S2, E and F) in Tg6 kidneys. The expression of E-cadherin increased slightly, but the phosphorylated Src level was not changed (Supplemental Figure S2E).

      Addition of Netrin-1 to Proximal Tubular Epithelial Cells in Culture or Overexpression in Vivo Increases Cilium Length

      Studies using hypomorphic mutant mice (Nphp3pcy/ko, Nphp9jck/jck, and PKD1RC/RC)
      • Hopp K.
      • Ward C.J.
      • Hommerding C.J.
      • Nasr S.H.
      • Tuan H.F.
      • Gainullin V.G.
      • Rossetti S.
      • Torres V.E.
      • Harris P.C.
      Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity.
      ,
      • Smith L.A.
      • Bukanov N.O.
      • Husson H.
      • Russo R.J.
      • Barry T.C.
      • Taylor A.L.
      • Beier D.R.
      • Ibraghimov-Beskrovnaya O.
      Development of polycystic kidney disease in juvenile cystic kidney mice: insights into pathogenesis, ciliary abnormalities, and common features with human disease.
      ,
      • Bergmann C.
      • Fliegauf M.
      • Bruchle N.O.
      • Frank V.
      • Olbrich H.
      • Kirschner J.
      • Schermer B.
      • Schmedding I.
      • Kispert A.
      • Kranzlin B.
      • Nurnberg G.
      • Becker C.
      • Grimm T.
      • Girschick G.
      • Lynch S.A.
      • Kelehan P.
      • Senderek J.
      • Neuhaus T.J.
      • Stallmach T.
      • Zentgraf H.
      • Nurnberg P.
      • Gretz N.
      • Lo C.
      • Lienkamp S.
      • Schafer T.
      • Walz G.
      • Benzing T.
      • Zerres K.
      • Omran H.
      Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia.
      show that the length of renal tubular cilia is increased compared with that of WT mice. This is proposed to play an important role in controlling cell proliferation and maintenance of cells in a differentiated state. To determine whether netrin-1 regulates cilium length, mouse proximal tubular epithelial cells were treated with netrin-1 (100 ng/mL) or vehicle in the presence or absence of serum. Serum induced cilium resorption, whereas the absence of serum induced cilium growth. As shown in Supplemental Figure S3, A and B, netrin-1 significantly increased cilium length in both the presence or the absence of serum compared with that in vehicle-treated controls. Consistent with in vitro observation, Tg6 transgenic mouse kidney tubule also showed increased cilium length compared with that in WT mice (Supplemental Figure S3, A and C).

      siRNA-Mediated Suppression of Transgene Expression Reduces Cyst Growth in Tg6 Transgenic Mice

      To determine directly whether increased expression of netrin-1 is responsible for cyst formation and growth and to exclude an insertional effect, at 4 weeks of age, siRNA or scrambled siRNA were injected intravenously twice a week into Tg6 animals for the following 4 weeks. As shown in Figure 4, scrambled siRNA infused Tg6 animal kidneys showed multiple cysts (Figure 4A) with increased kidney weight (Figure 4C). However, specific siRNA against chicken netrin-1 completely suppressed cyst formation (Figure 4B) and reduced kidney weight (Figure 4C). Scrambled siRNA-infused animal kidneys had extensive fibrosis (Figure 4, D and F), which was suppressed in specific siRNA infused animal kidneys (Figure 4, E and F). Knockdown of transgene expression was confirmed by quantifying chicken netrin-1 excretion in urine. As shown in Figure 4G, infusion with specific siRNA, but not with scrambled siRNA, completely suppressed excretion of netrin-1 in urine.
      Figure thumbnail gr4
      Figure 4Chicken netrin-1 siRNA infusion suppresses cyst growth in Tg6 animals. A and B: Photomicrograph of Tg6 animal kidneys from a scrambled siRNA (scr-siRNA)-infused mouse (A) and a specific siRNA-infused mouse (B). Specific siRNA infusion suppresses cyst growth. C: Netrin-1–specific siRNA infusion suppresses kidney growth. D: Trichrome-stained tissue section from scrambled siRNA-infused Tg6 mouse kidney, showing extensive fibrosis and a large number of cysts. E: Trichrome-stained tissue section from chicken netrin-1–specific siRNA-infused Tg6 mouse kidney, showing normal morphology. F: Quantification of fibrosis in the kidney. Administered Tg6 transgenic mice. G: Urine chicken netrin-1 excretion in scrambled siRNA-infused mouse and specific siRNA-infused mouse, measured by enzyme-linked immunosorbent assay. siRNA infusion completely suppressed the transgene chicken netrin-1 expression. Data are expressed as means ± SEM (C, F, and G). n = 6 to 8 (C, F, and G). ∗P < 0.05, ∗∗∗P < 0.001 versus wild type (WT); P < 0.05, †††P < 0.001 versus scr-siRNA. Scale bars = 100 μm (D and E).

