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Am J Physiol Renal Physiol 293: F1355-F1362, 2007. First published August 8, 2007; doi:10.1152/ajprenal.00003.2007
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Leptin is a coactivator of TGF-beta in unilateral ureteral obstructive kidney disease

Philipp Kümpers,1,* Faikah Gueler,1,* Song Rong,1 Michael Mengel,2 Irini Tossidou,1 Imke Peters,1 Hermann Haller,1 and Mario Schiffer1

1Department of Nephrology and 2Institute of Pathology, Hannover Medical School, Hannover, Germany

Submitted 3 January 2007 ; accepted in final form 7 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Progressive tubulointerstitial fibrosis is the common end point leading to end-stage renal disease in experimental and clinical settings. Since the peptide hormone leptin is involved not only in the regulation of obesity but also in the regulation of inflammation and fibrosis, we tested the hypothesis whether leptin deficiency has an impact on tubulointerstitial fibrosis in mice. Leptin-deficient (ob/ob) and leptin receptor-deficient mice (db/db) were exposed to 14 days of unilateral ureteral obstruction (UUO). The degree of fibrosis and inflammation was compared with that in sham-operated mice by performing immunohistochemistry, quantitative PCR, and Western blotting. We found that tubulointerstitial fibrosis was significantly reduced in the obstructed kidneys of ob/ob compared with db/db mice or control mice. Detailed analysis of infiltrating inflammatory cells by immunohistochemistry revealed a significant reduction of CD4+ cells at 14 days after UUO in both ob/ob and db/db mice. In contrast, we could not detect significant differences in CD8+ cells and macrophage content. Transforming growth factor (TGF)-beta mRNA levels, TGF-beta-induced Smad-2/3 activation, and the upregulation of downstream target genes were significantly reduced in ob/ob mice. In addition, we demonstrated that leptin could enhance TGF-beta signaling in normal rat kidney fibroblasts in vitro. We conclude that leptin can serve as a cofactor of TGF-beta activation and thus plays an important role in renal tubulointerstitial fibrosis. Therefore, selective blockade of the leptin axis might provide a therapeutic possibility to prevent or delay fibrotic kidney disease.

tubulointerstitial fibrosis; inflammation; transforming growth factor-beta; obesity

TUBULOINTERSTITIAL FIBROSIS characterizes the devastating final common pathway in most progressive renal diseases, regardless of their etiology (20, 24). Several cellular pathways, including fibroblast activation as well as tubular epithelial-mesenchymal transition (EMT), have been identified as the major avenues for the generation of the matrix-producing cells in diseased conditions. Among the many fibrogenic factors that regulate renal fibrosis, transforming growth factor-beta (TGF-beta) plays an essential role in a variety of kidney diseases, including diabetic nephropathy (19, 24, 37). After binding of TGF-beta to serine/threonine kinase receptors of both type II and type I, the activated type I receptor transmits intracellular signals through the phosphorylation of the receptor-associated R-Smads (Smad2/3). The activated R-Smads form complexes with the common pathway C-Smad (Smad4). Only this active complex can translocate to the nucleus, associate with specific DNA-binding partners, and initiate transcriptional responses of specific target genes (37).

Interstitial fibrosis can be induced in vivo in rodents by unilateral ureteral obstruction (UUO). This disease model is characterized by early macrophage infiltration, interstitial fibrosis, and tubular injury mainly induced by activation of local TGF-beta1 (2, 19). The increase of TGF-beta facilitates matrix accumulation by simultaneous activation of renal fibroblasts and tubular epithelial cells, which both contribute to exaggerated matrix protein synthesis (2).

Leptin, a peptide hormone of the long-chain helical cytokine family, is predominantly produced by white adipose cells (46). Leptin plays a crucial role in body weight regulation by inhibiting food intake via hypothalamic effects and stimulating energy expenditure in skeletal muscle cells (12). Maffei et al. (27) were the first to report a strong positive correlation between leptin levels and the body mass index in humans and rodents. However, the ubiquitous distribution of the functional long form of the leptin receptor Ob-Rb in almost all tissues underlies the pleiotropism of leptin (13, 36). Several authors have suggested an essential fibrogenic role for leptin in experimental liver fibrosis (18, 35, 39). In addition, recent studies in renal fibroblasts and glomerular endothelial cells have indicated that leptin might induce cellular proliferation and matrix expression through synergistic activation of the TGF-beta1 signaling system (21, 42). The significance of intact leptin signaling in renal fibrosis has not been studied so far.

Leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice share the same phenotype. Both strains lack hypothalamic inhibition of appetite, leading to obesity, insulin resistance, and type II diabetes (9, 46). Both ob/ob and, most commonly, db/db mice have been intensively investigated as experimental models of diabetic nephropathy (38). Furthermore, ob/ob mice display impaired cell-mediated immunity and a propensity to develop Th2-dominant immune responses (25). Ob/ob mice are protected from inflammation and fibrosis in different disease models, including autoimmune encephalomyelitis, nephrotoxic nephritis, and liver fibrosis (18, 29, 41). Consistently, leptin replacement restores the susceptibility to experimental damage in rodents and humans (43).

