IHG-1 Amplifies TGF-β1 Signaling and Is Increased in Renal Fibrosis
Madeline Murphy*,
Neil G. Docherty*,
Brenda Griffin*,
Jillian Howlin*,
Emmett McArdle*,
Ruth McMahon*,
Holger Schmid,
Matthias Kretzler,
Alejandra Droguett,
Sergio Mezzano,
Hugh R. Brady*,
Fiona Furlong*,
Catherine Godson* and
Finian Martin*
* UCD Diabetes Research Centre, Conway Institute, Schools of Medicine and Medical Science and Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin, Ireland; Medizinische Poliklinik, Ludwig-Maximilians-University of Munich, Munich, Germany; and Department of Nephrology, Universidad Austral, Valdivia, Chile
Correspondence: Dr. Madeline Murphy, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. Phone: +353-1-7166818; Fax: +353-1-7166713; E-mail: madeline.murphy{at}ucd.ie
Received for publication October 8, 2007.
Accepted for publication March 14, 2008.
Induced in high glucose-1 (IHG-1) is an evolutionarily conservedgene transcript upregulated by high extracellular glucose concentrations,but its function is unknown. Here, it is reported that the abundanceof IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-richrenal biopsies from patients with diabetic nephropathy comparedwith control subjects. In the diabetic nephropathy specimens,in situ hybridization localized IHG-1 to tubular epithelialcells along with TGF-β1 and activated Smad3, suggestinga possible role in the development of tubulointerstitial fibrosis.Supporting this possibility, IHG-1 mRNA and protein expressionalso increased with unilateral ureteral obstruction. In theHK-2 proximal tubule cell line, overexpression of IHG-1 increasedTGF-β1–stimulated expression of connective tissuegrowth factor and fibronectin. IHG-1 was found to amplify TGF-β1–mediatedtranscriptional activity by increasing and prolonging phosphorylationof Smad3. Conversely, inhibition of endogenous IHG-1 with smallinterference RNA suppressed transcriptional responses to TGF-β1.In summary, IHG-1, which increases in diabetic nephropathy,may enhance the actions of TGF-β1 and contribute to thedevelopment of tubulointerstitial fibrosis.
Diabetic nephropathy (DN) is a leading cause of kidney disease,accounting for more than one third of all new cases of end-stagerenal failure in Western society.1, 2 In DN, glomerulosclerosisprecedes and primes for progressive accumulation of extracellularmatrix in the interstitial space, resulting in the developmentof tubulointerstitial fibrosis (TIF).3 TIF is a final commonpathway of injury in DN and other renal diseases of diverseetiology; the extent of tubular fibrosis mirrors closely lossof renal function.3, 4
TGF-β1 plays a key role in regulating the pathologic changesof kidney disease, resulting in the development of TIF.3–5TGF-β1 mediates interstitial myofibroblast activation,a critical event in the pathogenesis of interstitial fibrosis,and also induces epithelial-to-mesenchymal transformation (EMT)of tubular epithelial cells into myofibroblast cells, furthercontributing to renal interstitial fibrogenesis.6, 7 TGF-β1mediates its effects principally via activation of Smad proteins.8–10TGF-β1 receptor activation triggers phosphorylation ofthe receptor-regulated Smad (R-Smad) 2 and 3.8–10 PhosphorylatedR-Smad proteins bind to Smad4 and accumulate in the nucleus,where they activate transcription. The inhibitory Smad (I-Smad)6 and 7 act in a negative feedback loop to inhibit TGF-β1activity by preventing phosphorylation and/or nuclear accumulationof R-Smad proteins.11
The critical role of Smad signaling in renal fibrogenesis isdemonstrated by a number of in vivo studies. Renal fibrosisdid not develop in Smad3 knockout mice with streptozotocin-induceddiabetes or after unilateral ureteric obstruction (UUO), anacute model of TIF.12, 13 In addition, overexpression of Smad7has been shown to protect against kidney fibrosis in a numberof animal models, including DN14 and UUO.15
In this article, we report that induced in high glucose-1 (IHG-1),a novel, highly conserved transcript, is associated with DNand UUO and is localized to renal tubules. Overexpression ofIHG-1 amplifies TGF-β1–induced transcriptional activationin kidney tubule cells and enhances Smad3 phosphorylation. Inhibitionof endogenous IHG-1 expression suppresses transcriptional responsesto TGF-β1 and Smad3 phosphorylation. These data suggestthat increased IHG-1 levels are likely to contribute to theTGF-β1–induced profibrotic changes in tubular cellsthat prime for TIF.