      Ovarian Hormone Is Responsible for the Suppression of Cyst Formation in Female Tg6 Netrin-1 Transgenic Animals

      A notable feature of netrin-1 Tg6 mice is the restriction of PKD development to male mice (Table 1). However, no change in transgene netrin-1 expression in the kidney in Tg6 males and females at 10 weeks of age was observed (Figure 5A). To determine whether ovarian hormones such as estrogen contribute to the suppression of cyst formation in netrin-1 transgenic female mice, female transgenic mice were ovariectomized or sham operated at the age of 5 weeks. As shown in Figure 5, sham-operated female mice did not develop cysts at 40 weeks. However, ovariectomized female mice developed cysts and increased kidney weight at 40 weeks (Figure 5, B and C). Consistent with increased kidney weight and cyst formation, kidney function decline was also seen in ovariectomized females compared with sham-operated female mice (Figure 5, D and E).
      Figure thumbnail gr5
      Figure 5Ovariectomy induces innumerable cyst formation in the netrin-1 transgenic mice (Tg6) after 40 weeks. A: Expression level of transgene chicken netrin-1 in male and female mice kidney at 10 weeks of age, measured by Western blot analysis. B: Photomicrograph showing cystic kidney in ovariectomized transgenic mouse. C: Kidney weight in sham-operated and ovariectomized female transgenic mice. D and E: Blood urea nitrogen (BUN) and serum creatinine levels in sham and ovariectomized mice. Data are expressed as means ± SEM (CE). n = 6 to 8 animals in each group (CE). ∗∗P < 0.01 versus sham. WT, wild type.

      Pkd1 Conditional Knockout Mice and Han:SPRD Rats Excrete a Large Amount of Netrin-1 and Show Increased Expression of Netrin-1 in the Cystic Kidney

      To determine whether netrin-1 is involved in cystogenesis in other rodent models of PKD, the renal expression and urinary excretion of netrin-1 in conditional Pkd1 knockout mice and in Han:SPRD rats were measured. As shown in Supplemental Figure S4, WT mice (Supplemental Figure S4, A and C) and rats (Supplemental Figure S4, B and D) showed a low level of netrin-1 in urine, which was increased severalfold in polycystin-1 mutant mice and in rat with cyst. Consistent with increased excretion, the expression of netrin-1 was also increased in the kidney compared with respective wild-type animals (Supplemental Figure S4, E and F).

      siRNA-Mediated Suppression of Netrin-1 Reduces Cyst Growth in Human PKD1 p.R3277C Knock-In and Pkd1 Knockout Animals in Vivo

      To confirm the role of netrin-1 in cyst formation and growth in established PKD animal models, Pkd1 knockout and PKD1 knock-in mice were treated with mouse netrin-1 siRNA or scrambled siRNA through tail vein at 10 weeks of age for the following 4 weeks. Administration of siRNA against netrin-1 suppressed cyst growth in the kidneys of human PKD1 p.R3277C knock-in animals (Figure 6, A–D) and Pkd1 knockout animals (Supplemental Figure S5, A–D). Netrin-1 expression and effect of siRNA on endogenous netrin-1 expression in cystic epithelium were confirmed by immunohistochemistry and by Western blot analysis in PKD1 knock-in animals (Figure 6, E–G and N) and Pkd1 knockout animals (Supplemental Figure S5, E–G and N), respectively. Trichrome staining showed increased fibrosis in both scrambled siRNA treated human PKD1 p.R3277C knock-in (Figure 6, I and K) and Pkd1 knockout (Supplemental Figure S5, I and K) compared with wild type (Figure 6H and Supplemental Figure S5H), which was reduced with netrin-1 siRNA infusion in human PKD1 p.R3277C knock-in mice (Figure 6, J and K) and Pkd1 knockout mice (Supplemental Figure S5, J and K). Structural improvement in the kidney was associated with improvement in kidney function, as measured by serum creatinine and blood urea nitrogen in siRNA administered PKD1 knock-in (Figure 6, L and M) and Pkd1 knockout (Supplemental Figure S5, L and M) compared with scrambled siRNA-treated animals.
      Figure thumbnail gr6
      Figure 6Mouse netrin-1 siRNA infusion suppresses cyst growth in human PKD1 knock-in animals. AC: Photomicrograph of periodic acid–Schiff (PAS)–stained wild-type (WT) mice (A), vehicle-treated PKD1 knock-in mice (B), and netrin-1 siRNA administered PKD1 knock-in mice (C). D: Specific siRNA infusion suppresses cyst growth, expressed as percentage of kidney weight (KW)/body weight (BW). EG: Immunohistochemical localization of netrin-1 in WT (E) and PKD1 knock-in mice treated with vehicle (F) and siRNA (G). HJ: Trichrome staining to assess fibrosis in WT (H) and PKD1 knock-in mice treated with scrambled siRNA (scr-siRNA; I) and netrin-1 siRNA (J). K: Quantification of fibrosis in the kidney in WT, scrambled siRNA, and siRNA-treated PKD1 knockout mice. L and M: Kidney function was determined by quantifying blood urea nitrogen (BUN; L) and serum creatinine (M) at the end of the experiments. N: Western blot analysis of netrin-1 excretion in urine. Data are expressed as means ± SEM (D and KM). n = 6 animals in each group (D and KM). ∗P < 0.05, ∗∗P < 0.01 versus WT; P < 0.05 versus scr-siRNA administered PKD1 knock-in mice. Scale bars = 100 μm (AC, EG, and HJ).