The aim of our study was to investigate whether deletion of leptin signaling leads to reduced tubulointerstitial fibrosis. Since functional short-form leptin receptors (Ob-Ra) have been demonstrated in the kidney (42), we performed UUO in both ob/ob and db/db mice compared with appropriate controls.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. We studied ob/ob mice at 8 wk and db/db mice at 7 wk of age, before the onset of diabetic glomerulopathy (9, 46). Male wild-type (WT), ob/ob, db/db, and heterozygous db/m mice (n = 5 per group) were purchased from the Charles River Laboratories (www.criver.com). The animals received a standard diet with water ad libitum. Serum creatinine levels were identical in the mice used compared with the appropriate control strains. However, weight was already notably different between ob/ob and C57Bl/6 wild-type mice (21.1 ± 1.2 vs. 50.6 ± 4.3 g) as well as db/m and db/db mice (22.1 ± 0.8 vs. 38.8 ± 1.9 g). Blood glucose level measurements revealed the beginning of hyperglycemia in the db/db group at the end of the experiment (at 9 wk of age: average 23.0 ± 3.3 mM). UUO was performed as follows: after induction of general anesthesia by application of Avertin (2.5%), the abdominal cavity was exposed via a midline incision and the left ureter was ligated at two points with 4-0 silk and dissected in between. Successful ureteral obstruction was later confirmed by observation of dilation of the renal pelvis and proximal ureter. Sham-operated mice of each strain (n = 5 per group) were used as controls. After 14 days of UUO, mice were euthanized. After anesthesia with Avertin (2.5%), a laparotomy was performed and urine was collected by puncturing the bladder with a 23-gauge needle. The abdominal aorta was then cannulated with a 23-gauge needle, and the organs were perfused with ice-cold lactated Ringer solution. Both kidneys were removed, cut in thirds, and then fixed for 20 h in 3.75% paraformaldehyde in Soerensen's phosphate buffer and embedded in paraffin for histological examination, snap frozen in isopentane (–40°C) for cryostat sectioning, or frozen in liquid nitrogen and stored at –80°C for protein chemistry and TaqMan PCR analysis.

Blood glucose levels were measured with the Glucometer Elite (Bayer, Leverkusen, Germany). Creatinine levels were measured using an automated method (Beckman analyzer; Beckman, Munich, Germany). All procedures were carried out according to guidelines from the American Physiological Society and were approved by local authorities.

Antibodies. Primary antibodies used for Western blotting and immunhistochemical studies were rabbit anti-phospho-Smad2 (Cell Signaling Technology, Beverly, MA), anti-mouse T lymphocytes (CD4 and CD8; BD Pharmingen, Heidelberg, Germany), rat anti-mouse monocytes/macrophages (F4/80; Serotec, Oxford, UK), rabbit anti-mouse fibronectin (Poesel&Lorei, Hanau, Germany), mouse anti-{alpha}-smooth muscle actin ({alpha}-SMA; DAKO, Hamburg, Germany), and rabbit anti-mouse beta-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit as secondary antibodies for Western blotting were purchased from Santa Cruz Biotechnology.

Histology. Histological analysis was carried out on 2-µm paraffin sections cut on a rotation microtome. Following a standard deparaffinization and staining protocol, periodic acid-Schiff (PAS)-, Trichrome-, and Sirius red-stained renal cortex sections were assessed with a digital camera (Axiocam; Zeiss, Jena, Germany) connected to a light microscope (Axioplan-2; Zeiss) by two independent investigators blinded to the study. Analysis of inflammation was done by semiquantitative scoring of the infiltrating cells in 10 randomly chosen, nonoverlapping fields of cortex and outer medulla in PAS-stained sections (x200 magnification) as follows: 0 = none, 1 = weak, 2 = moderate, 3 = high, and 4 = very high numbers of infiltrating cells. Fibrosis was examined in Sirius red-stained sections by fluorescence microscopy, using the Cy3 channel, and scored as follows: 0 = normal, thin tubular basement membrane; 1 = moderately thickened basement membrane; 2 = severely thickened basement membrane; 3 = severely thickened basement membrane plus a few interpositioned collagen fibrils; and 4 = severely thickened basement membrane plus numerous interpositioned collagen fibrils. Data are expressed as the mean score of 10 randomly chosen, nonoverlapping fields of cortex and outer medulla per section (x400 magnification). Fibrosis was additionally assessed in Trichrome-stained sections and scored as follows: 0 = none, 1 = weak, 2 = moderate, 3 = severe, and 4 = very severe fibrosis per section (x200 magnification).

Immunhistochemistry. Immunohistochemistry was performed on cryosections and paraffin sections. Nonspecific binding sites were blocked with 10% normal donkey serum (Jackson ImmunoResearch Laboratory, West Grove, PA) for 30 min. Sections were then incubated with the primary antibody for 1 h. All incubations were performed in a humidified chamber at room temperature. For fluorescent visualization of bound primary antibodies, cryostat sections were further incubated with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratory) for 1 h. On paraffin-embedded sections, a secondary goat anti-mouse antibody (Zytomed, Berlin, Germany) was visualized using diaminobenzidine (Zytomed). Sections were analyzed using a Zeiss Axioplan-2 imaging microscope with the computer program AxioVision 3.0 (Zeiss). Quantitative analysis of CD4-, CD8-, and F4/80-positive cells was done by counting the cell numbers in 10 randomly chosen, nonoverlapping fields (expressed as a mean) of cortex and outer medulla per section (x400 magnification). Semiquantitative analysis was scored as follows: 0 = none, 1 = weak, 2 = moderate, 3 = high, and 4 = very high expression. Semiquantitative analysis of {alpha}-SMA and fibronectin expression was done in 10 randomly chosen, nonoverlapping fields by scoring as follows: 0 = none, 1 = weak, 2 = moderate, 3 = high, and 4 = very high expression (x200 magnification).