IHG-1 Is a Conserved Gene Transcript Induced by High Extracellular Glucose
IHG-1 (NCBI accession no. AF110136), identified by suppressionsubtractive hybridization16–18 is homologous to THG1L(Genbank no. NM_017872, Unigene HS 353090).19, 20
The IHG-1 amino acid sequence contains a number of regions with>90% amino acid conservation between eukaryotic species (Figure 1A).This suggests that these regions are necessary for functionand therefore may represent or contain functional motifs; however,the only predicted functional domain to date is that of a mitochondriallocalization signal (Figure 1A). On discovery, therefore, IHG-1had no known function or functional classification.
Figure 1. IHG-1 is an evolutionarily conserved ubiquitously expressed transcript. (A) IHG-1 encodes an evolutionarily conserved protein. The predicted coding sequence of IHG-1 was aligned with homologous proteins from mouse, chicken, pufferfish, and mosquito. Conservation is indicated by text and background color: 90%, red; 70%, blue; 50%, yellow. Mitochondrial targeting and cleavage sequences that were predicted by the programs Target P server and MitoProt II 1.0a4 are boxed and labeled mTP. (B) IHG-1 expression in normal human tissues. Autoradiograph of IHG-1 mRNA levels analyzed by Northern blot (FirstChoice Human Blot 1; Ambion, Austin, TX). The blot contained 2 µg of poly A RNA from the following human tissues: brain (1), placenta (2), skeletal muscle (3), heart (4), kidney (5), pancreas (6), liver (7), lung (8), spleen (9), and colon (10). Bands at approximately 3 and 1.4 kb were detected after hybridization with the IHG-1 probe. (C) The influence of high ambient glucose on IHG-1 mRNA levels in human MC. MC were exposed to 5 mM glucose (lane 1) and 30 mM glucose (lane 2) for 7 d. (Left) Real-time analysis of IHG-1 expression. Data are presented as fold change after normalization to ribosomal RNA control. IHG-1 levels are increased approximately 29-fold after exposure to 30 mM glucose for 7 d. (Right) Autoradiograph of IHG-1 mRNA levels analyzed by Northern blot. A band of approximately 3 kb was detected after hybridization with the IHG-1 probe.
Analysis by Northern blot identified two IHG-1–relatedtranscripts of approximately 3 and 1.4 kb (Figure 1B) in 10human tissues, suggesting that IHG-1 was ubiquitously expressed.Although only the larger transcript (encoding IHG-1) was detectedas being induced in cultured mesangial cells (MC) treated withhigh extracellular glucose (Figure 1C), both transcripts aredetected in normal human kidney tissue, the smaller transcriptbeing more abundant (Figure 1B). Induction of IHG-1 mRNA expressionin primary human MC cultured in high glucose was confirmed byNorthern blotting and quantitative real-time reverse transcription–PCRwith primers directed against the open-reading frame (Figure 1C).
Increased Expression of IHG-1 Is Associated with Human DN
IHG-1 mRNA levels were significantly higher in tubule-rich microdissectedrenal biopsies from patients with DN (n = 13) as compared withthose taken from control kidney (mean 9.7-fold increase overcontrol; Figure 2A). Control tissue was from both individualswithout diabetes (n = 4) and from normal regions of tumor nephrectomies(n = 4).
Figure 2. Increased expression of IHG-1 mRNA is associated with DN. (A) IHG-1 mRNA levels are increased in DN. RNA was isolated from in tubule-rich microdissected renal biopsies taken from four living donors and four tumor nephrectomies (controls) and 11 patients with DN. Total RNA was reverse-transcribed into cDNA and analyzed for the expression of IHG-1 by quantitative real-time PCR (Taqman). Data are presented as fold change after normalization to ribosomal RNA control. Differences in means are significant (*P < 0.05). (B) IHG-1 expression in DN. In situ hybridization: (1) Sections obtained from patients with advanced DN showed marked IHG-1 staining in the tubules; (2) IHG-1 expression in normal kidney; no staining is seen; (3) IHG-1 sense probe; (4) TGF-β1 staining in DN showing strong tubular staining; (5) Southwestern (SW) showing activated Smad3 in tubules in DN; and (6) control for Smad3 (SW). (C) EGF but not TGF-β1 increases IHG-1 mRNA levels. RNA was isolated from HK-2 cells treated with EGF (10 ng/ml) or TGF-β1 (3 ng/ml) for 48 h. Total RNA was reverse-transcribed into cDNA and analyzed for the expression of IHG-1 by quantitative real-time PCR (Taqman). Data are presented as fold change after normalization to ribosomal RNA control ± SEM. Differences in means are significant (*P < 0.05).
