1. Volume 57, Issue 3, Pages 927-936 (March 2000)
Inhibition of monocyte chemoattractant protein-1 expression in tubular epithelium attenuates tubulointerstitial alteration in rat Goodpasture syndrome 
Hirokazu Okada, Kenshi Moriwaki, Raghuram Kalluri, Hiroe Imai, Shinichi Ban, Motohide Takahama, Hiromichi Suzuki 
Kidney International 
Volume 57, Issue 3, Pages (March 2000)
DOI: /j x
Copyright © 2000 International Society of Nephrology Terms and Conditions

2. Figure 1
Characterization of kidneys of Goodpasture syndrome (GPS) rats. (a) Day 28. Crescent formation was observed in some glomeruli, but no significant interstitial mononuclear cell infiltration was seen (HE stain, ×200). (b) Day 35. Prominent mononuclear cell infiltration in the interstitium appeared in parallel to the progression of glomerular and tubular damage (HE stain, ×200). (c) monocyte chemoattractant protein-1 (MCP-1) mRNA expression in the kidneys. Northern blot analysis with total RNA extracted from whole nephritic kidneys revealed that MCP-1 mRNA began to increase on day 21 and reached a peak level on day 35. A representative blot selected from three separate experiments is shown. (d) MCP-1 protein expression on day 28. MCP-1 is located in the glomeruli and in the tubular epithelium (×200).
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

3. Figure 2
Monocyte chemoattractant protein-1 (MCP-1) antisense oligodeoxynucleotide (ODN) application in vitro. (A) Northern blot analysis of MCP-1 mRNA in RPTC cells treated with antisense ODN. (B) Quantitative densitometric analysis of (A). Antisense ODN significantly lowered the MCP-1 mRNA level (lane 4 in A, column 4 in B) in relationship to the control level (lane 5 in A, column 5 in B). However, sense ODN-treated, mutated antisense ODN-treated and untreated RPTC cells strongly express MCP-1 mRNA (lanes 1 through 3, columns 1 through 3, respectively). A representative blot from three separate experiments is shown; densitometric data were obtained from all three blots.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

4. Figure 3
Distribution of FITC-conjugated ODN in the kidney at 12 hours after intravenous injection. (a) Normal kidney. FITC-conjugated ODN was observed in the tubular epithelium (×300). (b) Nephritic kidney of GPS rats on day 28. FITC-conjugated ODN was taken up by the tubular epithelium, and its distribution was substantially the same as in the normal kidney (×300). (c) The autofluorescence of nephritic kidney was negligible (×300). (d) Dual staining with TRITC-conjugated Tetragonolobus purpureas (TP) as a proximal tubular epithelium marker. FITC-conjugated ODN (in green) colocalized with TP (in orange) in the proximal tubular epithelium, yielding a yellow color and indicating that ODN was taken up by proximal tubular epithelial cells exclusively. TP, only, localized to the glomeruli, yielding an orange color (×300).
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

5. Figure 4
MCP-1 antisense ODN application in vivo. (A) Northern analysis of total RNA extracted from whole nephritic kidneys at 12 hours after the administration of ODN on day 28. (B) Quantitative densitometric analysis of (A). MCP-1 antisense ODN reduced the MCP-1 mRNA to control levels (lanes 3 and 4 in A, columns 3 and 4 in B). MCP-1 mRNA levels in the kidneys of sense ODN-treated GPS rats (lane 2 in A, column 2 in B) were similar to the untreated GPS rats (lane 1 in A, column 1 in B). A representative blot selected from four separate experiments is shown, and the densitometric data were obtained from these four blots. (C) RT-PCR detection of MCP-1 mRNA in the glomeruli isolated by serial sieving. The glomerular MCP-1 mRNA level was increased in the untreated GPS rats on day 28 when compared with the control rats (lanes 4 and 1). MCP-1 antisense or sense ODN did not affect the increased level of MCP-1 mRNA in the glomeruli (lanes 2–4). These are the representative data selected from three separate experiments.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

6. Figure 5
MCP-1 protein and mRNA expression in the kidney of GPS rats on day 37 after multiple injections of MCP-1 antisense ODN. (a) MCP-1 protein expression in the kidney of MCP-1-S rats. MCP-1 protein persisted in the tubular epithelium of the MCP-1-S rats (×200). (b) MCP-1 protein expression in the kidney of the MCP-1-AS group. The MCP-1 protein level was reduced in the tubular epithelium (×200). (c) In situ hybridization analysis of MCP-1 mRNA expression in the kidney of sense ODN-treated GPS rats on day 37. Using MCP-1 antisense probe, MCP-1 mRNA was detected in the tubular epithelium (arrows) and faintly in the glomeruli (×300). (d) The MCP-1 mRNA level in the kidney of antisense ODN-treated GPS rats on day 37. MCP-1 mRNA was not found in the tubular epithelium of these rats (×300). (e) In situ hybridization using the MCP-1 sense probe did not label the kidney of sense ODN-treated GPS rats on day 37 (×300). (f) Western blot analysis of protein extracts from the kidneys of ODN-treated GPS rats on day 37. The MCP-1 protein level was increased in the kidney of untreated animals, and the increased levels were not affected by treatment with sense ODN in the MCP-1-S rats. However, MCP-1 protein was not detected in the sample of the MCP-1-AS group and the control rats, suggesting that antisense ODN lowered MCP-1 protein expression in the nephritic kidneys.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