Protein chemistry. For protein isolation, frozen kidney tissue from each animal was homogenized in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, and 0.1% SDS) containing protease inhibitor (Complete Mini; Roche, Mannheim, Germany), 1 mM sodium orthovanadate, 50 mM NaF, and 200 µg/l okadaic acid. The samples were centrifuged for 15 min (14,000 g). The supernatant was aliquoted on the basis of the protein concentration measured using a Bradford protein assay (Pierce, Rockford, IL) and stored at –80°C. Fifty micrograms of protein per lane were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). After probing with primary antibodies, antigen-antibody complexes were detected with HRP-labeled anti-rabbit and anti-mouse antibodies, respectively, and visualized using enhanced chemiluminescence reagents (Pierce) according to the manufacturer's protocol. Quantification was done by measuring relative density compared with beta-tubulin (Quantify One; Bio-Rad).

RNA extraction and real-time quantitative PCR. Total mRNA was extracted using Trizol reagent (Invitrogen). For quantitative real-time PCR (qPCR), 1 µg of DNase-treated total RNA was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen), and qPCR was performed on an SDS 7700 system (Applied Biosystems, Darmstadt, Germany) using 10 ng of reverse-transcribed mRNA as a template, Rox dye as internal control (Invitrogen), FastStart Taq polymerase (Roche Diagnostics, Mannheim, Germany), and gene-specific primers in combination with SYBR green chemistry (Molecular Probes, Eugene, OR). PCR amplification was carried out for 10 min at 96°C and 40 cycles of 10 s at 95°C and 1 min at 60°C. beta-Actin served as internal reference gene for normalization. Primer sequences are available on request. Quantification was carried out using QGene software (30).

Cell culture. Normal rat kidney (NRK) fibroblasts (catalog no. 6509; American Type Culture Collection, Rockville, MD; kindly provided by Prof. L. Schäfer, University of Frankfurt, Frankfurt/Main, Germany) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin for 8–15 passages. To obtain quiescent NRK fibroblasts, cells were maintained in serum-starved DMEM supplemented with 0.5% heat-inactivated fetal calf serum for 24 h before the addition of recombinant mouse (rm)leptin (R&D Systems, Minneapolis, MN) or recombinant human (rh)TGF-beta1 (Cell Sciences, Canton, MA).

Statistical analysis. Data are means ± SE. Unpaired Mann-Whitney U testing (2 sided) was used after the Kruskal-Wallis test had been applied to show significant differences between different UUO groups. P < 0.05 was considered statistically significant. Data analysis was performed using SPSS software (www.spss.com).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Leptin deficiency but not leptin receptor deficiency results in an attenuated profibrotic response in kidneys after UUO. To study the effect of leptin deficiency on renal fibrosis, we performed UUO in WT mice, leptin-deficient ob/ob mice, leptin receptor-deficient db/db mice, and their littermates, db/m mice. Analysis was done 14 days after UUO. Examination of Sirius red- and Trichrome-stained renal cortex sections revealed dilated proximal tubules, interstitial expansion, ECM accumulation, and basement membrane thickening of both tubules and glomeruli in WT and ob/ob mice 14 days after UUO (Fig. 1). However, the degree of interstitial fibrosis in obstructed kidneys of WT, db/db, and db/m mice was much more pronounced compared with ob/ob mice, which exhibited significantly reduced basement membrane thickening, reduced interstitial expansion, and ECM deposition (Trichrome, P < 0.001; Sirius red, P < 0.05, respectively) (Fig. 1, k and l). No differences in tubular dilatation were evident among the groups. We also investigated the expression of {alpha}-SMA as a marker of activated myofibroblasts and that of fibronectin as a marker of ECM deposition by immunohistochemistry. As expected, expression of {alpha}-SMA was significantly increased in WT mice after UUO (Fig. 2 b). In contrast, expression of {alpha}-SMA was significantly attenuated in ob/ob mice (P < 0.005) (Fig. 2c). Analogous to the described histological findings, {alpha}-SMA expression after UUO was not attenuated in db/db mice (Fig. 2d). In addition, fibronectin deposition was solely reduced in ob/ob mice compared with WT, db/db, or db/m mice after 14 days of UUO (P < 0.005) (Fig. 2h).


Figure 1
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  		Fig. 1. Leptin deficiency protects from unilateral ureteral obstruction (UUO)-induced tissue fibrosis. Sirius red (a–e) and Masson-Goldner Trichrome stain (f–j) in representative renal cortex sections of C57Bl/6 (b and g), leptin-deficient ob/ob(c and h), normal heterozygous db/m (d and i), and leptin receptor-deficient db/db mice (e and j) after 14 days of UUO (b–e and g–j) compared with respective controls (a and f). Since fibrotic changes were not detectable in untreated mice of all groups, only 1 representative control panel for C57Bl/6 mice is shown. Leptin-deficient ob/ob mice (c and h) developed reduced fibrosis compared with all other groups. Semiquantitative scoring of Sirius red (k)- and Masson-Goldner Trichrome (l)-stained sections of control and obstructed kidneys of C57Bl/6 (solid bars), ob/ob (open bars), db/m (dark shaded bars), and db/db mice (light shaded bars) is shown after 14 days of UUO (n = 5 mice in each group). Scoring was done by 2 independent investigators blinded to the study. Results are means ± SE. *P < 0.05.

