BASIC RESEARCH

GDF15 Triggers Homeostatic Proliferation of Acid-Secreting Collecting Duct Cells

Jean Paul Duong Van Huyen, Lydie Cheval, May Bloch-Faure, Marie France Belair, Didier Heudes, Patrick Bruneval and Alain Doucet
JASN October 2008, 19 (10) 1965-1974; DOI: https://doi.org/10.1681/ASN.2007070781

Abstract

Although adult kidney cells are quiescent, enlargement of specific populations of epithelial cells occurs during repair and adaptive processes. A prerequisite to the development of regenerative therapeutics is to identify the mechanisms and factors that control the size of specific populations of renal cells. Unfortunately, in most cases, it is unknown whether the growth of cell populations results from transdifferentiation or proliferation and whether proliferating cells derive from epithelial cells or from circulating or resident progenitors. In this study, the mechanisms underlying the enlargement of the acid-secreting cell population in the mouse kidney collecting duct in response to metabolic acidosis was investigated. Acidosis led to two phases of proliferation that preferentially affected the acid-secreting cells of the outer medullary collecting duct. All proliferating cells displayed polarized expression of functional markers. The first phase of proliferation, which started within 24 h and peaked at day 3, was dependent on the overexpression of growth differentiation factor 15 (GDF15) and cyclin D1 and was abolished when phosphatidylinositol-3 kinase and mammalian target of rapamycin were inhibited. During this phase, cells mostly divided along the tubular axis, contributing to tubule lengthening. The second phase of proliferation was independent of GDF15 but was associated with induction of cyclin D3. During this phase, cells divided transversely. In summary, acid-secreting cells proliferate as the collecting duct adapts to metabolic acidosis, and GDF15 seems to be an important determinant of collecting duct lengthening.

Loss of kidney functions may be life-threatening and is always socially and economically costly. Reparative therapies triggering renewal of kidney epithelial cells would greatly improve the course of these pathologies. Identification of the mechanisms controlling proliferation of specific tubular renal cell populations is a prerequisite for developing such therapies. Unfortunately, little is known about tubular cell proliferation in the mature kidney.

In contrast to the juvenile kidney, in which active cell proliferation permits nephron growth in length,1 in adult kidney, tubular cells are quiescent under normal conditions.2 However, renal proximal tubule cell proliferation is triggered in postinjury repair after acute renal failure,3,4 ureteral obstruction, or five-sixths nephrectomy.5 Hyperplasia of specific tubule cell populations also occurs in physiologic adaptive processes. Early reports suggested that the abundance of collecting duct intercalated cells (IC), the acid-base transporting cells, increases homeostatically in response to changes in acid-base balance6 or to potassium depletion,7,8 but these results were not confirmed.9 More recently, the use of cell proliferation markers, such as incorporation of the bromodeoxyuridine (BrdU) into DNA or staining for proliferating cellular nuclear antigen (PCNA), demonstrated proliferation of distal tubule cells in response to increased sodium delivery10,11 and to potassium depletion.12 However, the issue of proliferation of collecting duct type A, acid secreting IC (AIC) during acidosis has not been re-addressed using this approach. Our first aim was to reinvestigate whether metabolic acidosis increases the population of AIC in the collecting duct and to determine the contribution of cell proliferation versus transdifferentiation on this effect and the differentiation status of proliferating cells.

The second aim was to evaluate the putative role of growth and differentiation factor 15 (GDF15) in the proliferation of AIC. GDF15, a member of the TGF-β superfamily, is synthesized as a 40-kD propeptide and undergoes cleavage of its N-ter to generate an active 30-kD disulfide-linked dimeric protein that is secreted.13 GDF15 is detected mainly in liver and placenta but is induced in many tissues in response to various stresses. Large-scale analysis of gene expression in mouse outer medullary collecting duct (OMCD) revealed tremendous overexpression of GDF15 mRNA during metabolic acidosis14 and potassium depletion.12