7. Figure 6
Renal histopathology of GPS rats on day 37 after repeated injections of MCP-1 antisense ODN. (a) Renal histology of the MCP-1-S rats. Remarkable glomerular and interstitial alterations were observed (HE stain, ×300). (b) Renal histology of the MCP-1-AS rats. Despite extensive glomerular damage, interstitial mononuclear cell infiltration was significantly attenuated (HE stain, ×300). (c) Glomerular hypercellularity and interstitial mononuclear cell infiltration index [mean cell number/10 glomeruli, and mean cell number/high-power (×200) cortical field, respectively]. Symbols are: (▪) glomeruli; () interstitium. No significant effects on glomerular hypercellularity were seen in either the MCP-1-AS or MCP-1-S rats when compared with the untreated rats. However, the number of mononuclear cells in the interstitium was significantly lower in the MCP-1-AS group than in either the MCP-1-S or untreated groups. (d) ED-1–positive monocyte/macrophages in the kidney of the MCP-1-S group. A number of ED-1–positive monocyte/macrophages were seen in the glomeruli and the interstitium (×300). (e) ED-1–positive monocyte/macrophages in the kidney of the MCP-1-AS group. The number of ED-1–positive monocyte/macrophages in the interstitium was significantly reduced (×300). (f) ED-1–positive monocyte/macrophage counts in the glomeruli (▪) and the interstitium () [mean ED-1 (+) cell number/10 glomeruli, and mean ED-1 (+) cell number/high-power (×200) cortical field, respectively]. Although the numbers of ED-1–positive monocyte/macrophages in the glomeruli were not different between the MCP-1-S and MCP-1-AS rats, the numbers of ED-1–positive monocyte/macrophages in the interstitium of the MCP-1-AS group were significantly lower than those of the MCP-1-S and untreated groups.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

8. Figure 6
Renal histopathology of GPS rats on day 37 after repeated injections of MCP-1 antisense ODN. (a) Renal histology of the MCP-1-S rats. Remarkable glomerular and interstitial alterations were observed (HE stain, ×300). (b) Renal histology of the MCP-1-AS rats. Despite extensive glomerular damage, interstitial mononuclear cell infiltration was significantly attenuated (HE stain, ×300). (c) Glomerular hypercellularity and interstitial mononuclear cell infiltration index [mean cell number/10 glomeruli, and mean cell number/high-power (×200) cortical field, respectively]. Symbols are: (▪) glomeruli; () interstitium. No significant effects on glomerular hypercellularity were seen in either the MCP-1-AS or MCP-1-S rats when compared with the untreated rats. However, the number of mononuclear cells in the interstitium was significantly lower in the MCP-1-AS group than in either the MCP-1-S or untreated groups. (d) ED-1–positive monocyte/macrophages in the kidney of the MCP-1-S group. A number of ED-1–positive monocyte/macrophages were seen in the glomeruli and the interstitium (×300). (e) ED-1–positive monocyte/macrophages in the kidney of the MCP-1-AS group. The number of ED-1–positive monocyte/macrophages in the interstitium was significantly reduced (×300). (f) ED-1–positive monocyte/macrophage counts in the glomeruli (▪) and the interstitium () [mean ED-1 (+) cell number/10 glomeruli, and mean ED-1 (+) cell number/high-power (×200) cortical field, respectively]. Although the numbers of ED-1–positive monocyte/macrophages in the glomeruli were not different between the MCP-1-S and MCP-1-AS rats, the numbers of ED-1–positive monocyte/macrophages in the interstitium of the MCP-1-AS group were significantly lower than those of the MCP-1-S and untreated groups.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

9. Figure 6
Renal histopathology of GPS rats on day 37 after repeated injections of MCP-1 antisense ODN. (a) Renal histology of the MCP-1-S rats. Remarkable glomerular and interstitial alterations were observed (HE stain, ×300). (b) Renal histology of the MCP-1-AS rats. Despite extensive glomerular damage, interstitial mononuclear cell infiltration was significantly attenuated (HE stain, ×300). (c) Glomerular hypercellularity and interstitial mononuclear cell infiltration index [mean cell number/10 glomeruli, and mean cell number/high-power (×200) cortical field, respectively]. Symbols are: (▪) glomeruli; () interstitium. No significant effects on glomerular hypercellularity were seen in either the MCP-1-AS or MCP-1-S rats when compared with the untreated rats. However, the number of mononuclear cells in the interstitium was significantly lower in the MCP-1-AS group than in either the MCP-1-S or untreated groups. (d) ED-1–positive monocyte/macrophages in the kidney of the MCP-1-S group. A number of ED-1–positive monocyte/macrophages were seen in the glomeruli and the interstitium (×300). (e) ED-1–positive monocyte/macrophages in the kidney of the MCP-1-AS group. The number of ED-1–positive monocyte/macrophages in the interstitium was significantly reduced (×300). (f) ED-1–positive monocyte/macrophage counts in the glomeruli (▪) and the interstitium () [mean ED-1 (+) cell number/10 glomeruli, and mean ED-1 (+) cell number/high-power (×200) cortical field, respectively]. Although the numbers of ED-1–positive monocyte/macrophages in the glomeruli were not different between the MCP-1-S and MCP-1-AS rats, the numbers of ED-1–positive monocyte/macrophages in the interstitium of the MCP-1-AS group were significantly lower than those of the MCP-1-S and untreated groups.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

10. Figure 7
Renal functional parameters of GPS rats on day 37 after multiple injections of MCP-1 antisense ODN. (a) Urinary protein excretion rate. Urinary protein excretion rate was not affected in either the MCP-1-AS or MCP-1-S groups when compared with the untreated group. (b) Creatinine clearance (CCr). The decrease in CCr of the MCP-1-AS group was significantly suppressed compared with the untreated and MCP-1-S groups.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions