The growth factor midkine regulates the renin-angiotensin system in mice

The renin-angiotensin system plays a pivotal role in regulating blood pressure and is involved in the pathogenesis of kidney disorders and other diseases. Here, we report that the growth factor midkine is what we believe to be a novel regulator of the renin-angiotensin system. The hypertension induced in mice by 5/6 nephrectomy was accompanied by renal damage and elevated plasma angiotensin II levels and was ameliorated by an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin receptor blocker. Notably, ACE activity in the lung, midkine expression in the lung, and midkine levels in the plasma were all increased after 5/6 nephrectomy. Exposure to midkine protein enhanced ACE expression in primary cultured human lung microvascular endothelial cells. Furthermore, hypertension was not induced and renal damage was less severe in midkine-deficient mice. Supplemental administration of midkine protein to midkine-deficient mice restored ACE expression in the lung and hypertension after 5/6 nephrectomy. Oxidative stress might be involved in midkine expression, since expression of NADH/NADPH oxidase–1, –2, and –4 was induced in the lung after 5/6 nephrectomy. Indeed, the antioxidative reagent tempol reduced midkine expression and plasma angiotensin II levels and consequently ameliorated hypertension. These results suggest that midkine regulates the renin-angiotensin system and mediates the kidney-lung interaction after 5/6 nephrectomy.

The renin-angiotensin system (RAS) is a hormonal cascade that functions in the homeostatic control of arterial pressure, tissue perfusion, and extracellular volume. Dysregulation of the RAS results in the pathogenesis of many diseases, including cardiovascular and renal disorders (1–3). The RAS is initiated by the regulated secretion of renin, which catalyzes the hydrolysis of Ang I from the N terminus of angiotensinogen. Ang I is in turn hydrolyzed by angiotensin-converting enzyme (ACE) to form Ang II, the primary active product of the RAS (4, 5). ACE is a zinc metallopeptidase widely distributed on the cell membrane of endothelial and epithelial cells (6). Ang II induces vasoconstriction and aldosterone release, leading to upregulation of blood pressure. It also exerts its vasoconstrictor effect on both the afferent and efferent arterioles, which may contribute to the onset and progression of chronic renal damage. Ang II may also directly contribute to the acceleration of renal damage by sustaining cell growth, inflammation, and fibrosis (7).

The growth factor midkine (MK; gene symbol, MDK) is implicated in cancer progression, neuronal survival and differentiation, and inflammation (8). MK is involved in the pathogeneses of tubulointerstitial damage induced by renal reperfusion and glomerular sclerosis associated with diabetes mellitus (9, 10). The finding of a recent report that angiotensinogen and renin expression was significantly elevated in the aorta of Mdk^–/– mice while ACE expression was significantly suppressed is of particular interest (11). However, Mdk^–/– mice develop normally (8), and there has been no report of systemic disturbance or organ disorders of Mdk^–/– mice. Therefore, the biological meaning of changes in the RAS molecules in the aorta of Mdk^–/– mice has remained obscure.

It is widely accepted that the RAS is involved in the pathogenesis of chronic kidney disease (CKD), and inhibitors of the RAS are the first choice of therapy for CKD (12–14). To investigate the molecular mechanisms regulating the RAS in CKD, we employed 5/6 nephrectomy in this study. 5/6 nephrectomized mice are a popular and useful model of CKD, since the remnant kidney model of progressive renal injury is characterized by systemic hypertension and glomerular hyperfiltration, the latter eventually causing glomerular sclerosis (15, 16). CKD accompanies multiple organ failure, the pathogenesis of which involves inter-organ cross-talk (17, 18). In this context, it is noteworthy that MK expression was induced in the lung by 5/6 nephrectomy, leading to elevation of ACE activity and plasma Ang II levels and subsequent hypertension in the present study. Our data therefore suggest that MK is a candidate mediator of inter-organ cross-talk in CKD.

MK is involved in RAS activation induced by 5/6 nephrectomy. Systolic and mean blood pressure were comparable in untreated Mdk^+/+ and Mdk^–/– mice (Figure 1, A and B). However, we found that 5/6 nephrectomy strikingly increased blood pressure in Mdk^+/+ mice but not in Mdk^–/– mice (Figure 1, A and B). The systolic and mean blood pressure of Mdk^+/+ mice strikingly increased after 2 weeks, but Mdk^–/– mice showed almost normal blood pressure, i.e., no significant increase (systolic blood pressure, 143 ± 11.6 mmHg in Mdk^+/+ mice vs. 119 ± 8.6 mmHg in Mdk^–/– mice; mean blood pressure, 104 ± 10.3 mmHg vs. 85 ± 6.7 mmHg). Consequently, systolic and mean blood pressures were significantly higher in Mdk^+/+ than in Mdk^–/– mice from 2 to 8 weeks (Figure 1, A and B).