RESULTS

Acidosis Induces Early Proliferation of IC

Combined immunofluorescence labeling of outer medullary sections of control mice kidney (Figure 1A) showed that all cells were stained either apically for aquaporin 2 (AQP2) or at their basal pole for kAE1, identifying them as principal cells (PC) and AIC, respectively. Electron micrographs (Figure 1C) confirmed that OMCD cells display structural characteristics of either PC (spherical nucleus, basolateral membrane infoldings, smooth apical membrane) or AIC (light cytoplasm, apical microplicae, higher number of mitochondria). This confirms that type B IC (BIC) and non-A non-B IC are not detectable in mouse OMCD.15 No cell was double-positive for AQP2 and kAE1. Analysis of 3-d acidotic mice (Figure 1, B and D) also demonstrated the exclusive presence of PC and AIC and revealed an apparent increase in the AIC proportion. Cell counting on kidney outer medulla sections stained for either AQP2 or kAE1 or on transmission electron microscopy (TEM) micrographs demonstrated increased proportion of AIC in 3-d acidotic mice (Table 1).

Figure 1.
Figure 1.

Identification of AIC and PC in control and acidotic mice OMCD. (A and B) Immunofluorescence of kidney outer medulla sections shows PC with red apical AQP2 labeling and AIC with green basolateral kAE1 staining. In control (A) and 3-d acidotic mice (B), OMCD were continuously lined by either AQP2- or kAE1-labeled cells, without any double-negative or double-positive cell. (C and D) Electron micrographs (bars = 5 μm) show clear PC with smooth apical membrane and interdigitations of basolateral membrane and lighter AIC with numerous mitochondria, apical membrane microplicae, and smooth basolateral membrane. A slight increase in AIC abundance was apparent in 3-d acidotic mice (B and D) as compared with controls (A and C). Magnification, ×2500 in C and D.

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Table 1.

Quantification of PC and AIC in control and acidotic mice OMCDa

Figure 2 shows co-stainings of outer medulla sections with three proliferation markers and anti-kAE1 antibody. In control mice, no staining was observed for PCNA (Figure 2A), Ki-67, or BrdU (data not shown). In contrast, in 3-d acidotic mice, OMCD cell staining was readily observed for PCNA (Figure 2, B and C), Ki-67 (Figure 2D), and BrdU (Figure 2, E and F). Most proliferating cells were positive for kAE1 (AIC, arrows), but some were kAE1-negative PC (Figure 2C, inset, arrowhead). Occasionally, mitotic figures were seen (Figure 2F). Similar conclusions were reached when outer medulla sections were co-stained for AQP2 and either Ki-67 (Figure 3, A through D), PCNA (Figure 3E), or BrdU (Figure 3F): No proliferating cells were detected in control mice (Figure 3A), and in 3-d acidotic mice, proliferation concerned preferentially AQP2-negative AIC (arrows) and occasionally PC (Figure 3D, arrowhead). Quantification of proliferation marker–positive cells confirmed that the proliferation rate of AIC exceeded that of PC (Table 2), thereby accounting for the enlargement of AIC population in acidotic mice. Proliferation of both AIC and PC appeared as early as 24 h after the onset of metabolic acidosis and peaked after 3 d (Figure 4).

Figure 2.
Figure 2.

(A through F) Co-localization of kAE1 and proliferation markers in OMCD from control mice (A) and 3-d acidotic mice (B through F). IC are characterized by basolateral kAE1 labeling (in brown in A through D; in red in E and F), and cells in cell cycle are labeled by anti-PCNA antibody (A through C, red), anti–Ki-67 antibody (D, red), or anti-BrdU antibody (E and F, brown). PCNA-positive cells were present in 3-d acidotic (B, *) but not in control mice (A). Proliferation markers PCNA (B and C), Ki-67 (D), and BrdU (E and F) are mainly present in kAE1-positive AIC (arrows) and occasionally in kAE1-negative PC (C, arrowhead). Occasionally, mitotic cells are seen (F, arrows). Plasma acid-base parameters of mice are shown in Figure 7.