IHG-1 expression in normal kidneys and in human DN was alsoassessed by means of in situ hybridization (ISH). Sections hybridizedwith sense probes showed no staining (e.g., Figure 2B, 3). Markedexpression of IHG-1 was observed in tubular epithelial cellsin biopsy specimens from patients with advanced DN (Figure 2B,1). TGF-β1 was also expressed abundantly in tubular epithelialcells in DN sections and seemed to have a similar pattern ofexpression as IHG-1 (Figure 2B, 4). In both cases, some tubulesstained more abundantly for these transcripts than others, mostlikely reflecting variations in cellular/regional responsesto disease stress. Activated Smad3 assessed by Southwesternanalysis was also observed to have a similar pattern of expressionin renal tubular cells in DN (Figure 2B, 5). Activated Smad3was localized to the nucleus as expected, whereas IHG-1 wasnot. These data suggest an association between expression ofthis gene and tubulointerstitial change in DN. This is an areacritical to disease progression and fibrosis and is associatedwith significant TGF-β1 presence and Smad3 function.21,22 IHG-1 expression was also increased and localized to renaltubules in progressive cases of the following fibrotic kidneydiseases: Systemic lupus erythematosus, IgA nephropathy, andmembranous nephropathy (S.M. et al., unpublished data). In contrastto Northern analysis and reverse transcription–PCR, IHG-1mRNA was not detected by ISH in normal kidney (Figure 2B, 2).This is most likely due to reduced sensitivity of the ISH technique.
Figure 3. IHG-1 expression is increased in rat UUO. (A) Histologic detection of TIF in UUO using Gomorri trichrome staining (magnification, x20) of a 10-d nonligated (NL; i) and ligated (L; ii) rat kidney illustrating mature collagen fibrils present in the expanded tubulointerstitium (arrows in ii). (B) Collagen 1 mRNA levels are increased in UUO. RNA was isolated from rat L and NL kidneys 3 (n = 6) and 10 d (n = 5) after UUO. Total RNA was reverse-transcribed into cDNA and analyzed for the expression of collagen I by quantitative real-time PCR (Taqman). Data are presented as fold change after normalization to ribosomal RNA control ± SEM. Differences in means are significant (*P < 0.05). (C) IHG-1 mRNA levels are increased in UUO. Experimental analysis and presentation of results is as in B. Differences in means are significant (P < 0.005). (D) IHG-1 protein levels are increased in UUO. Protein extracts were prepared from rat L and NL kidneys 3 (n = 3) and 10 d (n = 3) after UUO. Shown are Western analyses of these extracts probed for IHG-1.
EGF expression has been reported to be increased in experimentalDN23; in addition, high extracellular glucose transactivatesthe EGF receptor in proximal tubule cells.24 EGF receptor activationis believed to contribute to TIF.5 EGF but not TGF-β1 inducedthe expression of IHG-1 in HK-2 cells, a human proximal tubulecell line (Figure 2C). We previously reported both treatmentsto increase fibronectin expression and decrease E-cadherin expression,consistent with changes associated with fibrosis.7 When EGFand TGF-β1 were added in combination, IHG-1 expressionwas not stimulated.
IHG-1 Expression Is Increased in Rat Kidneys after UUO
We decided to use the UUO model of renal fibrosis to study furtherthe role of IHG-1 in TIF. TIF is the final pathway leading toend-stage renal disease in DN and in many chronic kidney diseases.21,22 Because fibrosis in this model does not occur secondary toa preexisting systemic disorder, it allowed us to examine whetherincreased expression of IHG-1 was a feature of TIF per se.