Figure 1

5/6 nephrectomy induces hypertension and renal damage via the RAS. (A and B) Blood pressure was measured at 0, 2, 4, 6, and 8 weeks after 5/6 nephrectomy. Systolic blood pressure (SBP) (A) and mean blood pressure (MBP) (B) were measured by the tail-cuff method. The mean and SD are represented by squares and bars, respectively, at each time point (Mdk^+/+: 0 weeks, n = 40; 2 weeks, n = 34; 4 weeks, n = 19; 8 weeks, n = 8; Mdk^–/–: 0 weeks, n = 26; 2 weeks, n = 23; 4 weeks, n = 13; 8 weeks, n = 4). ***P < 0.001 versus Mdk^–/– mice. (C) Representative glomerular histology shown by PAS staining. Scale bar: 50 μm. (D) Semiquantitative analysis of the glomerular sclerosis score. Data are shown as mean and SD (Mdk^+/+: 2 weeks, n = 5; 4 weeks, n = 4; 8 weeks, n = 4; Mdk^–/–: 2 weeks, n = 4; 4 weeks, n = 3; 8 weeks, n = 3). (E) Plasma Ang II concentration after 5/6 nephrectomy (Mdk^+/+: sham, n = 6; 2 weeks, n = 6; 4 weeks, n = 8; Mdk^–/–: sham, n = 6; 2 weeks, n = 7; 4 weeks, n = 5). *P < 0.05, **P < 0.01; ^†P < 0.01 versus sham Mdk^+/+. Nx, nephrectomy. (F and G) Effects of hydralazine, temocapril, and olmesartan on blood pressure. SBP (F) and MBP (G) were measured by the tail-cuff method (n = 3). ^‡P < 0.01 versus Mdk^+/+ mice. (H) Representative histology after treatment with hydralazine, temocapril, and olmesartan. The kidney specimens were stained with PAS. Tubular dilatation (black arrows), tubular cast formation (arrowheads), and tubular degeneration (white arrows) are indicated. Scale bar: 50 μm.

5/6 nephrectomy caused not only hypertension but also progressive renal failure. Blood urea nitrogen and serum creatinine levels gradually increased, and both parameters were significantly higher in Mdk^+/+ mice at 2 and 4 weeks after renal ablation (Table 1). Mdk^+/+ mice also exhibited more severe glomerular sclerosis, which is characterized by a marked deposition of extracellular matrix in the glomeruli and which occurred as early as 2 weeks after renal ablation (Figure 1C). Semiquantitative analysis of the glomerular sclerosis scores revealed significant differences between Mdk^+/+ and Mdk^–/– mice (Figure 1D). In addition, the tubulointerstitial damage was worse in Mdk^+/+ mice (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI37249DS1). Thus, tubular dilatation, cast formation in the tubular lumen, and tubular epithelial degeneration became apparent at 2 weeks after renal ablation and were more severe in Mdk^+/+ mice (Supplemental Figure 1, A–H). Interstitial fibrosis, as evidenced by collagen deposition revealed by Masson’s trichrome staining, was exhibited at 4 weeks and then more diffusely at 8 weeks, and the stained area became expanded in Mdk^+/+ mice (Supplemental Figure 1, I–P). These data collectively indicate that renal damage was more severe in Mdk^+/+ mice than in Mdk^–/– mice.

Table 1

Body weight, blood urea nitrogen, serum creatinine, and left kidney weight after 5/6 nephrectomy

These symptoms of hypertension and renal damage were attributable to the RAS, as (a) the hypertension was accompanied by elevated plasma Ang II concentration (Figure 1E); and (b) the ACE inhibitor temocapril and the angiotensin receptor blocker olmesartan reduced both blood pressure and renal tubulointerstitial damage, but the vasodilator hydralazine only ameliorated hypertension (Figure 1, F–H). Abnormal elevation of blood urea nitrogen and serum creatinine was ameliorated by administration of temocapril and olmesartan, but not hydralazine (Table 1). It is therefore conceivable that the RAS contributed to both hypertension and renal damage.