Figure 3.
Figure 3.

(A through F) Co-localization of AQP2 and proliferation markers in OMCD from control mice (A) and 3-d acidotic mice (B through F). PC show apical and cytoplasmic AQP2 labeling (A through D and F, red; E, brown), and collecting duct cells in cell cycle are labeled by anti–Ki-67 antibody (A through D, brown), anti-PCNA antibody (E, red), or anti-BrdU antibody (F, brown). Ki-67 labeling is increased in the collecting ducts from 3-d acidotic mice (B, *) whereas no Ki-67–positive cell is present in control mice (A). Proliferation markers Ki-67 (B through D), PCNA (E), and BrdU (F) are mainly present in AQP2-negative AIC (arrows) and occasionally in AQP2-positive PC (D, arrowhead).

Figure 4.
Figure 4.

Proliferating AIC and PC in control and acidotic mice OMCD. Outer medulla sections of kidneys from control (C) and 2- to 14-d acidotic mice (d2 through d14) were labeled with anti-AQP2 and anti–Ki-67 antibodies. In tubular sections with at least one AQP2-positive cell, Ki-67–positive and –negative cells with either positive (PC) or negative AQP2 staining (AIC) were counted. Two hundred cells were counted in each mouse, and the percentages of proliferating PC (▪) and AIC (□) were calculated. Data are means ± SEM from four to 10 mice. Statistical significance between acidotic and control mice was assessed by ANOVA analysis followed by unpaired t test: *P < 0.05; ***P < 0.001.

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Table 2.

Quantification of proliferating PC and AIC in control and acidotic mice OMCDa

Morphometric analysis of OMCD cross-sections on electron micrographs (Figure 5, A through C) revealed several acidosis-induced changes: (1) The cross-section epithelial surface area was slightly but significantly reduced in 3-d acidotic mice and returned to control level at day 14 (Figure 5D); (2) because the total number of cells varied in parallel with the epithelium surface (Figure 5E), the mean cell surface area remained constant in the three groups of mice (Figure 5F); and (3) changes in the total number of cells were accounted for by a decreased number of PC at day 3, which recovered at day 14, and an increased number of AIC at day 14 (Figure 5E).

Figure 5.
Figure 5.

Morphometric analysis of OMCD in control and acidotic mice. (A through C) TEM micrographs of OMCD from control and acidotic mice (bars = 5 μm). In a control mouse (A), the duct lumen is lined by a normal-sized AIC and three PC. In a 3-d acidotic mouse (B), the apparent AIC over PC number ratio is increased. In a 14-d acidotic mouse (C), the AIC cross-sectional surface area is apparently increased and the tubular lumen is dramatically narrowed. AIC enlargement is associated with appearance of cell dimples at the luminal surface (arrowheads), which are not associated with either tight junction or border between two cells. Arrows, cell junctions; arrowheads, cell dimples. (D) Quantification of the cross-section epithelium surface area of OMCD from control mice (□), 3-d acidotic mice (□), and 14-d acidotic mice (▪). (E) Number of AIC (▒) and PC (□) per OMCD cross-section from control and acidotic mice. (F) The cross-section surface area of cells (both AIC and PC) in the three groups of mice was calculated as the ratio of the epithelium surface area over the total number of cells in each OMCD section. Data are means ± SEM from three mice in each group. Statistical significance between acidotic and control mice was assessed by nonparametric Mann-Whitney test: *P < 0.05. Magnification, ×2500.

GDF15 and Acidosis-Induced Cell Proliferation

GDF15 mRNA was expressed at low level all along the nephron of control mice and was markedly induced in connecting tubules and collecting ducts from 3-d acidotic mice, mostly in OMCD (>40-fold increase; Figure 6A). In OMCD, expression of GDF15 mRNA increased already 24 h after the onset of acidosis, peaked at day 3, and decreased afterward (Figure 6B), a time course similar to that of AIC proliferation (see Figure 4).