Three days after UUO, tubular dilation and interstitial inflammationwas evident in the affected kidney. At 10 d after UUO, the contralateralnonligated kidneys of rats showed a staining pattern with Gomorritrichrome consistent with a normal renal morphology. The mesangialmatrix and brush border of proximal tubules was clearly evident(Figure 3A, i). In the ligated kidney, TIF was evident (thinarrow, Figure 3A, ii) in areas of expanded and inflamed tubulointerstitium.Discrete areas of heavy collagen deposition were particularlynotable in perivascular regions (thick arrow, Figure 3A, ii).Collagen I mRNA levels were also significantly increased inUUO both at day 3 (8.3-fold; Figure 3B) and at day 10 afterUUO (eight-fold; Figure 3B). IHG-1 mRNA levels were significantlyincreased in UUO both at day 3 (2.8-fold; Figure 3C), beforedevelopment of tubulointerstitial fibrosis, and were still significantlyincreased at day 10 after UUO (2.2-fold; Figure 3C), when thetubulointerstitial lesion was well advanced (Figure 3A, ii).IHG-1 protein was increased in UUO at both day 3 and day 10(Figure 3D).
IHG-1 Expression Amplifies TGF-β1 Signal Transduction
Similar to DN, activation of the TGF-β1 pathway in UUOis a pivotal event leading to development of TIF.5 Because IHG-1expression was increased in kidney tubules in advanced DN andin the rat model of TIF, we examined the impact of IHG-1 overexpressionon TGF-β1 signal transduction. Mv 1 Lu cells have beenwidely used to analyze transcriptional responses to TGF-β1.25–28IHG-1 overexpression significantly enhanced TGF-β1 mediatedtranscription from a transfected TGF-β1–sensitiveplasminogen activator inhibitor (PAI) promoter reporter construct.Levels of reporter gene expression were on average four-foldgreater in cells overexpressing IHG-1 after TGF-β1 stimulationas compared with mock-transfected cells (Figure 4A). IHG-1 overexpressionhad no effect on PAI promoter activity in the absence of TGF-β1stimulation.
Figure 4. IHG-1 overexpression amplifies TGF-β1 signal transduction. (A) IHG-1 enhances 3TP-Lux activity in response to TGF-β1. Mv 1 Lu cells were co-transfected with the 3TP-Lux reporter (A through C) and phRL-CMV, an internal control reporter driving the expression of Renilla luciferase (Promega, Madison, WI), with or without pcDNA6-IHG-1-V5 expression plasmid, as indicated. Firefly luciferase activity normalized to Renilla luciferase activity was determined as directed by the manufacturer (Promega). Where indicated, cells were stimulated with 5 ng/ml TGF-β1 24 h after transfection. Luciferase activity was measured 24 h later. The results shown are means ± SEM of at least three independent experiments. IHG-1 significantly enhances TGF-β1 induced luciferase activity (*P < 0.05). (B) IHG-1 enhances 3TP-Lux activity in response to TGF-β1 in HK-2 cells stably transfected with pIRESpuro3-IHG-1-V5. Stably transfected HK-2 cells were co-transfected with the 3TP-Lux reporter and phRL-CMV. Experimental analysis and presentation of results is as in A. IHG-1 significantly enhances TGF-β1–induced luciferase activity (P < 0.0001). (C) IHG-1 enhances 3TP-lux activity in response to Smad3 stimulation. Mv 1 lu cells were co-transfected with p3TP-lux reporter, phRL-CMV, and combinations of pcDNA6-IHG-1-V5 and pRK5-Smad3 as indicated. Experimental analysis and presentation of results is as in A. IHG-1 significantly enhances Smad3-induced luciferase activity. IHG-1 significantly enhances Smad3-induced luciferase activity (*P < 0.05)
To investigate whether IHG-1 modulated TGF-β1 activityin a similar manner in kidney proximal tubule cells, we generateda stable cell line overexpressing IHG-1 in human renal tubularHK-2 cells. IHG-1 overexpression also increased levels of reportergene expression after TGF-β1 stimulation in HK-2 cellsand again had no effect on PAI-1 promoter activity in the absenceof TGF-β1 (Figure 4B). These data suggest that IHG-1 proteinamplifies the activity of TGF-β1. Because TGF-β1 isbelieved to be the key mediator of TIF, these data suggest thatIHG-1 may contribute to kidney disease by amplifying TGF-β1action.