ACE levels are increased in the lung after 5/6 nephrectomy. Expression of the intrarenal angiotensinogen and renin was suppressed and ACE expression was unchanged in Mdk^+/+ mice, which was consistent with previous findings after 5/6 nephrectomy of rats (19, 20) (Supplemental Figure 2, A–C). In contrast to the kidney, the lung showed significant increases in ACE expression and its activity 2 and 4 weeks after renal ablation (Figure 2, A–E). ACE protein expression was localized to the pulmonary vascular endothelial cells and alveolar-capillary endothelial cells, consistent with a previous report (Figure 2D) (21).

Figure 2

ACE expression in the lung after 5/6 nephrectomy. (A) ACE protein was determined by Western blotting, and a representative result is shown. The lung tissues were obtained at the indicated time points. (B) Quantitative analysis of ACE protein expression using densitometry. Data are presented as mean and SD (n = 3). ^†P < 0.01; ^‡P < 0.05; *P < 0.05 and **P < 0.01 versus sham Mdk^+/+. (C) ACE mRNA was determined by real-time PCR and normalized to 18S mRNA. Data are presented as mean and SD (Mdk^+/+: sham, n = 5; 2 weeks, n = 5; 4 weeks, n = 3; Mdk^–/–: sham, n = 3; 2 weeks, n = 3; 4 weeks, n = 3). ^§P < 0.0001; ***P < 0.001 and ^#P < 0.0001 versus sham Mdk^+/+. (D) Immunohistochemical staining of lungs with mouse anti-ACE monoclonal antibody at 2 weeks. Upper panel: The first antibody used was anti–mouse ACE monoclonal antibody. Lower panel: Isotype-matched IgG was used as the first antibody. Scale bar: 50 μm. (E) ACE activity was determined by the ACE activity assay, as described in Methods. Data are presented as mean and SD (Mdk^+/+: sham, n = 5; 2 weeks, n = 5; 4 weeks, n = 5; Mdk^–/–: sham, n = 5; 2 weeks, n = 5; 4 weeks, n = 4). ^†P < 0.01; ^‡P < 0.05; **P < 0.01 versus sham Mdk^+/+.

Other organs that play major functions in the RAS were also examined for their expression of RAS components. Angiotensinogen mRNA expression was induced in the liver after 5/6 nephrectomy, but the expression level was not significantly different in Mdk^+/+ and Mdk^–/– mice (Supplemental Figure 3A). There was no difference in angiotensinogen protein levels between the two genotypes (Supplemental Figure 3, B and C), and renin mRNA expression was not detected in the liver (data not shown). Expression of ACE protein and MK protein was also not detected in the liver (Supplemental Figure 3, D and E). In the brain, mRNA expression of angiotensinogen and ACE did not change after 5/6 nephrectomy (Supplemental Figure 4, A and B), and the renin mRNA expression was undetectable (data not shown). ACE protein was also not detected in the brain (Supplemental Figure 4C). Furthermore, the mRNA expression of angiotensinogen, renin, and ACE did not change in the heart (Supplemental Figure 5). Therefore, it is most likely that the hypertension observed after 5/6 nephrectomy was due to activation of the lung ACE.

MK levels are increased in the lung, kidney, and plasma after 5/6 nephrectomy. MK expression was increased in the lung in association with an elevation in ACE expression (Figure 3, A–C). MK protein was localized to the endothelium of microvessels of the lung, as revealed by the use of thrombomodulin as a marker of the vascular endothelium (Figure 3D). MK expression was detected in alveolar-capillary endothelial cells but not in bronchial epithelial cells (Figure 3D).

Figure 3

MK expression in the lung and plasma after 5/6 nephrectomy. (A) Representative data from Western blotting for MK expression in the lung. (B) The intensity of MK bands on Western blotting was normalized to that of β-actin. Data are presented as mean and SD (n = 4). *P < 0.05 and **P < 0.01 versus sham. (C) MK mRNA expression in the lung was determined by real-time PCR and normalized to 18S mRNA. Data are presented as mean and SD (sham, n = 5; 2 weeks, n = 5; 4 weeks, n = 3). ***P < 0.001 versus sham. (D) Immunofluorescence staining of MK and thrombomodulin expression in the lung 2 weeks after 5/6 nephrectomy. Lower panels show negative controls using isotype-matched IgG as the first antibody. Arrowhead, bronchial epithelial cells; white arrows, alveolar-capillary endothelial cells. Scale bar: 50 μm. (E) Representative data from Western blotting for MK expression in plasma are shown. Lines under the blot indicate individual samples. (F) Western blot data for plasma MK were quantified using densitometry and are presented as mean and SD (sham, n = 4; 2 weeks, n = 5). **P < 0.01.