Figure 6.
Figure 6.

Expression of GDF15 mRNA in nephron segments of control and acidotic mice. (A) Expression profile of GDF15 mRNA along the nephron of control (□) and 3-d acidotic mice (▪). PCT, proximal convoluted tubule; PST, proximal straight tubule; cTAL, cortical thick ascending limb of Henle's loop; mTAL, outer medullary thick ascending limb of Henle's loop; DCT, distal convoluted tubule; CNT, connecting tubule; CCD, cortical collecting duct. (B) GDF15 mRNA expression in OMCD from control (C) and 1- to 14-d acidotic mice (d1 through d14). Data are means ± SEM from five to seven mice. Statistical significance between acidotic and control mice was assessed by ANOVA followed by unpaired t test: *P < 0.05; ***P < 0.001.

Compared with wild-type (WT) mice, proliferation of AIC was reduced drastically in 3-d acidotic GDF15−/− mice and was increased at day 14 (Figure 7A). Similar changes were observed for PC (data not shown). GDF15−/− mice displayed lower blood pH and bicarbonate concentration than WT mice at days 1 to 3 but afterward compensated their acidosis back toward WT level, concomitantly with the marked increase in AIC proliferation, whereas WT mice did not (Figure 7, B and C).

Figure 7.
Figure 7.

Effect of acidosis on cell proliferation, and acid-base parameters in WT (□) and GDF15−/− mice (▪). (A) Percentage of proliferation of AIC in control (C) and 3- and 14-d acidotic mice (d3 and d14). GDF15 deficiency reduces and postpones acidosis-induced proliferation of AIC. (B and C) Blood pH and plasma bicarbonate concentration in control and 1- to 14-d acidotic mice. GDF15−/− mice display more intense acidosis than WT at the early stages of the treatment and recover afterward to the same level of acidosis. Data are mean ± SEM from five to seven mice. Statistical significance between WT and GDF15−/− mice was assessed by unpaired t test: **P < 0.005.

Under basal conditions, OMCD from GDF15−/− and WT mice displayed similar morphologic features except a narrower lumen (in μm2± SE; WT 183 ± 8; GDF15−/− 52 ± 12; n = 3 in each group; P < 0.05), and a greater cross-section surface area of AIC (in μm2± SE; WT 104 ± 0; GDF15−/− 126 ± 13; n = 3 in each group; P < 0.05). In acidotic GDF15−/− mice, the cross-section epithelial surface area increased steadily from day 3 to day 14 (Figure 8A), whereas the total number of cells remained constant (Figure 8B). Therefore, the mean cross-section cell surface area increased in parallel with the epithelium surface area (Figure 8C). The constancy of the total number of cells was accounted for by a steady increase in AIC and a mirror decrease in PC (Figure 8B).

Figure 8.
Figure 8.

Morphometric analysis of OMCD in acidotic GDF15−/− mice. (A) Quantification of the cross-section epithelium surface area of OMCD from control mice (□), 3-d acidotic mice (□), and 14-d acidotic mice (▪). (B) Number of AIC (hatched bars) and PC (solid bars) per OMCD cross-section from control and acidotic mice. (C) The cross-section surface area of cells (both AIC and PC) in the three groups of mice was calculated as the ratio of the epithelium surface area over the total number of cells in each OMCD section. Data are means ± SEM from three mice in each group. Statistical significance between acidotic and control mice was assessed by nonparametric Mann-Whitney test: *P < 0.05.