Overexpressed Smad3 can activate a TGF-β1–sensitivepromoter in the absence of TGF-β1–induced Smad3 phosphorylation.It is suggested that under such conditions, multimerizationof Smad3 monomers induces activation.29 IHG-1 further enhancedactivation of the PAI-1 promoter by overexpressed Smad3, furthersuggesting that IHG-1 may modulate Smad signal transductionin more than one way (Figure 4C).
IHG-1 Expression Prolongs Smad3 Phosphorylation and Increases Fibronectin and Connective Tissue Growth Factor Expression in Response to TGF-β1
Smad-dependent responses to TGF-β1 are mediated by TGF-β1receptor–dependent Smad2 and/or Smad3 phosphorylation.10Overexpression of IHG-1 in stably transfected HK-2 cells resultedin increased levels of phosphorylated Smad3 after stimulationwith TGF-β1 (Figure 5A, right) as compared with mock-transfectedcells (Figure 5A, left): Higher levels of phosphorylated Smad3were seen in the IHG-1–overexpressing cells at each timepoint during the 180-min response observed, whereas total Smad3levels were unchanged by IHG-1 expression in this system (similarresults were obtained in HeLa cells; data not shown). Overexpressionof IHG-1 in HK-2 cells did not alter levels of pSmad2, p38,p42/44, or pAKT after stimulation with TGF-β1 (data notshown). These data suggest that IHG-1 facilitates increasedlevels and temporal prolongation of Smad3 phosphorylation. Itis proposed that induction of the profibrotic mediator connectivetissue growth factor (CTGF) by TGF-β1 is Smad3 dependent,30whereas TGF-β1–induced fibronectin expression maybe both Smad dependent and Smad independent.31–34 Overexpressionof IHG-1 after transduction of HK-2 cells resulted in increasedlevels of both CTGF (2.8-fold; Figure 5B) and fibronectin (2.6-fold;Figure 5C) protein after stimulation with TGF-β1 as comparedwith mock-transduced cells (CTGF 1.56-fold, fibronectin 1.48-fold).These data suggest that IHG-1 facilitated increases and prolongedphosphorylation of Smad3, enhancing transcriptional responsesto TGF-β1 along a fibrotic pathway.
Figure 5. IHG-1 overexpression increases levels of phosphorylated Smad3, CTGF, and fibronectin. (A, top) IHG-1 expression increases cellular phospho-Smad3 levels after TGF-β1 stimulation of HK-2 cells stably transfected with pIRESpuro3-IHG-1-V5. Protein extracts were prepared from stably transfected HK-2 cells in the presence or absence of TGF-β1 (5.0 ng/ml) stimulation for the times indicated. Shown are Western analyses of these extracts probed for V5, phospho-Smad3, and Smad3. The IHG-1-V5 fusion protein was detected using a V5 antibody. Results shown are representative blots. (Bottom) Alterations in phosphorylation for Smad3 levels were measured using densitometric analysis (n = 3). Shown are percentage of densitometric units ± SEM after normalization to total Smad3 protein. (B) IHG-1 expression increases CTGF levels after TGF-β1 stimulation. HK-2 cells were transduced by a lentivirus overexpressing IHG-1 (LLCIEP-IHG-1) and by a lentivirus with no insert (LLCIEP) for 24 h before serum starvation. Transduced cells were serum starved for 24 h before stimulation with TGF-β1. Protein extracts were prepared from transduced HK-2 cells in the presence or absence of TGF-β1 stimulation as indicated. Shown are Western analyses (left) of these extracts probed for CTGF, V5, and β-actin. The IHG-1-V5 fusion protein was detected using a V5 antibody. β-Actin was measured to control for equal loading of protein. Results shown are representative blots. Alterations in protein levels for CTGF were measured using densitometric analysis (right). IHG-1 enhanced TGF-β1–induced CTGF expression. Shown is fold change ± SEM after normalization to β-actin (n = 3). (C) IHG-1 expression increases fibronectin levels after TGF-β1 stimulation. Experimental analysis and presentation of results are as in A. Results shown are representative blots (left). Alterations in protein levels for fibronectin were measured using densitometric analysis (right). IHG-1 enhanced TGF-β1–induced fibronectin expression. Shown is fold change ± SEM after normalization to β-actin (n = 3).
