Mast cells decrease renal fibrosis in unilateral ureteral obstruction

http://www.kidney-international.org

original article

& 2009 International Society of Nephrology

Mast cells decrease renal fibrosis in unilateral ureteral obstruction Duk Hoon Kim1, Sang-Ok Moon1, Yu Jin Jung1, Ae Sin Lee1, Kyung Pyo Kang1, Tae Hwan Lee1, Sik Lee1, Ok Hee Chai2, Chang Ho Song2, Kyu Yun Jang3, Mi Jeong Sung4, Xin Zhang2,5, Sung Kwang Park1 and Won Kim1 1

Renal Regeneration Laboratory and Department of Internal Medicine, Chonbuk National University Medical School, Jeonju, South Korea; 2Department of Anatomy, Research Institute of Clinical Medicine, Chonbuk National University Medical School, Jeonju, South Korea; 3Department of Pathology, Chonbuk National University Medical School, Jeonju, South Korea; 4Food Function Research Division, Korea Food Research Institute, Song Nam, South Korea and 5Department of Anatomy, Medical College of Henan University of Science and Technology, Luoyang, Henan, China

Mast cells regulate both inflammatory responses and tissue repair in human diseases but there are conflicting reports on the role of these cells in the pathogenesis of various kidney diseases. Here we measured mast cell function in unilateral ureteral obstruction, a well-characterized model of renal fibrosis, using KitW/KitW
v mice genetically deficient in mast cells, wild-type mice, and deficient mice reconstituted by adoptive transfer with mast cells from the wild-type animals. Mast cell-deficient mice had higher levels of renal tubular damage, more stromal fibrosis, higher numbers of infiltrating ERHR3-positive macrophages and CD3-positive T cells, and higher tissue levels of profibrotic transforming growth factor-b1 than wild-type mice or mice reconstituted by adoptive transfer of mast cells 3 weeks after ureteral obstruction. Similarly, while wild-type and adoptively transferred mice had increased a-smooth muscle actin and decreased E-cadherin expression, which are indicators of epithelial-mesenchymal transition, the obstructed kidneys of the mast cell–deficient mice had significant attenuation of those indicators. Thus, our study suggests that mast cells protect the kidney against fibrosis by modulation of inflammatory cell infiltration and by transforming growth factor-b1-driven epithelial-to-mesenchymal transitions. Kidney International (2009) 75, 1031–1038; doi:10.1038/ki.2009.1; published online 25 February 2009 KEYWORDS: mast cells; renal fibrosis; ureteral obstruction

Correspondence: Sung Kwang Park, Renal Regeneration Laboratory and Department of Internal Medicine, Chonbuk National University Medical School, 634-18, Keum-Am dong, Jeonju 560-180, Korea. E-mail: [email protected] or Won Kim, Department of Internal Medicine, Chonbuk National University Medical School, 634-18, Keum-Am dong, Jeonju 560-180, Korea. E-mail: [email protected] Received 10 January 2008; revised 11 December 2008; accepted 16 December 2008; published online 25 February 2009 Kidney International (2009) 75, 1031–1038

Mast cells (MCs) have important roles in innate and adaptive immunity.1 The numbers of MCs increase at the sites of tissue injury and inflammation.2 After activation, MCs secrete various mediators, cytokines, and growth factors that mediate vascular permeability, inflammation, chemotaxis, and tissue destruction or tissue protection.3,4 MCs have been associated with conditions of persistent inflammation, such as multiple sclerosis,5 rheumatoid arthritis,6 inflammatory bowel disease,7 and ischemia-reperfusion injury.8 Thus, MCs regulate both inflammatory responses and tissue repair in human diseases.9 The numbers of MCs in the kidney increase in various human renal diseases, such as diabetic nephropathy,10 IgA nephropathy,11 rapid progressive glomerulonephritis,12 mesangial proliferative glomerulonephritis,13 and acute rejection.14 Experimental renal disease models have suggested that MCs may contribute to the progression of renal disease.9,15 Jones et al.15 showed that MC infiltration may contribute to progressive renal injury in the 5/6 nephrectomy model. Timoshanko et al.9 suggested that renal MCs can mediate crescentic glomerulonephritis by regulating adhesion molecules. In contrast, Miyazawa et al.16 suggested that MCs might have a protective role in puromycin aminonucleosideinduced nephrosis. Thus, there are conflicting data about the role of MCs in the pathogenesis of renal disease. Obstructive uropathy is an important cause of end-stage renal disease in infants and adults. Most forms of chronic renal disease progress to tubulointerstitial fibrosis, which is one of the major characteristics of obstructive nephropathy and correlates with a loss of renal function. In fact, the severity of tubulointerstitial changes is the best indicator of the progression of renal dysfunction.17,18 Mouse unilateral ureteral obstruction (UUO) has been used as a model of renal interstitial fibrosis, but the role of MCs in the UUO model has not been studied. To determine the influence of MCs on the tubulointerstitial fibrosis induced by UUO, we used MC-deficient WBB6F1-KitW/ KitW
v (KitW/KitW
v) mice to evaluate whether MCs have a protective effect on renal interstitial fibrosis. We found that 1031

original article

DH Kim et al.: Mast cells decrease renal fibrosis

MCs suppress the renal fibrotic processes and type I collagen deposition triggered by UUO. MCs decreased the number of ERHR3- and CD3-positive cells infiltrating the kidney tissue and the tissue level of transforming growth factor (TGF)-b1 in the UUO model. In an immunoblotting analysis, MCs also inhibited the expression of a-smooth muscle actin (a-SMA) and preserved the expression of E-cadherin in the obstructed kidneys. These findings indicate that MCs may have a beneficial effect on interstitial fibrosis in the UUO model. RESULTS MCs in sham-operated and obstructed kidneys

Protective effects of MCs on morphology in UUO

To determine the degree of tubular injury in the renal cortex of obstructed and sham-operated kidneys, we used three parameters: tubular dilatation, epithelial desquamation, and brush border loss. Three weeks after UUO, the obstructed kidneys of Kit þ / þ mice showed destruction of the renal tubules with significant mononuclear inflammatory infiltration and severe stromal fibrosis (Figure 1a and b). The obstructed kidneys of KitW/KitW
v mice showed relatively more damage to the renal tubular structure, more stromal fibrosis, and more inflammatory cell infiltration than those of Kit þ / þ mice (Figure 1c). The obstructed kidneys of MCreconstituted KitW/KitW
v mice showed fewer destructive changes to the renal tubular structure and less stromal fibrosis and stromal inflammatory cell infiltration than those of KitW/KitW
v mice (Figure 1d). The histological findings were supported by the semiquantitative histological score (Figure 1e). To evaluate the effect of UUO on kidney function, we measured the serum creatinine level in Kit þ / þ and KitW/KitW
v mice. The serum levels of creatinine were not significantly different between Kit þ / þ and KitW/KitW
v mice 3 weeks after UUO (0.32±0.05 mg/100 ml in Kit þ / þ mice; 0.35±0.04 mg/100 ml in KitW/KitW
v mice; n ¼ 5 in 1032

5

* Tubular injury score

4

†

3 2 1

v

tW R /K it W M eco -v C n in sti t Ki ut t W io /K n o it W f -

t +/+

Ki

Ki

am

0 Sh

To identify renal MCs in sham-operated or obstructed kidneys in congenic wild-type WBB6F1-Kit þ / þ (Kit þ / þ ) and MC-reconstituted KitW/KitW
v mice, we stained serial kidney sections with toluidine blue and an anti-tryptase antibody. Nearly all toluidine blue-stained MCs showed positive immunostaining for tryptase in kidneys from Kit þ / þ and MC-reconstituted KitW/KitW
v mice (Supplementary Figure 1a and b). Three weeks after UUO, the number of toluidine blue-positive and tryptase-positive MCs was higher in the obstructed kidneys of Kit þ / þ mice (0.5±0.1/mm2) than in the kidneys of sham-operated mice (0.1±0.1/mm2; Po0.025). No MCs were identified in the obstructed kidneys of the KitW/KitW
v mice. In contrast, a substantial number of toluidine blue-positive and tryptase-positive MCs had infiltrated the obstructed kidneys of MC-reconstituted KitW/KitW
v mice (0.4±0.1/mm2). The number of toluidine blue-positive and tryptase-positive MCs in sham-operated kidneys of MC-reconstituted KitW/KitW
v mice was 0.08±0.05/mm2. MCs were observed in the interstitium with a scattered distribution.

Figure 1 | MCs decrease renal tubulointerstitial damage after UUO. Formalin-fixed renal tissues were embedded in paraffin, cut into 5-mm sections, and stained with hematoxylin–eosin. (a–d) Representative pictures of tubulointerstitial lesions with glomeruli. Three weeks after UUO, more inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis were observed in KitW/KitW
v mice than in Kit þ / þ mice. In contrast, such lesions were significantly reduced in MC-reconstituted KitW/KitW
v mice. Bar ¼ 50 mm. (e) Semiquantitative scoring of tubular injury after UUO revealed more damage in KitW/KitW
v mice; n ¼ 6–8 for each experimental group. Data are expressed as means±s.d. *Po0.025 compared with UUO kidneys from Kit þ / þ mice; wPo0.025 compared with UUO kidneys from KitW/KitW
v mice. Kit þ / þ , congenic wild-type WBB6F1-Kit þ / þ mice; KitW/KitW
v, MC-deficient WBB6F1-KitW/KitW
v mice; MC, mast cell; Sham, sham-operated mice; UUO, unilateral uretal obstruction.

each group). Thus, these data show that MCs protect against tubulointerstitial histologic damage after UUO. UUO-induced renal fibrosis is ameliorated by MCs

To determine the effect of MCs on interstitial fibrosis in UUO, we stained slides with Masson’s trichrome and measured the deposition of blue-stained collagen. No fibrosis was observed in sham-operated kidneys in Kit þ / þ mice (Figure 2a). Collagen deposition was significantly higher in the obstructed kidneys of Kit þ / þ mice than in sham-operated Kidney International (2009) 75, 1031–1038

original article

DH Kim et al.: Mast cells decrease renal fibrosis

Increased infiltration of ERHR3-positive macrophages and CD3-positive T cells in kidneys of KitW/KitW
v mice

†

25 20 15 10 5

25

†

20 15 10

Changes in renal TGF-b1, Smad 2/3, and Smad 7 in KitW/KitW
v mice and MC-reconstituted KitW/KitW
v mice

5

v

R M eco t WC n v in sti Ki tut t W io /K n o it W f -

t +/+ Ki

tW /K i Ki

am

v

t +/+ Ki

tW /K i Ki

Sh

R M eco t WC n v in sti Ki tut t W io /K n o it W f -

0 am

0

*

30

Sh

Fibrotic area (%)

35

*

30

Type 1 collagen (%)

35

The numbers of ERHR3-positive monocytes/macrophages per unit area in KitW/KitW
v mouse kidneys were 1.5-fold higher than in kidneys from Kit þ / þ mice 2 and 3 weeks after UUO (Figure 3i). Reconstitution of MCs in KitW/KitW
v mice reduced this increase in the numbers of ERHR3-positive cells per unit area in kidneys by 90 and 60% at 2 and 3 weeks after UUO (Figure 3i). However, the numbers of ERHR3positive macrophages were not significantly changed in KitW/ KitW
v kidneys 1 week after UUO. The numbers of CD3-positive T cells per unit area in KitW/ KitW
v mouse kidneys were 3.3-fold, 1.6-fold, and 2.4fold higher than in kidneys from Kit þ / þ mice 1, 2, and 3 weeks after UUO (Figure 3j). Reconstitution of MCs in KitW/ KitW
v mice reduced this increase in the numbers of CD3positive T cells per unit area by 96 and 85% at 2 and 3 weeks after UUO (Figure 3j). Thus, the presence of MCs decreased the infiltration of monocytes/macrophages and CD3-positive T cells into UUO kidneys 2 and 3 weeks after UUO.

Figure 2 | MCs reduce renal interstitial fibrosis and type I collagen deposition in UUO kidneys. (a–d) Light-field photomicrographs of Masson’s trichrome–stained sections of sham-operated and UUO kidneys at 3 weeks after surgery. Collagen is visible as the blue stain. Bar ¼ 50 mm. (e) Percentage of area of tubulointerstitial fibrosis in Masson’s trichrome–stained sections. Eight randomly selected high-power fields were quantified and averaged to obtain the value for each animal; n ¼ 6–8 for each experimental group. (f) Percentage of area of type I collagen in the sham-operated and UUO kidneys 3 weeks after surgery. Bar graph shows the percentage of the positively stained area relative to the total field. n ¼ 6–8 for each experimental group. Data are expressed as mean±s.d. *Po0.025 compared with UUO kidneys from Kit þ / þ mice; wPo0.025 compared with UUO kidneys from KitW/KitW
v mice. Kit þ / þ , congenic wild-type WBB6F1-Kit þ / þ mice; KitW/KitW
v, MC-deficient WBB6F1-KitW/KitW
v mice; Sham, sham-operated mice.

mice 1, 2, and 3 weeks after UUO (Figure 2b). However, there were no significant differences in collagen deposition in the obstructed kidneys of Kit þ / þ and KitW/KitW
v mice 1 or 2 weeks after ureteral obstruction (data not shown). At 3 weeks, collagen deposition increased 1.5-fold in KitW/KitW
v kidneys compared with Kit þ / þ kidneys (Figure 2c and e). The reconstitution of MCs in KitW/KitW
v mice reversed the increase in interstitial renal fibrosis seen in the mice by approximately 60% (Figure 2d and e). Interstitial type I collagen expression in UUO kidneys in KitW/KitW
v mice was 1.4-fold higher than that in Kit þ / þ mice. Reconstitution of MCs in KitW/KitW
v mice reduced this increase in the deposition of interstitial type I collagen in kidneys by 44% (Figure 2f). Kidney International (2009) 75, 1031–1038

TGF-b1, Smad 2/3, and Smad 7 have pivotal roles in renal fibrosis in UUO.19 The tissue levels of TGF-b1 mRNA measured by real-time PCR were significantly higher in kidneys from KitW/KitW
v mice than in kidneys from Kit þ / þ mice 3 weeks after ureteral obstruction (Figure 4a). The TGFb1 mRNA level in kidneys from MC-reconstituted KitW/ KitW
v mice was significantly lower than in kidneys from KitW/KitW
v mice. Three weeks after ureteral obstruction, the tissue levels of active TGF-b1 were significantly increased in kidneys from KitW/KitW
v mice compared with those from Kit þ / þ mice as assessed by enzyme-linked immunosorbent assay (Figure 4b). Reconstitution of MCs in KitW/KitW
v mice significantly reversed the increase in tissue TGF-b1 level at 3 weeks after ureteral obstruction down to that seen in Kit þ / þ mice. These data suggest that MCs have a role in decreasing the tissue TGF-b1 level in the UUO mouse model. Downregulation of Smad 2/3 is associated with attenuated renal fibrosis in the UUO model, and Smad 7 negatively regulates renal fibrosis by inhibiting Smad 2/3 activation in vivo.20,21 Immunoblots of Smad 2/3 showed that renal Smad 2/3 was 1.4-fold higher in the obstructed kidneys of KitW/ KitW
v mice than in those of Kit þ / þ mice (Figure 4c). Smad 2/3 was approximately 66% lower in kidneys from MCreconstituted KitW/KitW
v mice 3 weeks after UUO than in UUO kidneys from KitW/KitW
v mice (Figure 4c). After 3 weeks, Smad 7 was approximately 46% lower in UUO kidneys from KitW/KitW
v mice than in UUO kidneys from Kit þ / þ mice. Smad 7 in kidneys from MC-reconstituted KitW/KitW
v mice increased approximately 1.3-fold compared with kidneys from KitW/KitW
v mice (Figure 4d). These results suggest that MCs restored the UUO-induced levels of Smad 2/3 and Smad 7 to the levels seen in Kit þ / þ mice. 1033