Entry of quiescent cells into early G1 requires transcriptional activation of cyclins D.16 Cyclin D2 mRNA was not detected in OMCD of either control or acidotic mice. Cyclin D1 showed early overexpression (days 1 to 2) in WT mice and returned toward control level after. Overexpression of cyclin D1 mRNA was blunted in GDF15−/− mice (Figure 9). Cyclin D3 displayed delayed overexpression (day 14) in WT mice. In GDF15−/− mice, overexpression of cyclin D3 was evident earlier, as soon as day 3 (Figure 9). Expression of the V1d subunit of H-ATPase, considered as representative of the acid-base regulon,14 increased gradually during acidosis in WT and GDF15−/− mice (Figure 9), indicating that cell proliferation and induction of acid-base regulon are independent. p53, known to control the transcription of GDF15,17 showed early overexpression at day 1 in WT but not GDF15−/− mice (Figure 9).

Figure 9.
Figure 9.

Acidosis-induced gene expression pattern in OMCD from WT (□) and GDF15−/− mice (▪). mRNA expression of cyclins D1 and D3, V1d subunit of vacuolar H-ATPase (ATP1V1d), and p53 in OMCD from control (C) and 1- to 14-d acidotic mice (d1 through d14). Data are means ± SEM from five to seven mice. Statistical significance was assessed by ANOVA followed by unpaired t test: *P < 0.05, acidosis versus control; §P < 0.05, GDF15−/− versus WT.

Because expression of cyclin D1 and G1 cell-cycle progression are regulated by phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling in cancer epithelial cells,18,19 we investigated the involvement of this pathway in the early phase of acidosis-induced proliferation. Administration of the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin abolished the early AIC proliferation (day 2) and the overexpression of cyclin D1 (Figure 10).

Figure 10.
Figure 10.

Effect of PI3K and mTOR inhibition on cyclin D1 expression and cell proliferation. (A) Expression of cyclin D1 mRNA in OMCD from control and 2-d acidotic mice treated or not (C) with PI3K inhibitor LY294002 (LY, 30 mg/kg once) or mTOR inhibitor rapamycin (Ra, 0.2 mg/kg per d). (B) Percentage of proliferation of AIC (AQP2-negative, Ki-67–positive cells) in the same groups of mice. Data are means ± SEM from three to six mice. Statistical significance was assessed by ANOVA followed by unpaired t test: *P < 0.05, acidotic versus control.

DISCUSSION

Acidosis Increases AIC Population via Proliferation of Fully Differentiated Cells

Early reports suggested that the AIC population grows during metabolic acidosis,6 but later this finding was not confirmed.9 Meanwhile, Schwartz and Al Awqati20 developed the concept of cell plasticity (transdifferentiation) to account for homeostatic adaptation of the rabbit cortical collecting duct to metabolic acidosis. They interpreted this adaptation to acidosis as representing conversion of BIC to AIC and demonstrated the role of hensin in that process.20,21 Our results (Table 2) demonstrate an enlargement of the AIC population in OMCD during acidosis that is accounted for by preferential proliferation of AIC and not transdifferentiation: (1) We confirmed that mouse OMCD contains neither BIC nor non-A non-B IC,15 the potential precursors of AIC in the plasticity process, and (2) proliferating IC display basolateral expression of the AIC marker kAE1 (Figure 2). This conclusion does not preclude the possibility that transdifferentiation may participate in adaptation of the cortical collecting duct, which contains both BIC and non-A non-B IC.

It is commonly accepted that terminally differentiated epithelial cells can no longer enter cell cycle and divide. Accordingly, the turnover of permanently renewing epithelia, such as the intestine or the skin, proceeds through division of progenitor cells that differentiate afterward during their migration from intestinal crypts to the apex of the villi or from the epidermis basal layer to the skin surface. In epithelial cells with high proliferating capacity, such as hepatocytes during liver regeneration, reappearance of fetal markers22 suggests that they undergo partial dedifferentiation at the G0/G1 transition. Postinjury regeneration of renal tubules also relies on proliferation of partially dedifferentiated epithelial cells and, possibly, of progenitors.3,23–25 In contrast, our results show proliferation of cells displaying normal polarized expression of specific functional markers. In some instances, mitotic cells with basolateral kAE1 labeling were seen. Proliferation of fully differentiated cells in the normal kidney was documented by another group.1,11 Teleologically, it is significant that in adaptive processes, cell proliferation does not alter the integrity of the epithelium barrier so as to maintain the tubular function. In contrast, when the integrity of the epithelium is compromised beforehand, as in postinjury repair, proliferation may rely on the recruitment of progenitor cells and/or on cell dedifferentiation.