Histological evidence of lung damage, i.e., due to edema and degeneration of alveolar cells, was not observed after 5/6 nephrectomy in the Mdk^+/+ and Mdk^–/– mice (Supplemental Figure 6A). Increases in macrophage and neutrophil infiltration into the lung were also not observed after 5/6 nephrectomy in the two genotypes (Supplemental Figure 6, B–E). These results indicated that the increase in MK expression in the lung after 5/6 nephrectomy was not due to leukocytes.

MK expression was also significantly elevated in the kidney at both the protein and mRNA levels 2 and 4 weeks after renal ablation (Supplemental Figure 7, A–C). Immunohistochemical analysis revealed that MK protein was mainly localized in the tubular epithelium (Supplemental Figure 8A). This result is consistent with previous reports in which MK was expressed in the kidney after ischemia/reperfusion injury and its associated massive leukocyte infiltration (22). We also detected a substantial increase in macrophage infiltration into the kidney after 5/6 nephrectomy, and this increase was significantly higher in Mdk^+/+ mice than Mdk^–/– mice (Supplemental Figure 8, B and C). It is known that MK is expressed by activated macrophages (23, 24). Thus, it is conceivable that the increase in MK expression in the kidney after 5/6 nephrectomy was due to enhanced expression in both the tubular epithelium and infiltrating macrophages.

Since MK is a secreted protein, and its expression was induced by 5/6 nephrectomy in the kidney and lung (Figure 3, A–C, and Supplemental Figure 7, A–C), we next examined plasma MK levels. As shown in Figure 3, E and F, plasma MK levels were indeed increased 2 weeks after 5/6 nephrectomy.

Exogenous MK induces ACE expression. If MK is required for ACE expression in the lung and hypertension, supplementary administration of MK might also affect these symptoms. To clarify this issue, we administered exogenous MK continuously through an osmotic pump into Mdk^–/– mice after 5/6 nephrectomy. This administration was found to restore hypertension and ACE expression in the lung (Figure 4, A–C). We also administered pleiotrophin (PTN; also called HB-GAM), which shows 50% homology with MK (8), to Mdk^–/– mice after 5/6 nephrectomy. However, exogenous PTN neither induced hypertension nor increased ACE expression in the lung (Figure 4, A, D, and E). These data support the specificity of MK with respect to its involvement in ACE expression and blood pressure regulation.

Figure 4

Effects of exogenous MK and PTN in Mdk^–/– mice on blood pressure and ACE expression in the lung. (A) SBP was measured by the tail-cuff method at 0 and 2 weeks after 5/6 nephrectomy (n = 5). **P < 0.01 versus untreated Mdk^–/– mice (0 weeks); ^#P < 0.01, rh-MK versus saline. rh-PTN, recombinant human PTN. (B) ACE expression in the lung of Mdk^–/– mice treated with MK and saline. Western blot data are shown. (C) The data in B were quantified using densitometry and are presented as mean and SD. ^†P < 0.01. (D) ACE expression in the lung of Mdk^–/– mice treated with rh-PTN and saline. Western blot data are shown. (E) The data in D were quantified using densitometry and are presented as mean and SD.

Furthermore, exogenous MK protein on primary cultured human lung microvascular endothelial cells (HMVEC-L) significantly enhanced ACE expression, suggesting that ACE is one of the targets of MK in the lung (Figure 5, A and B). When Ang I was added to the culture medium of the lung endothelial cells treated with MK and heparin for 36 hours, Ang I was converted to Ang II in a time-dependent manner, while cells treated with heparin alone did not show such a conversion (Figure 5C). These results suggest that MK is a potent inducer of Ang II through upregulation of ACE expression in lung endothelial cells.

Figure 5

Mechanism of ACE induction by MK in HMVEC-L. (A) ACE expression in HMVEC-L treated with exogenous MK protein (100 ng/ml) and heparin (20 μg/ml). Lower panels: For PKC inhibition, BIS (5 μM) was added 1 hour before a 36-hour treatment with rh-MK plus heparin. Western blot data are shown. (B) The data in A were quantified using densitometry and are presented as mean and SD (n = 3). *P < 0.05 and **P < 0.01 versus 0 hours. (C) Conversion from Ang I to Ang II by the cells treated with MK. HMVEC-L cells were treated with exogenous MK protein (100 ng/ml) and heparin (20 μg/ml) or heparin (20 μg/ml) for 36 hours. Ang I (500 pM) was then added to the medium and incubated for the indicated times (n = 5). (D) Phospho-PKC expression in HMVEC-L treated with exogenous MK protein (100 ng/ml) and heparin (20 μg/ml). Western blot data are shown. (E) The data in D (left panel) were quantified using densitometry and are presented as mean and SD (n = 3). *P < 0.05 and **P < 0.01 versus 0 hours. (F) Representative data from Western blotting for phospho-PKC expression in the lung. The intensity of phospho-PKC bands on Western blotting was normalized to that of β-actin (n = 5). ^§P < 0.05 versus sham Mdk^+/+. Western blotting results are not shown. ^†P < 0.01; ^‡P < 0.05.