original article

CD3

ERHR3

DH Kim et al.: Mast cells decrease renal fibrosis

Kit+/+

i

40

ERHR3-positive cells/400X field

Sham

30

KitW/KitW-v

Sham Kit+/+ KitW/KitW-v Reconstitution of MC in KitW/KitW-v

Reconstitution of MC in KitW/KitW-v

* ** ‡

‡

20 10 0

CD3-positive cells/400X field

1 week

6

Sham Kit+/+ KitW/KitW-v Reconstitution of MC in KitW/KitW-v

4 2

2 weeks

3 weeks

** **

† †

**

0 1 week

2 weeks

3 weeks

Figure 3 | MCs decrease the number of infiltrating ERHR3- and CD3-positive cells in UUO kidneys. Representative light micrographs of ERHR3 staining (a–d, arrows) and CD3 staining (e–h, open arrows); the sections were cut from kidneys taken from sham-operated, Kit þ / þ , KitW/KitW
v, and MC-reconstituted KitW/KitW
v mice 3 weeks after surgery and are counterstained with hematoxylin. Bar ¼ 50 mm. The number of infiltrating ERHR3-positive cells (i) and CD3-positive cells (j) in the sham-operated and UUO kidneys at 1, 2, and 3 weeks after surgery; n ¼ 6–8 for each experimental group. Data are expressed as means±s.d. *Po0.025 compared with UUO kidneys from Kit þ / þ mice; **Po0.01 compared with UUO kidneys from Kit þ / þ mice; wPo0.025 compared with UUO kidneys from KitW/KitW
v mice; zPo0.01 compared with UUO kidneys from KitW/KitW
v mice. Kit þ / þ , congenic wild-type WBB6F1-Kit þ / þ mice; KitW/KitW
v, MC-deficient WBB6F1-KitW/KitW
v mice; Sham, sham-operated mice.

MCs preserve E-cadherin and inhibit a-SMA expression

Because TGF-b1 has an important regulatory role in the epithelial-to-mesenchymal transition (EMT) in renal fibrosis, and the epithelial adhesion receptor E-cadherin and a-SMA are the molecular hallmarks of EMT, we next examined the effects of MCs on the expression of E-cadherin and a-SMA. Ureteral obstruction in KitW/KitW
v mice suppressed Ecadherin by approximately 55% and dramatically increased a-SMA by approximately 1.8-fold compared with the levels in Kit þ / þ mice (Figure 5), a shift that is consistent with tubular EMT.22 UUO kidneys from MC-reconstituted KitW/KitW
v mice had preserved E-cadherin expression and inhibited a-SMA induction compared with UUO kidneys 1034

from KitW/KitW
v mice (Figure 5). This suggests that MCs decrease the occurrence of tubular EMT, a key event in the pathogenesis of renal interstitial fibrosis. The effect of MCs in cytokine expression after UUO

Cytokines released from infiltrated monocytes/macrophages or T cells have been implicated in kidney inflammation in the UUO model.23 To evaluate changes in the expression profiles of cytokines in kidney, we used LINCOplex to measure the kidney tissue levels of monocyte chemoattractant protein (MCP)-1, interferon (IFN)-g, and interleukin (IL)-10 in kidneys from Kit þ / þ , KitW/KitW
v, and MC-reconstituted KitW/KitW
v mice 3 weeks after ureteral obstruction. Kidney International (2009) 75, 1031–1038

original article

DH Kim et al.: Mast cells decrease renal fibrosis

100 50

0

0 Sh

v

tW /K it W Re -v M co C ns in tit Ki uti t W on /K o it W f -

Smad 7

Actin

Actin

4

*

1.2 †

3 2 1 0

1.0 Relative ratio

Relative ratio

5

Reconstitution of MC in KitW/KitW-v

Sham

Smad 2/3

Kit+/+

UUO Reconstitution of MC in KitW/KitW-v

KitW/KitW-v

Kit+/+

Sham

UUO

KitW/KitW-v

t +/+

Ki

Ki

Sh

am

20

v

40

150

t +/+

60

200

Ki

80

†

Ki tW /K it W Re -v M co C ns in tit Ki uti t W on /K o it W f -

Tissue TGF-β1 in kidney (pg/mg)

100

*

250

†

120

am

Relative TGF-β1 mRNA expression

300

*

140

0.8 0.6

†

*

0.4 0.2 0.0

Figure 4 | Effect of MCs on TGF-b1 tissue levels and TGF-b1-induced Smad 2/3 and Smad 7 in UUO and sham-operated kidneys. (a) TGF-b1 mRNA levels in sham-operated or UUO kidneys 3 weeks after the operation as determined by real-time PCR. Results were similar in three independent experiments. (b) The tissue level of activated TGF-b1 was quantified by enzyme-linked immunosorbent assay in sham-operated or UUO kidneys 3 weeks after surgery. Results were similar in three independent experiments. (c–d) Immunoblot analyses of Smad 2/3 (c) and Smad 7 (d) in UUO kidneys. Blots were probed with Smad 2/3 or Smad 7 antibodies. The membrane was stripped and reprobed with an anti-actin antibody to verify equal protein loading. Results were similar in three independent experiments. Densitometric analyses are presented below the blots as the relative ratio of Smad 2/3 or Smad 7 to actin. The relative ratio measured in sham-operated kidneys is arbitrarily presented as 1. Data are expressed as mean±s.d. *Po0.025 compared with UUO kidneys from Kit þ / þ mice; wPo0.025 compared with UUO kidneys from KitW/KitW
v mice. Kit þ / þ , congenic wild-type WBB6F1-Kit þ / þ mice; KitW/KitW
v, MCdeficient WBB6F1-KitW/KitW
v mice; Sham, sham-operated mice.

However, there were no significant differences in MCP-1, IFN-g, or IL-10 between kidneys from Kit þ / þ and KitW/ KitW
v mice (data not shown). DISCUSSION

Conflicting data have been published about the antifibrotic effect of MCs in a renal fibrosis model. In this study, we showed that MCs decrease interstitial renal fibrosis in a UUO mouse model. The evidence that MCs have an antifibrotic effect is that renal fibrosis, shown with Masson’s trichrome stain, and collagen type I deposits in obstructed kidneys were more extensive in MC-deficient KitW/KitW
v mice than in Kit þ / þ control mice (Figure 2). Reconstitution of MCs in KitW/KitW
v mice resulted in less interstitial fibrosis and collagen type 1 deposition into the obstructed kidneys. Furthermore, we found that MCs protect against renal Kidney International (2009) 75, 1031–1038

structural damage in obstructed kidneys. The obstructed kidneys of KitW/KitW
v mice showed relatively more damage to the renal tubular structure than the obstructed Kit þ / þ kidneys (Figure 1c). The obstructed kidneys of MCreconstituted KitW/KitW
v mice showed fewer destructive changes to the renal tubular structure and less stromal fibrosis and stromal inflammatory cell infiltration than those in KitW/KitW
v mice. Because TGF-b1 is the main mediator of fibrosis, the origin of the TGF-b1 causing renal fibrosis in the UUO model is important. It has been shown that infiltrating inflammatory cells, such as monocytes/macrophages, may release cytokines such as TGF-b1 that can induce collagen synthesis by fibroblasts and cause further fibrosis.24 Supporting this, decreased interstitial macrophage infiltration correlates with reduced tubulointerstitial fibrosis in the 1035

original article

DH Kim et al.: Mast cells decrease renal fibrosis

Actin

Actin 5

1.0

4

0.8 0.6 †

0.4

*

0.2 0.0

Relative ratio

1.2

kitW/kitW-v

kit+/+

Sham

Reconstitution of MC in kitW/kitW-v

kitW/kitW-v

kit+/+

Sham

α-SMA

Relative ratio

E-cadherin

Reconstitution of MC in kitW/kitW-v

UUO

UUO

*

3

†

2 1 0

m

a Sh

/+

+ kit

-v W

C M -v of itW kit W/ n k tio W / kit itu it st in k n co Re

am

Sh

-v W

+

+/

kit

C M -v of itW n k tio W / kit itu it st in k n co Re kit

W/

Figure 5 | Immunoblotting analyses for E-cadherin and a-SMA. E-cadherin expression (a) was significantly decreased and a-SMA expression (b) was significantly increased in KitW/KitW
v kidneys compared with Kit þ / þ kidneys. In contrast, reconstitution of MCs in KitW/KitW
v mice preserved E-cadherin expression and inhibited a-SMA induction in the obstructed kidney compared with UUO kidneys from KitW/KitW
v mice. The relative ratio measured in sham-operated kidneys is arbitrarily presented as 1. Data are expressed as mean±s.d. *Po0.025 compared with UUO kidneys from Kit þ / þ mice; wPo0.025 compared with UUO kidneys from KitW/KitW
v mice. Kit þ / þ , congenic wild-type WBB6F1-Kit þ / þ mice; KitW/KitW
v, MC-deficient WBB6F1-KitW/KitW
v mice; Sham, sham-operated mice.

UUO model.25 Our data show that MCs decrease both the infiltration of ERHR3-positive macrophages and the tissue levels of TGF-b1 in obstructed kidney (Figure 3). Therefore, these data suggest that MCs may decrease the production of TGF-b1 and its release from ERHR3-positive macrophages in the UUO model. MCs produce and release several chemical mediators.26 Some of these mediators can modulate biological responses by regulating cytokine expression or inflammatory cell infiltration.27 Timoshanko et al.9 measured renal mRNA levels of chemokines such as RANTES (regulated upon activation, normal T cell, expressed, and secreted), macrophage inflammatory proteins, MCP-1, and IFN-inducible protein-10 in wild-type mice and MC-deficient mice in the crescentic glomerulonephritis model and suggested that MCs aggravate crescentic glomerulonephritis by regulating the intercellular adhesion molecule (ICAM)-1 and P-selectin expression in kidney. Miyazawa et al.28 found that MC-deficient mice expressed higher levels of TGF-b1 mRNA than wildtype mice in the chronic glomerular disease model, whereas IL-10 and IFN-g mRNA levels did not differ significantly between MC-deficient and wild-type mice. They therefore suggested that MCs may have a protective role in this model. We similarly showed that there were no significant differences in MCP-1, IFN-g, and IL-10 in kidney between Kit þ / þ and KitW/KitW
v mice, and our data also showed that MCs decrease the tissue levels of TGF-b1 in obstructed kidneys from Kit þ / þ and MC-reconstituted KitW/KitW
v mice. 1036

These results suggest that MCs may downregulate TGF-b1 expression in models of chronic glomerular disease or renal fibrosis. Because TGF-b1 is the main inducer of EMT, the increased expression of TGF-b1 in the obstructed kidneys could be involved in the loss of the epithelial phenotype and the acquisition of the mesenchymal phenotype. Our data show that obstructed kidneys in Kit þ / þ mice express less Ecadherin and more a-SMA than the kidneys of shamoperated mice, and this suppression of E-cadherin and induction of a-SMA is greater in the kidneys of KitW/KitW
v mice than in those of Kit þ / þ mice. Reconstitution of MCs in KitW/KitW
v mice preserved E-cadherin expression and inhibited a-SMA induction in UUO kidneys. Therefore, the regulation of MCs might decrease the effect of EMT in ureteral obstruction. Another controversial aspect of the role of MCs in kidney disease is whether there are MCs in kidney. Hochegger et al.4 have detected MCs not in kidney but in regional lymph nodes in the anti-glomerular basement membrane glomerulonephritis model and suggested that MCs can regulate inflammatory responses in regional lymph nodes. In this study, we found that MCs are located in the UUO kidney from Kit þ / þ and MC-reconstituted KitW/KitW
v mice. Thus, MCs in the kidney may regulate the renal inflammatory response in the UUO model. All of these data from the UUO model suggest that MCs have a protective role in interstitial fibrosis, but future studies Kidney International (2009) 75, 1031–1038

DH Kim et al.: Mast cells decrease renal fibrosis

will be needed to investigate whether MCs can be used therapeutically for the treatment of renal disease. MATERIALS AND METHODS Animal experiments: UUO model Our animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Chonbuk National University Medical School. Genetically MC-deficient KitW/KitW
v mice and Kit þ / þ mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Male Kit þ / þ mice (20–30 g body weight, n ¼ 8), KitW/KitW
v mice (20–30 g body weight, n ¼ 7), and MC-reconstituted KitW/KitW
v mice (20–30 g body weight, n ¼ 6) were used in these experiments. In mice anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), renal interstitial fibrosis was induced as previously described.29 Briefly, an incision was made in the midline of the abdomen, and the left proximal ureter was exposed and was ligated at two separate locations using 3–0 silk. In sham-operated Kit þ / þ mice, the ureters were exposed and manipulated but not ligated. In preliminary experiments, we examined the renal cortex 1, 3, and 5 weeks after UUO. Because the histopathological score (described below) was most appropriate for assessing renal fibrosis at 3 weeks after ureteral obstruction, we evaluated the effects of UUO at this time. Three weeks after UUO, the mice were killed and the unilaterally obstructed kidney was harvested and cut in half. One half was fixed in formalin for subsequent immunohistochemical staining, and the remaining tissue was stored at
201C for subsequent protein isolation. Reconstitution of MCs in MC-deficient KitW/KitW
v mice The MC deficiency of KitW/KitW
v mice was systemically repaired as described.30 Bone marrow cultured mast cells were obtained by culturing bone marrow cells from femurs of wild-type Kit þ / þ mice for 4–5 weeks in Dulbecco’s modified Eagle’s medium (Sigma Chemical Co., St Louis, MO, USA) supplemented 20% with WEHI3-conditioned medium (containing IL-3); at this time over 95% of the cells were identified as immature MCs by May–Grunwald–Giemsa staining. For MC reconstitution studies, bone marrow cultured mast cells were transferred by tail-vein injection (1  107 cells in 200 ml of Dulbecco’s modified Eagle’s medium) into MCdeficient KitW/KitW
v mice, and the recipients were used for experiments 18 weeks later. MCs were identified in kidney by toluidine blue staining Histology and immunohistochemistry Portions of sham-operated and UUO kidneys were fixed in 10% neutral buffered formalin and embedded in paraffin. The tissue was sectioned at 5 mm and stained with hematoxylin–eosin stain for light microscopic analysis. The presence of interstitial fibrosis was assessed in slides stained with Masson’s trichrome. Toluidine blue staining was performed by immersing kidney sections in 0.1% toluidine blue (Sigma Chemical Co.) for 5 min at room temperature. MCs were quantified in 20 randomly selected kidney sections, and the amount was expressed as the number of MCs in a  200 magnification field. Immunostaining for anti-tryptase (Abcam, Cambridge, UK), anti-mouse type I collagen (SouthernBiotech, Birmingham, AL, USA), anti-CD3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-ERHR3 (BMA, Augst, Switzerland) was also used to stain paraffin sections as described previously.29 Digital images of the immunostaining were obtained with a Zeiss Z1 microscope (Carl Zeiss, Go¨ttingen, Germany). The area positive for type I collagen was evaluated from the unit area (  200 Kidney International (2009) 75, 1031–1038

original article

magnification field) and expressed as a percentage per unit area using a digital image analysis program (AnalySIS, Soft Imaging System, Mu¨nster, Germany). Histopathological scoring Histopathology was scored in the cortex by a scorer blinded to treatment group. Tubular injury was assessed by grading tubular dilatation, epithelial desquamation, and brush border loss in 10 randomly chosen, nonoverlapping fields (  200 magnification) using methods previously described.31 Briefly, the lesions were graded on a scale from 0 to 4: 0 ¼ normal; 1 ¼ mild, involvement of less than 25% of the cortex; 2 ¼ moderate, involvement of 25–50% of the cortex; 3 ¼ severe, involvement of 50–75% of the cortex; 4 ¼ extensive damage involving more than 75% of the cortex. Immunoblotting Immunoblotting was performed as previously described.32 Primary antibodies to Smad 2/3 and Smad 7 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The antibody to E-cadherin was purchased from BD Biosciences (Bedford, UK), and those to actin and a-SMA were purchased from Sigma-Aldrich (St Louis, MO, USA). Signals were analyzed by densitometric scanning (LAS-3000; Fuji Film, Tokyo, Japan). Densitometric analyses are presented as the ratio relative to actin. Enzyme-linked immunosorbent assay of TGF-b1 The tissue concentrations of TGF-b1 in kidney were determined using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA) 3 weeks after the UUO operation.29 In all cases, a standard curve was constructed from the standards provided by the manufacturer. Real-time quantitative RT-PCR Total RNA from each kidney sample was isolated using TRIzol solution (GIBCO Invitrogen, Grand Island, NY, USA) and quantified by the ratio of absorption at 260 nm to that at 280 nm. For each sample, 1 mg of total RNA was reverse transcribed into firststrand cDNA in a 20-ml reaction system (Roche Diagnostics, Mannheim, Germany) at 551C for 30 min. Quantitative RT-PCR analysis was performed with the Light-Cycler FastStart DNA Master SYBR Green I (Roche Diagnostics). For real-time PCR, the following primers were used: TGF-b1: forward, 50 -GAGAGCCCTGGATAC CAACTATTG-30 ; reverse, 50 -GTGTGTCCAGGCTCCAAATATAG-30 . GAPDH (internal control): forward, 50 -AGAACATCATCCCTG CATCC-30 ; reverse, 50 -CCTGCTTCACCACCTTCTTG-30 . Cytokine analysis Tissue MCP-1, IFN-g, and IL-10 concentrations from kidney were measured in triplicate by using a Mouse Cytokine Lincoplex kit (Linco Research Inc., St Charles, MI, USA) 1, 2, and 3 weeks after ureteral obstruction. Kidney from Kit þ / þ , KitW/KitW
v and MCreconstituted KitW/KitW
v mice were analyzed in the same assay. In all cases, a standard curve was constructed from the standards provided by the manufacturer. Statistical analysis Data are expressed as means±standard deviation. Mean comparisons between two groups were examined for significant differences using the Mann–Whitney U-test, with Po0.025 indicating statistical significance. 1037

original article

DISCLOSURE

DH Kim et al.: Mast cells decrease renal fibrosis

14.

All the authors declared no competing interests. 15.

ACKNOWLEDGMENTS

This paper was supported by the Fund of the Chonbuk National University Hospital Research Institute of Clinical Medicine (2008, W.K.). This work was supported by grants from the National Research Laboratory Program, Ministry of Science and Technology (ROA-2004000-10292-0, S.K.P), and in part by a grant from the Post-doc Program, Chonbuk National University (2006, W.K.). SUPPLEMENTARY MATERIAL Figure S1. Photomicrographs of mast cells within the kidney from MC-reconstituted KitW/KitW
v mice 3 weeks after ureteral obstruction. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki REFERENCES 1. Boyce JA. Mast cells: beyond IgE. J Allergy Clin Immunol 2003; 111: 24–32; quiz 33. 2. Metz M, Grimbaldeston MA, Nakae S et al. Mast cells in the promotion and limitation of chronic inflammation. Immunol Rev 2007; 217: 304–328. 3. Gordon JR, Galli SJ. Mast cells as a source of both preformed and immunologically inducible TNF-alpha/cachectin. Nature 1990; 346: 274–276. 4. Hochegger K, Siebenhaar F, Vielhauer V et al. Role of mast cells in experimental anti-glomerular basement membrane glomerulonephritis. Eur J Immunol 2005; 35: 3074–3082. 5. Secor VH, Secor WE, Gutekunst CA et al. Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis. J Exp Med 2000; 191: 813–822. 6. Lee DM, Friend DS, Gurish MF et al. Mast cells: a cellular link between autoantibodies and inflammatory arthritis. Science 2002; 297: 1689–1692. 7. He SH. Key role of mast cells and their major secretory products in inflammatory bowel disease. World J Gastroenterol 2004; 10: 309–318. 8. Abonia JP, Friend DS, Austen WG et al. Mast cell protease 5 mediates ischemia-reperfusion injury of mouse skeletal muscle. J Immunol 2005; 174: 7285–7291. 9. Timoshanko JR, Kitching R, Semple TJ et al. A pathogenetic role for mast cells in experimental crescentic glomerulonephritis. J Am Soc Nephrol 2006; 17: 150–159. 10. Ruger BM, Hasan Q, Greenhill NS et al. Mast cells and type VIII collagen in human diabetic nephropathy. Diabetologia 1996; 39: 1215–1222. 11. Ehara T, Shigematsu H. Contribution of mast cells to the tubulointerstitial lesions in IgA nephritis. Kidney Int 1998; 54: 1675–1683. 12. Toth T, Toth-Jakatics R, Jimi S et al. Mast cells in rapidly progressive glomerulonephritis. J Am Soc Nephrol 1999; 10: 1498–1505. 13. Danilewicz M, Wagrowska-Danilewicz M. Immunohistochemical analysis of the interstitial mast cells in rebiopsied patients with idiopathic mesangial proliferative glomerulonephritis. Pol J Pathol 2005; 56: 63–68.

1038

16.

17. 18.

19.

20.

21.

22.

23. 24.

25. 26. 27.

28.

29.

30.

31.

32.

Lajoie G, Nadasdy T, Laszik Z et al. Mast cells in acute cellular rejection of human renal allografts. Mod Pathol 1996; 9: 1118–1125. Jones SE, Kelly DJ, Cox AJ et al. Mast cell infiltration and chemokine expression in progressive renal disease. Kidney Int 2003; 64: 906–913. Miyazawa S, Hotta O, Doi N et al. Role of mast cells in the development of renal fibrosis: use of mast cell-deficient rats. Kidney Int 2004; 65: 2228–2237. Nath KA. Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis 1992; 20: 1–17. Risdon RA, Sloper JC, De Wardener HE. Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet 1968; 2: 363–366. Sato M, Muragaki Y, Saika S et al. Targeted disruption of TGF-beta1/ Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. J Clin Invest 2003; 112: 1486–1494. Inazaki K, Kanamaru Y, Kojima Y et al. Smad3 deficiency attenuates renal fibrosis, inflammation, and apoptosis after unilateral ureteral obstruction. Kidney Int 2004; 66: 597–604. Fukasawa H, Yamamoto T, Togawa A et al. Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice. Proc Natl Acad Sci USA 2004; 101: 8687–8692. Park SH, Choi MJ, Song IK et al. Erythropoietin decreases renal fibrosis in mice with ureteral obstruction: role of inhibiting TGF-beta-induced epithelial-to-mesenchymal transition. J Am Soc Nephrol 2007; 18: 1497–1507. Manucha W. Biochemical-molecular markers in unilateral ureteral obstruction. Biocell 2007; 31: 1–12. Schreiner GF, Harris KP, Purkerson ML et al. Immunological aspects of acute ureteral obstruction: immune cell infiltrate in the kidney. Kidney Int 1988; 34: 487–493. Schanstra JP, Neau E, Drogoz P et al. In vivo bradykinin B2 receptor activation reduces renal fibrosis. J Clin Invest 2002; 110: 371–379. Galli SJ. New concepts about the mast cell. N Engl J Med 1993; 328: 257–265. Lukacs NW, Hogaboam C, Chensue SW et al. Type 1/type 2 cytokine paradigm and the progression of pulmonary fibrosis. Chest 2001; 120: 5S–8S. Miyazawa S, Hotta O, Doi N et al. Role of mast cells in the development of renal fibrosis: use of mast cell-deficient rats. Kidney International 2004; 65: 2228–2237. Kim W, Moon SO, Lee SY et al. COMP-angiopoietin-1 ameliorates renal fibrosis in a unilateral ureteral obstruction model. J Am Soc Nephrol 2006; 17: 2474–2483. Yu M, Tsai M, Tam SY et al. Mast cells can promote the development of multiple features of chronic asthma in mice. J Clin Invest 2006; 116: 1633–1641. Rouschop KM, Sewnath ME, Claessen N et al. CD44 deficiency increases tubular damage but reduces renal fibrosis in obstructive nephropathy. J Am Soc Nephrol 2004; 15: 674–686. Sung MJ, Kim W, Ahn SY et al. Protective effect of a-lipoic acid in lipopolysaccharide-induced endothelial fractalkine expression. Circ Res 2005; 97: 880–890.

Kidney International (2009) 75, 1031–1038