AIC Proliferation Contributes to Acid-Base Homeostasis

Collecting ducts play a major role in acid-base homeostasis by controlling bicarbonate, proton, and ammonium excretion. Thus, loss-of-function mutations of proteins involved in collecting duct proton secretion are responsible for metabolic acidosis.26 Reciprocally, diet-induced acidosis promotes homeostatic adaptations of collecting ducts, including targeting of acid-base transporters from intracellular pools to the plasma membrane27 and transcriptional induction of genes involved in acid-base metabolism.14 Our results show that AIC proliferation also participates in early homeostatic adaptation of the collecting ducts. As a matter of fact, reduced AIC proliferation observed in 1- to 3-d GDF15−/− acidotic mice was associated with more pronounced acidosis. Conversely, acid-base parameters in GDF15−/− mice returned to WT level at day 14, when proliferation was triggered back (Figure 7, B and C).

Mechanisms Underlying Proliferation of AIC

In WT mice, the high proliferation rate of AIC observed at days 2 and 3 (Figure 4) and the lack of variation of their cross-section number at day 3 (Figure 5E) indicate that AIC divided mainly along the tubular axis (longitudinal proliferation), which thereby should lengthen OMCD. Given that during the same period PC proliferation rate was weaker (Figure 4), tubule lengthening required the longitudinal migration of some PC, accounting for the reduction of their cross-section number (Figure 5E). Conversely, from day 3 to day 14, proliferation of AIC decreased drastically (Figure 4), whereas their cross-section number increased (Figure 5E), indicating that dividing AIC were oriented mainly parallel to the cross-section plane of OMCD (transverse proliferation). Transverse proliferation was also evidenced by the marked increase in the number of AIC doublets, without interposed PC in cross-sections from day 3 to day 14 acidotic mice (data not shown). Again, PC proliferation was slower during that period (Figure 4), suggesting that increased cross-section number of PC (Figure 5E) might have been accounted for by elongation of PC along the tubule axis. This cell remodeling is compatible with the decreased cross-section surface area of PC observed at day 14 (data not shown). Longitudinal AIC proliferation is concomitant with overexpression of GDF15 (Figure 6B) and cyclin D1 (Figure 9), whereas transverse proliferation appears later and concomitantly with cyclin D3 overexpression (Figure 9). Figure 11A schematizes the proposed cell proliferation, migration, and remodeling processes induced by acidosis in WT mice. Longitudinal cell proliferation and subsequent lengthening of OMCD in acidotic mice, in absence of lengthening of neighboring thick ascending limbs (i.e., without increasing the height of the outer medulla) requires that OMCD become sinuous in acidotic mice, which was commonly observed during microdissection (Figure 11B).

Figure 11.
Figure 11.

Longitudinal and transverse proliferation of AIC. (A) Model picturing morphometric changes observed in OMCD between control (C) and day 3 and day 14 (d3 and d14) WT acidotic mice. For the sake of clarity and because the proliferation rate of AIC (dark) exceeded that of PC (light), only divisions of AIC are shown in this simplified model. From C to d3, longitudinal division (arrows) of A and D AIC lengthens the tubule, whereas migration of PC from one ring of cells to the next, newly formed one decreases the number of cells per ring. These changes account for the decreased cross-section number of PC and epithelia surface area and the maintenance of the cross-section surface area of individual cells. From d3 to d14, transverse division of A and E AIC (arrows) and remodeling of PC account for increased cross section number of AIC and decrease cross-section number of PC. (B) Light microscopy photographs of OMCD and mTAL microdissected from control and acidotic mice. OMCD from acidotic mice were sinuous, whereas mTAL remained rectilinear.