 

Figure 2
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  		Fig. 2. Expression of {alpha}-smooth muscle actin ({alpha}-SMA) and fibronectin is reduced in the obstructed kidney of leptin-deficient ob/ob mice after UUO. Immunohistochemistry using anti-{alpha}-SMA (a–e) and anti-fibronectin antibodies (f–j) shows reduced activation of myofibroblasts and ECM deposition in obstructed kidneys of leptin-deficient ob/ob mice (c and h) compared with all other groups after UUO. Representative renal cortex sections of C57Bl/6 (b and g), ob/ob (c and h), db/m (d and i), and db/db mice (e and j) after 14 days of UUO were compared with respective controls (a and f). Since no changes in {alpha}-SMA and fibronectin staining were detectable in untreated mice, only 1 representative control (C57Bl/6) is shown (a and f). Semiquantitative analysis of anti-{alpha}-SMA (k)- and anti-fibronectin (l)-stained sections of control kidneys and obstructed kidneys of C57Bl/6 (solid bars), ob/ob (open bars), db/m (dark shaded bars), and db/db mice (light shaded bars) are shown after 14 days of UUO (n = 5 in each group). Scoring was done by 2 independent investigators blinded to the study. Results are means ± SE. *P < 0.05. Western blot analysis of total kidney lysates (m) reveals reduced expression of {alpha}-SMA in obstructed kidneys of ob/ob compared with C57Bl/6 and db/db mice 14 days after UUO.

 
In summary, histology and immunohistochemistry demonstrated markedly attenuated fibrosis, {alpha}-SMA, and fibronectin expression in ob/ob mice compared with db/db mice and respective controls. These data indicate that leptin deficiency but not deletion of the long-form leptin receptor Ob-Rb protects mice from matrix accumulation and tubulointerstitial fibrosis after UUO.

Effects of leptin signaling on inflammation and composition of inflammatory infiltrates in kidneys after UUO. Since the UUO model directly links the inflammatory and profibrotic response, we examined whether leptin signaling has an influence on inflammatory cell infiltration in this model. Differences in leukocyte infiltration after UUO were semiquantitatively scored on PAS-stained sections. Obstructed kidneys of C57Bl/6, db/db, and db/m mice showed a homogenous inflammatory cell infiltration of the renal interstitium (Fig. 3). In contrast, we found significantly less inflammation in renal cortex sections of ob/ob mice after UUO (P < 0.005) (Fig. 3f).


Figure 3
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  		Fig. 3. Reduced renal inflammation in the obstructed kidney of leptin-deficient ob/ob mice after UUO. Periodic-acid Schiff (PAS) stain (a–e) shows reduced inflammation in kidneys of leptin-deficient ob/ob mice (c) compared with all other groups after UUO compared with untreated controls (a). Representative renal cortex sections of C57Bl/6 (b), ob/ob (c), db/m (d), and db/db mice (e) after 14 days of UUO were compared with respective controls (a). Since inflammatory changes were not detectable in untreated mice, only 1 representative control (C57Bl/6) is shown. Semiquantitative scoring of inflammation (f), anti-F4/80-positive monocytes/macrophages (g), anti-CD4 (h), and anti-CD8 (i) of control kidneys (n = 5 in each group) and obstructed kidneys (solid bars), ob/ob (open bars), db/m (dark shaded bars), and db/db mice (light shaded bars) is shown after 14 days of UUO. Scoring was done by 2 independent investigators blinded to the study. Results are means ± SE. *P < 0.05.

 
To characterize the cellular composition of the infiltrates in this model, we performed immunohistochemistry with specific antibodies for monocytes/macrophages (F4/80) and lymphocyte subsets (CD4+ and CD8+). Interestingly, significant quantitative differences were detected among CD4+ lymphocytes (Fig. 3). CD4+ cells were twofold higher in controls compared with ob/ob and db/db mice after UUO (both P < 0.001). No significant differences were observed in CD8+ lymphocytes and monocytes/macrophages among groups (Fig. 3i). These data indicate that recruitment of CD4+ cells to sites of tissue inflammation requires leptin and the intact Ob-Rb.

TGF-beta activation is reduced in leptin-deficient mice. Since TGF-beta is the central cytokine mediating UUO-induced tubulointerstitial fibrosis, we analyzed whether the advantageous response of leptin deficiency is based on a reduced activation of local TGF-beta. When we measured TGF-beta1 mRNA levels in whole kidney lysates after UUO, we detected reduced TGF-beta1 expression levels solely in ob/ob mice (Fig. 4 a). Next, we performed Western blot analysis of whole kidney protein lysates. Obstructed kidneys of WT mice showed a strong and uniform phosphorylation response of Smad2/3. In contrast, Smad2/3 phosphorylation was significantly reduced in whole kidney lysates of ob/ob mice (Fig. 4b). The Smad2/3 phosphorylation response was similar in db/db mice compared with db/m mice (Fig. 4b), indicating a coactivating role for leptin in TGF-beta activation in vivo. Analysis of mRNA expression levels of classic TGF-beta downstream target genes in whole kidney tissue revealed significantly reduced expression levels of plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), matrix metalloproteinase-2 (MMP-2), and MMP-9 in ob/ob mice compared with all other groups after UUO (P < 0.005) (Fig. 5). Together, these data indicate a link between leptin and TGF-beta in vivo.


Figure 4
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  		Fig. 4. Reduced transforming growth factor-beta (TGF-beta) activation and Smad2/3 phosphorylation in kidneys of leptin-deficient ob/ob mice after UUO. A: quantitative real-time PCR demonstrates relative induction of TGF-beta mRNA expression from kidney samples after 14 of UUO (compared with untreated controls of respective strains, n = 5 each group) in C57Bl/6 wild-type (WT), ob/ob, db/m, and db/db mice. B: Western blot analysis (top) demonstrates levels of phosphorylated Smad2/3 (p-Smad2/3) in whole kidney lysates of control and obstructed kidneys 14 days (d14) after UUO in C57Bl/6 and ob/ob mice. Densitometric analysis (bottom) demonstrates relative expression levels of p-Smad2/3 normalized for beta-tubulin content in C57Bl/6 and ob/ob mice. C: Western blot analysis (top) of p-Smad2/3 in whole kidney lysates of control and obstructed kidneys of db/m and db/db mice 14 days after UUO. Densitometric analysis (bottom) demonstrates expression levels of p-Smad2/3 normalized for beta-tubulin content in db/m and db/db mice. *P < 0.05.

 

Figure 5
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  		Fig. 5. Quantitative real-time PCR demonstrates reduced activation of TGF-beta-dependent target genes in obstructed kidneys of leptin-deficient ob/ob mice. Quantitative real-time PCR demonstrates relative mRNA induction in kidney tissue after 14 days of UUO (compared with untreated controls of the respective strain) of TGF-beta target genes plasminogen activator inhibitor-1 (PAI-1; a), connective tissue growth factor (CTGF; b); matrix metalloproteinase-2 (MMP-2; c), and MMP-9 (d) in kidneys from C57Bl/6 (WT), ob/ob, db/m, and db/db mice (n = 5 each group). mRNA content was normalized using murine beta-actin. *P < 0.05.

 
Leptin augments TGF-beta signaling in renal fibroblast in vitro. Next, we examined the potential role of leptin as a cofactor for TGF-beta activation in vitro. NRK fibroblasts were treated with a single dose of rmLeptin (100 ng/ml), rhTGF-beta1 (5 ng/ml), or a combination of both. Stimulation with leptin alone resulted in a slight phosphorylation response of Smad2/3. Interestingly, combination treatment of leptin and TGF-beta resulted in an enhanced Smad2/3 phosphorylation response compared with TGF-beta alone (Fig. 6, a and b). These data indicate that leptin can serve as a cofactor of TGF-beta activation in vitro.


Figure 6
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  		Fig. 6. Leptin induces Smad2/3 phosphorylation and enhances TGF-beta-induced Smad signaling in rat kidney fibroblasts. Normal rat kidney fibroblasts (NRK-49F) were stimulated for 1 and 24 h with a single dose of leptin (100 ng/ml), TGF-beta1 (5 ng/ml), or a combination of both compared with untreated controls. Representative Western blot (A) and densitometric analysis of 2 independent experiments (B) reveals a slight increase in phosphorylation of Smad2/3 after leptin treatment and a marked enhancement of TGF-beta1-induced Smad2/3 phosphorylation in the presence of leptin 1 h after stimulation compared with TGF-beta1 alone. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Progressive fibrosis is the hallmark of end-organ failure in the kidney, since functional parenchymal cells are replaced by nonfunctional scar tissue. In the past, remarkable progress was made in slowing down the progression of chronic renal failure by such second-line therapeutic approaches as consequent blood pressure control. However, normalization of blood pressure alone is not efficient to prevent disease progression or reverse existing fibrosis. TGF-beta is a powerful mediator of profibrotic responses in the kidney. TGF-beta-induced tissue fibrosis is mediated by direct induction of matrix molecules, activation of resident fibroblasts, and EMT (2, 24). In the past, the blockade of TGF-beta activation was proven to be a successful approach in preventing and even reversing matrix accumulation and fibrosis in animal models (5, 47). Especially in UUO and diabetic kidney disease, the role of TGF-beta is very well defined as the key mediator of hypertrophic and prosclerotic changes (19, 33). However, TGF-beta is a difficult target molecule for ablative therapy because of its pleiotropic effects on various tissues.

In addition to its central and peripheral metabolic effects on peripheral tissues, paracrine effects of leptin on TGF-beta synthesis and synthesis of the TGF-beta type II receptor have been described previously (15, 23, 42). Rat kidney interstitial fibroblasts respond to leptin treatments in vitro with increased mitogenesis and collagen expression (21). A similar effect was described in a model of hepatic fibrogenesis, where leptin induced collagen-I expression independently of TGF-beta in human stellate cells (40). Likewise, leptin induced collagen-I expression in isolated mesangial cells of db/db mice. These direct effects of leptin are often potentiated by direct induction of the TGF-beta type II receptor, leading to a more efficient TGF-beta action as demonstrated in human stellate cells and mesangial cells (15, 39). Recently, Wolf et al. (42) demonstrated elevated TGF-beta mRNA and subsequently increased matrix expression in glomerular endothelial cells after leptin treatment in vivo and in vitro.

In the present study we have demonstrated that leptin deficiency, but not leptin receptor deficiency, leads to significantly less inflammation, tissue damage, fibrotic changes, and matrix accumulation in ob/ob mice after UUO compared with db/db mice and respective controls. We also demonstrated markedly less TGF-beta activation in leptin-deficient ob/ob compared with leptin receptor-deficient db/db mice after UUO. These results are well in line with our finding of markedly reduced mRNA levels of TGF-beta, Smad2/3 phosphorylation (i.e., TGF-beta activation) in ob/ob compared with db/db mice after UUO. Since db/db mice lack the functional long form of the leptin receptor (Ob-Rb), it is conceivable that either signaling via the short form of the receptor (Ob-Ra) or leptin per se as a cofactor of TGF-beta activation might enhance interstitial fibrosis after UUO. We can demonstrate that leptin alone induces phosphorylation of Smad-2/3 in renal fibroblasts in vitro. In addition, leptin in combination with TGF-beta1 leads to enhanced Smad phosphorylation.