Loss of IHG-1 Expression Inhibits TGF-β1 Signal Transduction
To determine whether endogenously expressed IHG-1 modulatesTGF-β1 signal transduction, we used small interfering RNA(siRNA) to achieve selective knockdown of IHG-1 in HK-2 cells.IHG-1–directed siRNA (10 nM) led to an eight-fold decreasein IHG-1 expression in transfected HK-2 cells as compared withcells transfected with scrambled siRNA (Figure 6A), whereasit had no effect on the expression of nontarget transcript glyceraldehyde-3-phosphatedehydrogenase (Figure 6B). Transfection of IHG-1 siRNA alsoled to a consistent 50% decrease in PAI-luciferase reportergene expression after TGF-β1 stimulation (Figure 6C) anddecreased levels and time of presence of phosphorylated Smad3(Figure 6D) after TGF-β1 stimulation, when compared withscrambled siRNA-transfected cells. These data demonstrate thatendogenous IHG-1 potentially plays an active role in regulatingthe level of TGF-β1 signaling responses in renal tubularepithelial cells.
Figure 6. siRNA directed against IHG-1 inhibits TGF-β1 activity. (A) HK-2 cells were transfected with either siRNA targeted against IHG-1 or scrambled siRNA. Total RNA was reverse-transcribed into cDNA and analyzed for the expression of IHG-1 by quantitative real-time PCR (Taqman). Data are presented as fold change after normalization to ribosomal RNA control. The results shown are means ± SEM of at least three independent experiments. IHG-1 mRNA is significantly decreased; differences in means are significant (*P < 0.05). (B) Total RNA was reverse-transcribed into cDNA and analyzed for the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by quantitative real-time PCR (Taqman). Experimental analysis and presentation of results are as in A. No change in GAPDH mRNA levels was observed on transfection with IHG-1–specific siRNA. (C) HK-2 cells were co-transfected with the 3TP-Lux reporter, phRL-CMV, and either siRNA targeted against IHG-1 or scrambled siRNA. Firefly luciferase activity normalized to Renilla luciferase activity was determined as directed by the manufacturer (Promega). Where indicated, cells were stimulated with 5 ng/ml TGF-β1 24 h after transfection. Luciferase activity was measured 24 h later. The results shown are means ± SEM of a representative experiment of three independent analyses. In all cases, siRNA directed against IHG-1 decreased TGF-β1 induction of the 3TP-lux reporter by at least 50%. (D) Loss of IHG-1 expression inhibits Smad3 phosphorylation. HK-2 cells were transfected with siRNA for IHG-1 or scrambled siRNA in serum-free medium for 24 h, before TGF-β1 stimulation. Protein extracts were prepared from these cells after TGF-β1 (5.0 ng/ml) stimulation for the times indicated. Shown are Western analyses (top) of these extracts probed for phospho-Smad3 and β-actin. β-Actin was analyzed to control for equal loading of protein. Results shown are representative blots (n = 3). (Bottom) Alterations in phosphorylation for Smad3 levels were measured using densitometric analysis (n = 3). Shown are percentage of densitometric units ± SEM.
We previously reported the identification of a novel gene transcript,IHG-1, in an in vitro screen for genes associated with developmentof DN.15, 16 IHG-1 transcript levels were significantly upregulatedin MC cultured in high glucose conditions, leading us to investigatewhether the expression of this gene also occurred in human DN.IHG-1 transcript levels were significantly increased in tubule-richmicrodissected renal biopsies from patients with DN, with clearexpression being localized to the tubule in the diabetic kidney.The expression pattern was similar to that of TGF-β1 andof activated Smad3. TGF-β1 is believed to be the key mediatorof fibrosis in the kidney.5, 21, 22, 35 Increased activity ofTGF-β1 in the tubulointerstitium resulted in increasedexpression and accumulation of extracellular matrix proteins,resulting in compartment-specific pathologic matrix remodelingand scarring.5
In advanced DN, we hypothesized that increased IHG-1 levelsare likely to contribute to the TGF-β1–induced profibroticchanges in tubular cells that prime for TIF. The significantincrease in expression of IHG-1 in the UUO model adds furthersupport to our hypothesis that IHG-1 is a mediator of TIF. UUOleads directly to TIF, in contrast to DN, in which changes inthe glomeruli come first and lead to the development of thetubulointerstitial lesion. Increased expression of IHG-1 inthis model of renal fibrosis suggests that this novel gene maycontribute to TIF per se and may not be restricted to DN. Observationsof increased IHG-1 expression in the tubules in other fibroticdiseases add further weight to our hypothesis.