GDF15 and Longitudinal Proliferation of AIC

In acidotic GDF15−/− mice, the early proliferation of AIC was reduced (Figure 7A), but their cross-section number was increased (Figure 8B), indicating transverse proliferation. Early transverse proliferation of AIC was associated with overexpression of cyclin D3, which was evident earlier in GDF15−/− mice than in WT mice (Figure 9).

On the basis of the marked induction of GDF15 expression in regenerating liver, Lee et al.28 first proposed that GDF15 might control cell proliferation, but they failed to demonstrate decreased liver regeneration in GDF15−/− mice; however, they evaluated hepatocyte proliferation 1 to 7 wk after partial hepatectomy, whereas they observed increased expression of GDF15 within hours. Given our results showing early effect of GDF15 in the kidney, it is likely that they missed the period when GDF15 controls liver cell proliferation.

Altogether, our results suggest that GDF15 may control longitudinal proliferation of AIC and point to a role of cyclin D1 in this mechanism. GDF15 also prevented transverse proliferation, because overexpression of cyclin D3 (Figure 9) and transverse proliferation were triggered earlier in GDF15−/− mice than in WT mice. Thus, GDF15 seems more important to determine the orientation of the mitotic spindle along the OMCD axis than to induce growth itself.

GDF15-dependent proliferation of AIC and overexpression of cyclin D1 were prevented by inhibition of the PI3K/Akt/mTOR pathway (Figure 10), but whether this pathway is directly activated by GDF15 remains unknown. Interestingly, Akt was recently reported to play a major role in the orientation of the mitotic spindle in Drosophila embryo.29

Also unknown is the mechanism triggering GDF15 expression during acidosis. GDF15 promoter contains a p53 response element,17 and p53-dependent induction of GDF15 has been reported in several systems and conditions; however, early overexpression of p53 observed in OMCD of acidotic mice is likely not responsible for induction of GDF15 because it was blunted in GDF15−/− mice (Figure 9). Overexpression of p53 might rather be a consequence of GDF15-induced proliferation and serve as gatekeeper of cell division.30 p53-independent induction of GDF15 has been reported in kidney, lung, and liver.31,32 Alternately, acidosis might induce directly GDF15. As a matter of fact, metabolic acidosis and GDF15 overexpression are also associated in mice after potassium depletion12 and liver resection.28,33

Acidosis and OMCD Hypertrophy

Cell hypertrophy is defined as cell enlargement as a result of increased protein/DNA ratio. It is either dependent or independent on cell cycle.16 Cell cycle–independent hypertrophy results from inhibition of protein degradation in quiescent (G0) cells. In contrast, cell cycle–dependent hypertrophy results from entry in the cell cycle and blockade before progression through the S phase, before DNA replication.

Hypertrophy could not be evaluated directly, because this would require evaluating protein/DNA ratios in AIC and PC separately. Alternately, increased cell volume can be considered as presumptive evidence of cell hypertrophy, but cell volume could not be determined either. As a last and questionable resource, we used the cross-section epithelium surface area/number of cells ratio as an index of hypertrophy. This approximation entailed errors because cell remodeling may modify cross-section surface area but not cell volume (as proposed for PC in Figure 11); however, this bias is circumvented in part when considering AIC and PC together, because axial elongation of one cell type is supposed to palliate transverse invasion by the other cell type.

Provided these biases do not induce redhibitory errors, the constancy of the mean cross-section cell surface area in control and acidotic WT mice (Figure 5F) suggests that OMCD do not hypertrophy during acidosis. In contrast, in acidotic GDF15−/− mice, mean cross-section cell surface increased steadily (Figure 8C), suggesting cell hypertrophy. Thus, GDF15 seems to prevent acidosis-induced cell hypertrophy in OMCD. An anti-hypertrophy role of GDF15 was reported in the mouse heart, where overexpression of GDF15 protected cardiomyocytes against pressure overload–induced hypertrophy.34 Increased cross-section surface area of OMCD and of AIC and decreased lumen surface area observed in control GDF15−/− mice suggest that GDF15 may also play its anti-hypertrophy role during kidney development.