We also have demonstrated that mRNA expression of typical downstream targets of TGF-beta such as PAI-1 and CTGF accumulate in UUO but are significantly less induced in the obstructed ob/ob kidneys. PAI-1 promotes net proteolysis via inhibition of tissue-type plasminogen inhibitor (t-PA) and urokinase-type plasminogen activator (u-PA). Both t-PA and u-PA lead to activation of plasmin, which leads to fibrinolysis. Therefore, PAI-1 induction is a solid marker of ongoing tissue fibrosis. PAI-1 is upregulated in almost all known renal diseases, including UUO (10). PAI-1 knockout mice develop less tubulointerstitial fibrosis after UUO than their control littermates. Interestingly, this benefit results from less cellular infiltration as well as reduced mRNA levels for TGF-beta and collagens but is not caused by an antiproteolytic effect via inhibition of t-PA or u-PA (32).

CTGF is mainly expressed in tubular cells in the kidney. It has been shown to induce EMT in tubuloepithelial cells, and its expression also can serve as a biomarker of ongoing kidney fibrosis (4, 7). Furthermore, CTGF antisense treatment has been shown to attenuate renal fibrosis after UUO (45). In addition, we detected less accumulation of MMP-2 and MMP-9 mRNA in ob/ob mice after UUO. This also could be explained by the lack of TGF-beta activation, since MMPs are described target genes of TGF-beta (1). The classic function of MMPs is the attenuation of fibrosis by degradation of matrix; however, recent studies have revealed some effects of MMP-2 and MMP-9 that might even lead to a more aggravated profibrotic response. Transgenic overexpression of MMP-2 in tubular cells leads to direct induction of renal injury and interstitial collagen (8). In addition, the activation of MMP-9 promotes degradation of the tubular basement membrane, which is a critical key step leading to kidney fibrosis via the induction of EMT (43, 44).

Ob/ob and db/db mice are widely accepted as classic disease models of type 2 diabetes. Both animal models display glomerular features similar to human diabetic glomerulopathy. However, tubulointerstital fibrosis, the key event leading to end-stage renal failure in human diabetic nephropathy, is rarely reported in db/db mice but virtually absent in ob/ob mice (3, 38). Based on our results, we hypothesize that the lack of leptin-mediated signaling responses is due to a missing cofactor necessary for local TGF-beta activation that leads to fibrosis of the tubulointerstitial compartment in the latter model. Moreover, Gunduz et al. (14) reported an increase in glomerular TGF-beta activation after leptin infusion, indicating a close regulatory connection of leptin and the TGF-beta axis. However, the TGF-beta activation in glomerular cells seems to occur independently of leptin, since local glomerular synthesis and activation of TGF-beta and CTGF has been demonstrated in both model systems in the diabetic state (5, 34).

Despite the fact that we detected fewer inflammatory cells in the obstructed kidneys of ob/ob mice, we detected no difference in macrophage infiltration. Macrophages play a central role after tissue injury, since they produce numerous factors that promote activation of resident fibroblasts and synthesis of matrix. Macrophages of ob/ob mice display some phenotypic alterations (i.e., reduced phagocytosis capacity and increased IL-6 levels), but these have so far not directly been linked to fibrogenesis (22, 28). However, recently published evidence suggests that in the absence of active TGF-beta, macrophages may be ineffective in promoting fibrosis (26, 31).

In our model we found decreased numbers of CD4+ cells in both ob/ob and db/db mice. This is in line with previous results showing that patients suffering from leptin deficiency had reduced numbers of circulating CD4+ cells and impaired T cell proliferation (11). However, since db/db mice are not protected from renal fibrosis, reduced numbers of CD4+ cells in ob/ob and db/db mice are probably not primary effectors in UUO. In general, the impact of TGF-beta on immune cells and the immune-modulatory response is determined by the type of inflammatory microenvironment and the cell type. This is especially exemplified in the case of CD4+ cells. Naive cells and Th1 cells respond to TGF-beta1 with a suppressed proliferative response, whereas TGF-beta1 has little or no effect on the proliferation of Th2 cells (17). Moreover, TGF-beta has direct immunomodulatory functions by influencing the conversion of naive infiltrating CD4+ cells to regulatory CD4+CD25+ cells (6). Thus the difference in CD4+ cell number in the leptin-deficient mice could be explained in part by a lack of local TGF-beta activation. However, we can certainly speculate that tissue infiltration of CD4+ cells partially requires an intact leptin-leptin receptor axis. Our data are further supported by observations in experimental models of hepatic fibrosis demonstrating a reduced inflammatory and profibrotic response in the absence of leptin (16, 35).

In summary, we have provided evidence that leptin per se or leptin signaling via the short-form receptor (Ob-Ra) functions as an important coactivator of local TGF-beta in the kidney. Targeting leptin signaling in inflammatory or profibrotic disease states of the kidney could be a novel approach to prevent tubulointerstital fibrosis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by an Emmy Noether Scholarship granted by Deutsche Forschungsgemeinschaft (Schi 587/2) to M. Schiffer.


   ACKNOWLEDGMENTS
 
We thank Melanie Paschy, Yvonne Nicolai, Herle Chlebusch, and Kerstin Bankes for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Schiffer, Dept. of Nephrology, Medical School Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany (e-mail: schiffer.mario{at}mh-hannover.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* P. Kümpers and F. Gueler contributed equally to this work. Back


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Benbow U, Brinckerhoff CE. The AP-1 site and MMP gene regulation: what is all the fuss about? Matrix Biol 15: 519–526, 1997.[CrossRef][Web of Science][Medline]
  2. Bottinger EP, Bitzer M. TGF-beta signaling in renal disease. J Am Soc Nephrol 13: 2600–2610, 2002.[Abstract/Free Full Text]
  3. Breyer MD, Bottinger E, Brosius FC, Coffman TM, Fogo A, Harris RC, Heilig CW, Sharma K. Diabetic nephropathy: of mice and men. Adv Chronic Kidney Dis 12: 128–145, 2005.[CrossRef][Web of Science][Medline]
  4. Burns WC, Twigg SM, Forbes JM, Pete J, Tikellis C, Thallas-Bonke V, Thomas MC, Cooper ME, Kantharidis P. Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to-mesenchymal transition: implications for diabetic renal disease. J Am Soc Nephrol 17: 2484–2494, 2006.[Abstract/Free Full Text]
  5. Chen S, Iglesias-de la Cruz MC, Jim B, Hong SW, Isono M, Ziyadeh FN. Reversibility of established diabetic glomerulopathy by anti-TGF-beta antibodies in db/db mice. Biochem Biophys Res Commun 300: 16–22, 2003.[CrossRef][Web of Science][Medline]
  6. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+. J Exp Med 198: 1875–1886, 2003.[Abstract/Free Full Text]
  7. Cheng O, Thuillier R, Sampson E, Schultz G, Ruiz P, Zhang X, Yuen PS, Mannon RB. Connective tissue growth factor is a biomarker and mediator of kidney allograft fibrosis. Am J Transplant 6: 2292–2306, 2006.[CrossRef][Web of Science][Medline]
  8. Cheng S, Pollock AS, Mahimkar R, Olson JL, Lovett DH. Matrix metalloproteinase 2 and basement membrane integrity: a unifying mechanism for progressive renal injury. FASEB J 20: 1898–1900, 2006.[Abstract/Free Full Text]
  9. Chua SC Jr, Chung WK, Wu-Peng XS, Zhang Y, Liu SM, Tartaglia L, Leibel RL. Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science 271: 994–996, 1996.[Abstract]
  10. Eddy AA. Plasminogen activator inhibitor-1 and the kidney. Am J Physiol Renal Physiol 283: F209–F220, 2002.[Abstract/Free Full Text]
  11. Farooqi IS, Matarese G, Lord GM, Keogh JM, Lawrence E, Agwu C, Sanna V, Jebb SA, Perna F, Fontana S, Lechler RI, DePaoli AM, O'Rahilly S. Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest 110: 1093–1103, 2002.[CrossRef][Web of Science][Medline]
  12. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 395: 763–770, 1998.[CrossRef][Medline]
  13. Fruhbeck G. Intracellular signalling pathways activated by leptin. Biochem J 393: 7–20, 2006.[CrossRef][Web of Science][Medline]
  14. Gunduz Z, Dursun N, Akgun H, Ozturk F, Okur H, Koc N. Renal effects of long-term leptin infusion and preventive role of losartan treatment in rats. Regul Pept 132: 59–66, 2005.[CrossRef][Web of Science][Medline]
  15. Han DC, Isono M, Chen S, Casaretto A, Hong SW, Wolf G, Ziyadeh FN. Leptin stimulates type I collagen production in db/db mesangial cells: glucose uptake and TGF-beta type II receptor expression. Kidney Int 59: 1315–1323, 2001.[CrossRef][Web of Science][Medline]
  16. Honda H, Ikejima K, Hirose M, Yoshikawa M, Lang T, Enomoto N, Kitamura T, Takei Y, Sato N. Leptin is required for fibrogenic responses induced by thioacetamide in the murine liver. Hepatology 36: 12–21, 2002.[CrossRef][Web of Science]
  17. Huber S, Schramm C, Lehr HA, Mann A, Schmitt S, Becker C, Protschka M, Galle PR, Neurath MF, Blessing M. Cutting edge: TGF-beta signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells. J Immunol 173: 6526–6531, 2004.[Abstract/Free Full Text]
  18. Ikejima K, Takei Y, Honda H, Hirose M, Yoshikawa M, Zhang YJ, Lang T, Fukuda T, Yamashina S, Kitamura T, Sato N. Leptin receptor-mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat. Gastroenterology 122: 1399–1410, 2002.[CrossRef][Web of Science]
  19. Inazaki K, Kanamaru Y, Kojima Y, Sueyoshi N, Okumura K, Kaneko K, Yamashiro Y, Ogawa H, Nakao A. Smad3 deficiency attenuates renal fibrosis, inflammation, and apoptosis after unilateral ureteral obstruction. Kidney Int 66: 597–604, 2004.[CrossRef][Web of Science][Medline]
  20. Klahr S, Schreiner G, Ichikawa I. The progression of renal disease. N Engl J Med 318: 1657–1666, 1988.[Abstract]
  21. Lee CI, Guh JY, Chen HC, Lin KH, Yang YL, Hung WC, Lai YH, Chuang LY. Leptin and connective tissue growth factor in advanced glycation end-product-induced effects in NRK-49F cells. J Cell Biochem 93: 940–950, 2004.[CrossRef][Web of Science][Medline]
  22. Lee FY, Li Y, Yang EK, Yang SQ, Lin HZ, Trush MA, Dannenberg AJ, Diehl AM. Phenotypic abnormalities in macrophages from leptin-deficient, obese mice. Am J Physiol Cell Physiol 276: C386–C394, 1999.[Abstract/Free Full Text]