Although the initiating stresses in DN and UUO are different,the development of TIF is associated with common cytokine/growthfactor stimuli.5 For instance, both conditions have been successfullytreated experimentally with bone morphogenic protein-7.36, 37EGF receptor activation has been implicated in tubulointerstitialfibrogenesis.5 It is transactivated by high extracellular glucose24and has been proposed to assist in the selective survival ofa transdifferentiated, profibrotic cell type.7 One of the waysin which it may facilitate the profibrotic effects on tubulecells may be via induction of IHG-1 expression.
Our investigations of IHG-1's effect on responses to TGF-β1stimulation in renal tubule cells clearly points to IHG-1'sbeing an amplifier of TGF-β1 action in the tubule. Itsoverexpression-induced increases in luciferase reporter activityfrom a TGF-β1 responsive region of the PAI-1 promoter.PAI-1 is strongly induced in various kidney pathologies, includingDN and UUO, and is considered an important factor in the developmentof renal fibrosis.21, 22, 38 IHG-1 had no effect on basal levelsof reporter expression, suggesting that signal transductionmust be first initiated for IHG-1 to mediate its effects. ActivatedSmad3 co-localized with IHG-1 in DN, suggesting that IHG-1 mightinfluence TGF-β1 signaling by targeting Smad3.
TGF-β1 stimulation of epithelial cells causes a transientphosphorylation of R-Smad evident within 10 min, peaking between30 and 60 min and persisting for up to 5 h.10, 39 Both R-Smadand Smad nuclear accumulation are maintained only when receptorsare active.8–11 As soon as receptor activity decreases,R-Smad phosphorylation decreases and nuclear accumulation islost. Dephosphorylation is proposed as the main mechanism ofdeactivation.8–11 The majority of phospho-Smad are believedto be recycled after dephosphorylation, phospho-Smad3 by thephosphatase PPM1A.10, 40 The early and sustained increase inSmad3 phosphorylation in epithelial cells overexpressing IHG-lcoupled with the rapid loss of phosphorylation with IHG-1 knockdownsuggests that IHG-1 may function by inhibiting the activityof a phosphatase, which may target either the activated receptor(e.g., GADD34)41 or the R-Smad itself. How this may functionwill be the subject of our future investigations. It is alsopossible that IHG-1 may modulate the activity of the I-Smad,Smad7, which not only targets TGF-β1 receptors for dephosphorylationbut also both receptors and R-Smad for proteosomal degradation.11The dephosphorylation machinery is believed to be active andin place from onset to termination of signaling; thereby, signaltransduction and consequent gene transcription relies on a dynamicbalance between phosphorylation and dephosphorylation.8–11Increased expression of IHG-1, leading to increased phosphorylationof Smad3, may tip this balance in favor of fibrosis.
Smad3 is required for TGF-β1–induced fibrosis.12,13, 42 An increasing body of evidence demonstrates that decreasingTGF-β1 signaling through blocking Smad3 can protect againstfibrosis both in vivo and in vitro.12, 13, 38 It has been reportedthat renal fibrosis did not develop in Smad3 knockout mice withstreptozotocin-induced diabetes or after UUO. In addition, cellsfrom these mice failed to undergo an EMT when stimulated withTGF-β1.12, 13 Interestingly, we detected no amplificationof Smad2 activation by IHG-1. Why the effect of IHG-1 is Smad3specific remains unknown and will be the subject of our futureinvestigations.
Expression of CTGF, a profibrotic mediator, is increased inthe tubular epithelium by TGF-β1 and is proposed to playa key role in renal fibrogenesis.43 Overexpression of IHG-1increased TGF-β1–induced CTGF expression, furtherstrengthening our hypothesis that IHG-1 contributes to the developmentof TIF. IHG-1 expression also increased TGF-β1–inducedfibronectin expression, which has been reported to be inducedby both Smad-dependent and Smad-independent pathways.31–34Our data suggest that IHG-1 amplifies TGF-β1–inducedfibronectin expression by a Smad-dependent mechanism; however,there is also the possibility that this induction is Smad dependentand indirect, mediated, for instance, by CTGF.