Along with the increase in ACE expression, phosphorylation levels of PKC were also increased in primary cultured HMVEC-L after exposure to exogenous MK (Figure 5, D and E). This result suggests that MK upregulates ACE expression through activation of PKC. This idea was further supported by three lines of evidence. First, bisindolylmaleimide I (BIS), a PKC-specific inhibitor, blocked the MK-mediated increase in ACE expression (Figure 5, A and B). Second, PKC phosphorylation was significantly increased in the lungs of Mdk^+/+ but not Mdk^–/– mice after 5/6 nephrectomy (Figure 5F). Third, consistent with previous reports (25), the increase in ACE expression in primary cultured HMVEC-L was also induced by PMA, a PKC activator, and was blocked by BIS (Supplemental Figure 9).

Oxidant stress induces MK expression in the lung after 5/6 nephrectomy. Finally, the mechanism of MK induction in the lung by 5/6 nephrectomy was investigated. The NADPH oxidases (Nox’s) are superoxide-generating enzymes that release superoxide by electron transfer from NADPH to oxygen. Increased production of ROS has been implicated in various pathologies, including hypertension, atherosclerosis, diabetes, and CKD (26, 27). In the present study, Nox1, -2, and -4 mRNA expression in the lungs of Mdk^+/+ mice was found to be significantly increased at 2 and 4 weeks after renal ablation compared with the levels in the sham-operated animals, suggesting that oxidant stress was generated in the lung; in contrast, the expression of Nox1, -2, and -4 mRNA was unchanged in the Mdk^–/– mice (Figure 6A). A cell membrane–permeable radical scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol), reduced MK expression to normal levels in the lung (Figure 6B); plasma Ang II levels (Figure 1E) and blood pressure (Figure 6C) were also reduced. Tempol also ameliorated glomerular sclerosis and tubulointerstitial damage (Figure 6D) and improved renal function, i.e., significantly reduced blood urea nitrogen and serum creatinine levels (Table 1). These results suggest that MK expression was induced by oxidative stress in the lung after 5/6 nephrectomy. Tempol also reduced MK expression in the kidney (Supplemental Figure 7, D and E).

Figure 6

The mechanism of MK induction in the lung. (A) Time course of Nox1, -2, and -4 expression in the lung after 5/6 nephrectomy. Expression of Nox1, -2, and -4 mRNA was determined by real-time PCR. Data are presented as mean and SD (Mdk^+/+: sham, n = 5; 2 weeks, n = 5; 4 weeks, n = 3; Mdk^–/–: sham, n = 3; 2 weeks, n = 3; 4 weeks, n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 versus sham Mdk^+/+. (B) Effect of tempol on MK expression in the lung. Tempol mixed in drinking water was administered to Mdk^+/+ mice after 5/6 nephrectomy. MK protein expression was determined by Western blotting (upper panel). The intensity of MK bands on Western blotting was normalized to that of β-actin (lower panel). Data are presented as mean and SD (n = 5). (C) Effect of tempol on blood pressure after 5/6 nephrectomy. SBP was measured by the tail-cuff method. Data are presented as mean and SD (n = 6). (D) Effect of tempol on renal damage. PAS staining of a kidney specimen 2 weeks after 5/6 nephrectomy in Mdk^+/+ mice that received tempol treatment. The tubulointerstitial damage was slight compared with that in the mice without tempol treatment (Figure 1C). Scale bar: 20 μm. ^†P < 0.05; ^‡P < 0.01.

Our study demonstrated that Mdk^–/– mice had almost normal blood pressure after 5/6 nephrectomy, while wild-type mice showed marked hypertension. This hypertension was ameliorated by RAS-related inhibitors and indeed was accompanied by elevated plasma Ang II levels. Surprisingly, ACE activity was enhanced in the lung, whereas RAS components were not activated in other organs. Plasma MK levels and MK expression in the lung and kidney were elevated. Supplementary MK administration to Mdk^–/– mice restored lung ACE expression and hypertension. MK also induced ACE expression and consequently conversion from Ang I to Ang II in primary cultured lung microvascular endothelial cells. We therefore concluded that MK-mediated ACE induction in the lung is critical for hypertension induced by 5/6 nephrectomy (Figure 7).

Figure 7

Schematic diagram showing the possible relationship between MK and the RAS cascade that promotes hypertension and renal damage.

Inter-organ interactions involving the kidney have recently been highlighted. Regarding factors affecting lung function after acute kidney injury, several cytokines, including IL-6, IL-1β, and TNF-α, have been suggested as candidates (28, 29). Such results contribute to our understanding of the high mortality associated with pulmonary complications following acute kidney injury. CKD has also been linked with damage in other organs, especially with cardiovascular damage (so-called cardiorenal syndrome) (30). It is particularly interesting that RAS components are increased in the heart and brain of subtotally nephrectomized rats (20, 31) and that Ang II amounts are increased in the isolated perfused hind limbs of uremic rats (32). Inhibitors of the RAS, e.g., angiotensin receptor blockers and ACE inhibitors, are indeed the first choice of therapy for CKD (33). Our results clearly show that the lung is a promising target in the cross-talk between the kidney and other organs and suggest that MK is a candidate mediator for pulmonary and other organ complications associated with CKD.

Regarding the cross-talk between the kidney and lung in 5/6 nephrectomy, our study has also provided an insight into the underlying mechanism. We found that Nox1, -2, and -4 were induced in the lung and that tempol reduced MK and plasma Ang II levels. Nox mediates the initial reaction of 3 successive reduction products of molecular oxygen, i.e., superoxide (O₂^•–), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH^•). Since tempol is a membrane-permeable and metal-independent superoxide dismutase mimetic that is specific for superoxide anion (O₂^•–) (34, 35), tempol may target ROS initiated by Nox in the lung. To the best of our knowledge, this is the first study to show that 5/6 nephrectomy induces oxidative stress in the lung. We have previously reported that oxidative stress induces MK expression (10, 22). Therefore, it is conceivable that Nox-mediated ROS production leads to an induction of MK expression in the lung (Figure 7). As ROS have very short half-lives (36), it is not likely that the ROS themselves travel between the kidney and lung. Furthermore, it is widely accepted that Ang II induces Nox expression (37). Based on this background data, we speculate that the following molecular circuit is established after circulating MK induces ACE expression in the lung: Ang II induces Nox expression, which in turn initiates ROS production and subsequently MK and ACE expression (Figure 7).

RAS-related inhibitors, but not hydralazine, ameliorated renal damage in the present study. It has also been reported that systemically administered Ang II worsens renal function (38–40). Therefore, the RAS might play at least a partial role in renal damage. In addition, we found a significant elevation of macrophage infiltration into the kidney in wild-type mice compared with Mdk^–/– mice in the present study. This finding is consistent with our previous report showing MK-mediated macrophage recruitment in neointima formation (41). Therefore, MK-mediated inflammation in the kidney may contribute to the renal damage after 5/6 nephrectomy (Figure 7). On the other hand, in the context of inflammation and hypertension, it is important to note that inflammatory cell infiltration into the kidney does not always induce hypertension (42, 43). Furthermore, the local RAS in organs other than the lung was not activated in the present study. Thus, the relationship between intrarenal inflammation and hypertension after 5/6 nephrectomy remains to be verified.

View Supplemental data

We thank Norihiko Suzuki, Naoko Asano, Yuriko Sawa, and Kayoko Miyata for their excellent technical assistance. This work was supported in part by the 21st Century COE Program and Global COE program, Ministry of Education, Culture, Sports, Science, and Technology, Japan.

Address correspondence to: Kenji Kadomatsu, Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Phone: 81-52-744-2059; Fax: 81-52-744-2065; E-mail: kkadoma@med.nagoya-u.ac.jp.

Conflict of interest: The authors have declared that no conflict interest exists.

Nonstandard abbreviations used: ACE, angiotensin-converting enzyme; BIS, bisindolylmaleimide I; CKD, chronic kidney disease; HMVEC-L, human lung microvascular endothelial cell(s); MK, midkine; Nox, NADPH oxidase; PTN, pleiotrophin; RAS, renin-angiotensin system; rh-MK, recombinant human MK.

Reference information: J. Clin. Invest.119:1616–1625 (2009). doi:10.1172/JCI37249

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