  23. Leung JC, Chan LY, Tang SC, Chu KM, Lai KN. Leptin induces TGF-beta synthesis through functional leptin receptor expressed by human peritoneal mesothelial cell. Kidney Int 69: 2078–2086, 2006.[CrossRef][Web of Science][Medline]
  24. Liu Y. Renal fibrosis: new insights into the pathogenesis and therapeutics. Kidney Int 69: 213–217, 2006.[CrossRef][Web of Science][Medline]
  25. Lord GM, Matarese G, Howard JK, Baker RJ, Bloom SR, Lechler RI. Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature 394: 897–901, 1998.[CrossRef][Medline]
  26. Ma LJ, Yang H, Gaspert A, Carlesso G, Barty MM, Davidson JM, Sheppard D, Fogo AB. Transforming growth factor-beta-dependent and -independent pathways of induction of tubulointerstitial fibrosis in beta6–/– mice. Am J Pathol 163: 1261–1273, 2003.[Abstract/Free Full Text]
  27. Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1: 1155–1161, 1995.[CrossRef][Web of Science][Medline]
  28. Mancuso P, Huffnagle GB, Olszewski MA, Phipps J, Peters-Golden M. Leptin corrects host defense defects after acute starvation in murine pneumococcal pneumonia. Am J Respir Crit Care Med 173: 212–218, 2006.[Abstract/Free Full Text]
  29. Matarese G, Di Giacomo A, Sanna V, Lord GM, Howard JK, Di Tuoro A, Bloom SR, Lechler RI, Zappacosta S, Fontana S. Requirement for leptin in the induction and progression of autoimmune encephalomyelitis. J Immunol 166: 5909–5916, 2001.[Abstract/Free Full Text]
  30. Muller PY, Janovjak H, Miserez AR, Dobbie Z. Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques 32: 1372–1379, 2002.[Web of Science][Medline]
  31. Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, Sheppard D. The integrin {alpha}vbeta6 binds and activates latent TGFbeta1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96: 319–328, 1999.[CrossRef][Web of Science][Medline]
  32. Oda T, Jung YO, Kim HS, Cai X, Lopez-Guisa JM, Ikeda Y, Eddy AA. PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction. Kidney Int 60: 587–596, 2001.[CrossRef][Web of Science][Medline]
  33. Reeves WB, Andreoli TE. Transforming growth factor beta contributes to progressive diabetic nephropathy. Proc Natl Acad Sci USA 97: 7667–7669, 2000.[Free Full Text]
  34. Roestenberg P, van Nieuwenhoven FA, Joles JA, Trischberger C, Martens PP, Oliver N, Aten J, Hoppener JW, Goldschmeding R. Temporal expression profile and distribution pattern indicate a role of connective tissue growth factor (CTGF/CCN-2) in diabetic nephropathy in mice. Am J Physiol Renal Physiol 290: F1344–F1354, 2006.[Abstract/Free Full Text]
  35. Sahai A, Malladi P, Pan X, Paul R, Melin-Aldana H, Green RM, Whitington PF. Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin. Am J Physiol Gastrointest Liver Physiol 287: G1035–G1043, 2004.[Abstract/Free Full Text]
  36. Sanchez-Margalet V, Martin-Romero C, Santos-Alvarez J, Goberna R, Najib S, Gonzalez-Yanes C. Role of leptin as an immunomodulator of blood mononuclear cells: mechanisms of action. Clin Exp Immunol 133: 11–19, 2003.[CrossRef][Web of Science][Medline]
  37. Schiffer M, von Gersdorff G, Bitzer M, Susztak K, Bottinger EP. Smad proteins and transforming growth factor-beta signaling. Kidney Int Suppl 77: S45–S52, 2000.[CrossRef][Medline]
  38. Susztak K, Sharma K, Schiffer M, McCue P, Ciccone E, Bottinger EP. Genomic strategies for diabetic nephropathy. J Am Soc Nephrol 14: S271–S278, 2003.[Abstract/Free Full Text]
  39. Tang M, Potter JJ, Mezey E. Leptin enhances the effect of transforming growth factor beta in increasing type I collagen formation. Biochem Biophys Res Commun 297: 906–911, 2002.[CrossRef][Web of Science][Medline]
  40. Tang M, Potter JJ, Mezey E. Activation of the human {alpha}1(I) collagen promoter by leptin is not mediated by transforming growth factor beta responsive elements. Biochem Biophys Res Commun 312: 629–633, 2003.[CrossRef][Web of Science][Medline]
  41. Tarzi RM, Cook HT, Jackson I, Pusey CD, Lord GM. Leptin-deficient mice are protected from accelerated nephrotoxic nephritis. Am J Pathol 164: 385–390, 2004.[Abstract/Free Full Text]
  42. Wolf G, Hamann A, Han DC, Helmchen U, Thaiss F, Ziyadeh FN, Stahl RA. Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis. Kidney Int 56: 860–872, 1999.[CrossRef][Web of Science][Medline]
  43. Yang J, Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. Am J Pathol 159: 1465–1475, 2001.[Abstract/Free Full Text]
  44. Yang J, Shultz RW, Mars WM, Wegner RE, Li Y, Dai C, Nejak K, Liu Y. Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy. J Clin Invest 110: 1525–1538, 2002.[CrossRef][Web of Science][Medline]
  45. Yokoi H, Mukoyama M, Nagae T, Mori K, Suganami T, Sawai K, Yoshioka T, Koshikawa M, Nishida T, Takigawa M, Sugawara A, Nakao K. Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis. J Am Soc Nephrol 15: 1430–1440, 2004.[Abstract/Free Full Text]
  46. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425–432, 1994.[CrossRef][Medline]
  47. Ziyadeh FN. Mediators of diabetic renal disease: the case for TGF-beta as the major mediator. J Am Soc Nephrol 15, Suppl 1: S55–S57, 2004.[Abstract/Free Full Text]



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