      Discussion

      PKD is a developmental genetic disorder that causes progressive loss of kidney function. The mechanism by which PKD pathogenesis occurs is not fully understood. The current study describes a pathogenic role of netrin-1 in the development of PKD using a transgenic mouse model. One of the transgenic strains that produces large amounts of netrin-1 in the tubular epithelial cells developed numerous cysts in the kidney by the age of 4 weeks, and growth of the cysts expanded rapidly along with age. Cyst development was associated with increased excretion of cAMP, albuminuria, and polyuria. Netrin-1 also induced increased cell proliferation that was associated with p53 inactivation, increased activation of ERK and focal adhesion kinase, and dedifferentiation marker vimentin in the kidney. Inhibition of the chicken netrin-1 in transgenic mice and mouse netrin-1 in PKD mice model using siRNA suppressed cyst growth, suggesting that chronic up-regulation of netrin-1 causes innumerable cyst formation in the kidney. Thus, these data demonstrate a possible new therapeutic pathway for the treatment of PKD.
      Several morphogenic or guidance cues, including netrin 1, that are normally expressed only during embryogenesis are shown to be induced after kidney injury.
      • Wang W.
      • Reeves W.B.
      • Ramesh G.
      Netrin-1 and kidney injury, I: netrin-1 protects against ischemia-reperfusion injury of the kidney.
      ,
      • Wang W.
      • Reeves W.B.
      • Ramesh G.
      Netrin-1 increases proliferation and migration of renal proximal tubular epithelial cells via the UNC5B receptor.
      ,
      • Reeves W.B.
      • Kwon O.
      • Ramesh G.
      Netrin-1 and kidney injury, II: netrin-1 is an early biomarker of acute kidney injury.
      ,
      • Ramesh G.
      • Krawczeski C.D.
      • Woo J.G.
      • Wang Y.
      • Devarajan P.
      Urinary netrin-1 is an early predictive biomarker of acute kidney injury after cardiac surgery.
      The expression of netrin-1 is transient and returns to normal levels once tubular epithelial cells are regenerated or kidney recovers from injury, suggesting that netrin-1 expression in the adult kidney is well regulated, which is required for regeneration of the kidney tubules after an acute injury (Figure 7A). However, chronic up-regulation of netrin-1 proximal tubular epithelium led to uncontrolled proliferation and maintenance of an undifferentiated state (Figures 3 and 7, B and C). Moreover, cAMP excretion was also increased in netrin-1 overexpressed transgenic mice, which is known to increase cell proliferation by stimulating ERK signaling and promote cyst growth.
      • Yamaguchi T.
      • Nagao S.
      • Wallace D.P.
      • Belibi F.A.
      • Cowley B.D.
      • Pelling J.C.
      • Grantham J.J.
      Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys.
      Consistent with earlier reports,
      • Wang W.
      • Reeves W.B.
      • Ramesh G.
      Netrin-1 increases proliferation and migration of renal proximal tubular epithelial cells via the UNC5B receptor.
      these studies show that netrin-1 overexpression activated ERK in the Tg6 kidney compared with WT (Figure 3). In addition, netrin-1 overexpression increased cilium length in tubular epithelial cells (Supplemental Figure S3). In support of this, a recent study reported that cilia were elongated in kidneys from ADPKD patients and in both Pkd1 and Pkd2 knockout mice. Reduction of cilium length in mouse models of autosomal polycystic kidney disease delayed kidney cystogenesis and reduced cell proliferation.
      • Shao L.
      • El-Jouni W.
      • Kong F.
      • Ramesh J.
      • Kumar R.S.
      • Shen X.
      • Ren J.
      • Devendra S.
      • Dorschel A.
      • Wu M.
      • Barrera I.
      • Tabari A.
      • Hu K.
      • Haque N.
      • Yambayev I.
      • Li S.
      • Kumar A.
      • Behera T.R.
      • McDonough G.
      • Furuichi M.
      • Xifaras M.
      • Lu T.
      • Alhayaza R.M.
      • Miyabayashi K.
      • Fan Q.
      • Ajay A.K.
      • Zhou J.
      Genetic reduction of cilium length by targeting intraflagellar transport 88 protein impedes kidney and liver cyst formation in mouse models of autosomal polycystic kidney disease.
      These data suggest that elongation of cilium length by netrin-1 may influence the tubular epithelial cell properties by inducing dedifferentiation and/or cell proliferation and stimulating cyst growth through cAMP-mediated ERK or Akt pathways.
      Figure thumbnail gr7
      Figure 7Model depicting the proposed mechanism for netrin-1–mediated cyst formation in the kidney. A: Schematic representation of the role of netrin-1 in epithelial cell proliferation and migration required for effective regeneration of injured tubules in adult kidney, which was observed in Tg3 mice. B: In Tg6 strain, excess netrin-1 induced uncontrolled epithelial cell proliferation and maintained an undifferentiated state that was suppressed by the presence of ovarian hormones in females. C: However, if netrin-1 was produced in an uncontrolled manner during development, epithelial cells maintained their undifferentiated state through suppression of cAMP-dependent inactivation of p53, which led to the arrest of terminal differentiation. Continued epithelial cell proliferation led to the formation of innumerable cysts in the kidney.
      Netrin-1 is up-regulated in tubular epithelium of Pkd1 knockout (B6.129S4-Pkd1tm2Ggg/J) and human PKD1 p.R3277C knock-in mice. Inhibition of netrin-1 reduced cyst growth, suggesting that netrin-1 contributes to increased cyst growth in Pkd1 knockout and PKD1 knock-in mice. However, it is not clear how PKD1 inactivation or deletion causes up-regulation of netrin-1 in tubular epithelium, where netrin-1 is not normally expressed. One possibility is that extreme overexpression of nertrin-1 may disturb maturation and trafficking of these proteins. However, netrin-1 overexpression did not alter polycystin-1 and polycystin-2 mRNA expression in the kidney. Recently, a netrin-2–like gene was cloned on chromosome 16p13.3, the region containing the Pkd1 gene.
      • Van Raay T.J.
      • Foskett S.M.
      • Connors T.D.
      • Klinger K.W.
      • Landes G.M.
      • Burn T.C.
      The NTN2L gene encoding a novel human netrin maps to the autosomal dominant polycystic kidney disease region on chromosome 16p13.3.
      However, mutations in this gene have not yet been linked to human PKD. PKD1 gene prevents the immortalized proliferation of renal tubular epithelial cells via p53 induction,
      • Nishio S.
      • Hatano M.
      • Nagata M.
      • Horie S.
      • Koike T.
      • Tokuhisa T.
      • Mochizuki T.
      Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation.
      and p53 expression is associated with terminal differentiation in renal epithelial cells.
      • Saifudeen Z.
      • Dipp S.
      • El-Dahr S.S.
      A role for p53 in terminal epithelial cell differentiation.
      In addition, p53 knockout mice show abnormal kidney morphology with cyst formation.
      • Saifudeen Z.
      • Dipp S.
      • El-Dahr S.S.
      A role for p53 in terminal epithelial cell differentiation.
      The p53 activation by phosphorylation was known to induce cell cycle arrest and apoptosis. Moderate overexpression of netrin-1 or deletion of receptor uncoordinated-5b homolog suppresses p53 activation by inhibiting its phosphorylation in the kidney of Tg3 mice.
      • Rajasundari A.
      • Pays L.
      • Mehlen P.
      • Ramesh G.
      Netrin-1 overexpression in kidney proximal tubular epithelium ameliorates cisplatin nephrotoxicity.
      ,
      • Tanikawa C.
      • Matsuda K.
      • Fukuda S.
      • Nakamura Y.
      • Arakawa H.
      p53RDL1 regulates p53-dependent apoptosis.
      ,
      • Mehlen P.
      • Bredesen D.E.
      The dependence receptor hypothesis.
      ,
      • Ranganathan P.
      • Jayakumar C.
      • Navankasattusas S.
      • Li D.Y.
      • Kim I.M.
      • Ramesh G.
      UNC5B receptor deletion exacerbates tissue injury in response to AKI.
      Surprisingly, p53 phosphorylation was completely absent and high levels of p53 protein accumulated in Tg6 mice kidneys, suggesting that p53 accumulates in an inactive form that may lead to increased survival of renal epithelial cells and maintenance of an undifferentiated state that leads to cyst growth in the kidney (Figure 7C).
      Netrin-1 overexpression induced sex-specific cyst formation because only male animals developed cysts compared with age-matched females (Table 1). However, there is no significant production of netrin-1 observed between males and females (Figure 4A). The reason behind this observed sex difference is unknown. Moreover, so far there is no report available explaining sexual dimorphism of netrin-1 and its regulation and cellular function in health and disease states. The siRNA-mediated netrin-1 knockdown shows that netrin-1 does directly mediate cyst formation and growth observed in male transgenic mice. Therefore, additional factors in male mice may interact with netrin-1 to enhance cystogenesis or, alternatively, cyst growth may be suppressed in female mice by sex hormones. Surprisingly, ovariectomized females developed cysts at 40 weeks after ovariectomy, suggesting that ovarian hormone may be responsible for the observed sex differences in cyst development. Male patients with ADPKD typically show a larger cyst burden and reach end-stage renal disease on average at an earlier age compared with female patients with ADPKD. Although it was suggested that the large kidney size and abnormal fatty acid oxidation
      • Menezes L.F.
      • Lin C.C.
      • Zhou F.
      • Germino G.G.
      Fatty acid oxidation is impaired in an orthologous mouse model of autosomal dominant polycystic kidney disease.
      in males may be a contributing factor for such accelerated cyst growth, clear mechanisms have not been identified.
      • Grantham J.J.
      Clinical practice: autosomal dominant polycystic kidney disease.
      ,
      • Harris P.C.
      • Torres V.E.
      Polycystic kidney disease.
      ,
      • Torres V.E.
      • Chapman A.B.
      • Perrone R.D.
      • Bae K.T.
      • Abebe K.Z.
      • Bost J.E.
      • Miskulin D.C.
      • Steinman T.I.
      • Braun W.E.
      • Winklhofer F.T.
      • Hogan M.C.
      • Oskoui F.R.
      • Kelleher C.
      • Masoumi A.
      • Glockner J.
      • Halin N.J.
      • Martin D.R.
      • Remer E.
      • Patel N.
      • Pedrosa I.
      • Wetzel L.H.
      • Thompson P.A.
      • Miller J.P.
      • Meyers C.M.
      • Schrier R.W.
      • Group H.P.S.
      Analysis of baseline parameters in the HALT polycystic kidney disease trials.
      This study suggests that female sex hormones may contribute, to some extent, to the observed difference in cyst growth. More studies are needed to understand the role of netrin-1 in mediating sex-specific regulation of biological function in males and females.
      In summary, this study demonstrated that excessive and chronic overexpression of netrin-1 in tubular epithelial cells induces cyst formation in the kidney in a sex-specific manner. Cyst formation was associated with cell proliferation, inactivation of p53, and enhanced activation of cAMP and ERK pathways. siRNA-mediated suppression of netrin-1 expression completely suppressed cyst growth, suggesting the direct role of netrin-1 in cyst formation in the kidney. Further studies are needed to determine the signaling pathways that lead to increased netrin-1 expression in cystic epithelium and its role in human PKD.

      Author Contributions

      R.M. conceived and designed research, performed experiments, analyzed data, prepared figures, and wrote the manuscript; R.M. and M.T. interpreted results; R.M., M.T., A.D.K., and P.C.H. edited and revised the manuscript; R.M., M.T., Y.L., A.D.K., and P.C.H. approved the final version of the article.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1Tg6 netrin-1 transgenic mice develop proteinuria at 10 weeks of age. Kidney function was assessed by measuring serum creatinine and albuminuria at 10 weeks. A: Serum creatinine is significantly increased in Tg6 mice compared with that in wild type (WT) at 10 weeks of age. B and C: Urine volume (B) and urine albumin excretion (C) are significantly increased in Tg6 animals compared with those in WT and Tg3 animals. Data are expressed as means ± SEM (AC). n = 6 mice (AC). ∗P < 0.05 versus WT; P < 0.05 versus Tg3.
      Figure thumbnail figs2
      Supplemental Figure S2Gene expression in wild-type (WT) and netrin-1 transgenic animal kidneys at 10 weeks of age. A: PKD gene mRNA expression in Tg6 kidney compared with WT at 10 weeks of age. B and C: PCR array analysis of transcription factor expression in WT and Tg6 kidney. Several transcription factors are up-regulated, but many others are down-regulated significantly. D: RT-PCR analysis of profibrotic gene expression in WT and Tg6 animal kidney at 10 weeks of age. E: Western blot analysis of different kinases and dedifferentiation marker vimentin in WT and netrin-1 transgenic mouse kidneys. F: Densitometric quantification of vimentin is significantly increased in Tg6 mouse kidney compared with WT and Tg3 mouse kidney. Levels are expressed as fold over WT after normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are expressed as means ± SEM (AD and F). n = 5 to 6 mice (AD and F). ∗P < 0.05 versus WT; P < 0.05 versus Tg3 mice. CTGF, connective tissue growth factor; TGF-β1, transforming growth factor-β1.
      Figure thumbnail figs3
      Supplemental Figure S3Netrin-1 overexpression increases cilium length in Tg6 netrin-1 transgenic mouse kidney. A: Mouse proximal tubular epithelial cells (TKPTS) were treated with netrin-1 (100 ng/mL) in the presence or absence of serum (5%) for 72 hours and then stained for acetylated tubulin. B: Cell cilium length quantified using Olympus microscope with Cellsens Standard software (version 1.14). Cilium length expressed as micrometer. A and C: WT and netrin-1 transgenic mouse kidney were stained with anti-acetylated tubulin, and cilium length quantified using Cellsens Standard software and Olympus microscope. Cilium length expressed as micrometer. Data are expressed as means ± SEM (B and C). n = 3 (B and C).∗P < 0.05 versus wild-type (WT) mice; ∗∗P < 0.01 versus vehicle controls. Scale bars = 20 μm (A).
      Figure thumbnail figs4
      Supplemental Figure S4Polycystin-1 mutant mouse and rat show increased expression of netrin-1 in cystic kidney and excretion in urine. Polycystin-1 gene was deleted as described in . The 12-week–old cystic mouse (A, C, and E) and rat (B, D, and F) urine and kidney were used to determine netrin-1 excretion and expression by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Urine from cystic mouse and rat showed a significant increase in netrin-1 excretion compared with wild-type (WT) animals. Similarly, cystic kidney showed increased expression of netrin-1 compared with respective WT animal kidney. Data are expressed as means ± SEM (AD). n = 5 to 6 (AD). ∗∗∗P < 0.001 versus WT. Scale bars = 100 μm (E and F).
      Figure thumbnail figs5
      Supplemental Figure S5Mouse netrin-1 siRNA infusion reduces cyst growth in Pkd1 knockout (KO) animals. AC: Photomicrograph of periodic acid–Schiff (PAS)–stained wild-type (WT) mice (A), scrambled siRNA (scr-siRNA) treated Pkd1 knockout mice (B), and netrin-1 siRNA administered Pkd1 knockout (C) mice. D: Specific siRNA infusion suppresses cyst growth, expressed as percentage of kidney weight (KW)/body weight (BW). EG: Immunohistochemical localization of netrin-1 in WT (E) and Pkd1 knockout mice treated with scr-siRNA (F) and siRNA (G). HJ: Trichrome staining to assess fibrosis in WT (H) and Pkd1 knockout mice treated with scr-siRNA (I) and siRNA (J). K: Quantification of fibrosis in the kidney. L and M: Kidney function was determined by quantifying blood urea nitrogen (BUN; L) and serum creatinine (M) at the end of the experiments. N: Western blot analysis of netrin-1 excretion in urine. Data are expressed as means ± SEM (D and KM). n = 6 (D and KM). ∗P < 0.05, ∗∗P < 0.01 versus WT; P < 0.05 versus scr-siRNA administered Pkd1 knockout mice. Scale bar = 100 μm (AC, EG, and HJ).

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