Suppression of IHG-1 expression using siRNA led to reduced TGF-β1induction of the PAI promoter activation and reduced levelsof Smad3 phosphorylation, suggesting that IHG-1 has an importantrole in promoting TGF-β1 responses in renal tubular cells,and given the ubiquitous nature of tissue IHG-1 expression,this may be a general phenomenon. Thus, we describe IHG-1, anovel protein-encoding transcript, whose increased expressionis associated with renal tubular elements in human DN and inkidney tissue in the rat UUO model of interstitial fibrosis.We show, on overexpression, that IHG-1 is a novel amplifierof a TGF-β1 transcriptional response possibly through increasingand/or maintaining phosphorylated Smad3 protein levels afterreceptor activation by TGF-β1. In addition, knockdown ofendogenous IHG-1 blunts Smad3 phosphorylation and a TGF-β1transcriptional response. Considering TGF-β1's centralrole in the development of fibrotic renal disease, IHG-1 maywell constitute a novel profibrotic mediator.
IHG-1 cDNA Assembly
Database searching was performed using BLAST.44 Suppressionsubtractive hybridization analysis16–18 yielded a 198-bpcDNA fragment that we have called IHG-1 (Genbank accession no.AF110136). A sequence identical to UniGene cluster HS353090that encoded a complete open-reading frame of 894 bp was generatedby expressed sequence tag walking.45
Northern Blot Analysis and Real-Time (Taqman) PCR
Northern blots were performed using formaldehyde denaturationaccording to standard protocols. Transcript levels were determinedby quantitative real-time Taqman PCR, as described previously.46Probe and primer sequences were Pre-Developed Assay Reagent(PDAR kit; Applied Biosystems, Foster City, CA).
Human DN Kidney Biopsies
Human biopsy segments were obtained from patients after informedconsent and with the approval of their local ethical committees.47ISH and Southwestern analyses were as described previously.48,49 Ureteric obstruction was performed in rats anaesthetizedusing isofluorane inhalation. Following laparotomy, the proximalportion of the left ureter was ligated with a 6/0 silk, thelaparotomy closed and animals allowed to recover for 3 days(n = 6) or 10 days (n = 5). Animals were then anaesthetizedand underwent a second laparotomy.
Transfection
Stably transfected cell lines were generated with plasmid pIRESpuro3and pIRESpuro3-IHG-1-V5 (Clontech, Paola Alto, CA). Transfectionof HK-2 cells with siRNA (Dharmacon, Chicago, IL; SMARTpoolreagent) was as described.50
Recombinant Lentivirus Production
HEK 293T cells were transfected with (pCMVR8.9), (pMD.2G), andLLCIEP or IHG-1-V5-LLCIEP using a calcium phosphate transfectionkit (Invitrogen, Paisley, UK).
S.M. is a recipient of Fondecyt 1080083. This research was fundedby the Wellcome Trust (M.M., F.M., and C.G.) and Science FoundationIreland (M.M., F.M., and C.G.). N.G.D., B.G., H.R.B., F.M.,and C.G. are recipients of grants from the Health Research Boardof Ireland. C.G. is a member of EU EICOSANOX consortium (ISHM-CT-2004-005033).
We express our thanks to Catherine Moss, Laura Connole, andJanet McCormack (Transcriptomic Unit, Conway Institute, UCD,Dublin, Ireland) for performing real-time PCR analyses. We acknowledgereceipt of reagents from Dr. Rik Derynck (University of California,San Francisco, CA), Dr. Joan Massague (Sloan Kettering Institute,New York, NY), and Dr. Ed Leof (Mayo Clinic College of Medicine,Rochester, MN). Renal biopsy profiling was performed in theERCB.47
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
J.H.'s current affiliation is Division of Experimental Pathology,Lund University, University Hospital, Malmö, Sweden. M.K.'scurrent affiliation is Division of Nephrology, Department ofInternal Medicine, University of Michigan, Ann Arbor, Michigan.
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Top
Abstract
Introduction
R8.9), (pMD.2G), and LLCIEP or IHG-1-V5-LLCIEP using a calcium phosphate transfection kit (Invitrogen, Paisley, UK). 