In kidney, both cell cycle–dependent and –independent mechanisms of hypertrophy have been described.35 Because OMCD hypertrophy in GDF15−/− mice is concomitant with cell proliferation, it is likely to be dependent on cell cycle.

In summary, this study demonstrated that acidosis induces homeostatic proliferation of acid-secreting cells in the mouse collecting duct according to a two-phase process: Early longitudinal proliferation is associated with overexpression of GDF15 and cyclin D1 and with activity of the PI3K/Akt/mTOR pathway, whereas late transverse proliferation is independent of GDF15 and depends on cyclin D3. We propose that GDF15 is an important determinant of collecting duct lengthening, through the control of the orientation of the mitotic spindle, which may also play a role during development.

CONCISE METHODS

Animals

Reproduction couples of GDF15 gene targeted mice (GDF15−/−) in the strain background C57BL/6/129/SvJ were provided by Dr. Se-Jin Lee (John Hopkins University, Baltimore, MD). Animals were backcrossed with C57BL/6J mice (Charles River, L'Arbresle, France) and genotyped by single PCR. Control mice were either WT littermates of GDF15 null mice or C57BL/6 mice. There was no difference in any parameter analyzed between the two types of controls. Animals were maintained on a standard diet (A04; SAFE, Epinay, France). Metabolic acidosis was induced by feeding the mice an NH4Cl-supplemented diet (5 g of powdered A04 diet, 0.2 g of agar agar, and 6 ml of 0.7 M NH4Cl) for 1 to 14 d. Acidosis was verified before the mice were killed by measurement of blood acid-base parameters (AVL Compact 1; AVL Instruments, Eragny, France). Some mice were administered an injection of BrdU (0.1 mg/g body wt in PBS, intraperitoneally) 18 and 4 h before being killed. In vivo inhibition of PI3K and mTOR was achieved as described previously with LY29400236 and rapamycin,37 respectively. LY294002 (Calbiochem, Nottingham, UK) was administered once (30 mg/kg in DMSO 50%, intraperitoneally) at the onset of NH4Cl feeding, and animals were killed 2 d later (day 2). Rapamycin (MPBio, Illkirch, France) was injected twice (0.2 mg/kg per d in 3% DMSO, intraperitoneally), at the onset of the treatment and 1 d latter, and animals were killed on day 2. Nonacidotic control mice were treated with LY294002 or rapamycin using the same protocols. All animal experiments were performed in accordance with the French legislation.

Immunohistochemistry

Kidneys were fixed by in situ perfusion, removed, sliced into four sections, and fixed in formalin for an additional 18 h before paraffin embedding. Double immunohistochemistry was performed on 5-μm-thick kidney sections with pairs of the following antibodies: Monoclonal anti-AQP2 antibody (1:800012), polyclonal anti-kAE1 antibody (1:400; provided by Dr. Alper), monoclonal anti–Ki-67 antibody (clone TEC-3, 1:25; DAKO, Trappes, France), monoclonal anti-PCNA antibody (PC10 clone, 1:200; DAKO), and monoclonal anti-BrdU antibody (clone BU-33, 1:200; Sigma, St. Quentin Fallavier, France), using either a three-step streptavidin-biotin method or ENVISION amplification kit (DAKO), with previous antigen unmasking procedure.12,38 Secondary anti-rabbit (1:400), anti-mouse (1:200), and anti-rat (1:300) biotinylated antibodies and streptavidin-peroxidase amplification ELITE and ABC-phosphatase kits were from Vector laboratories (Burlingame, CA). Diaminobenzidine (DAKO) and Fast-Red (DAKO) were used as chromogens. Secondary anti-mouse Cy3 antibody (1:800; Amersham, Les Ulis, France) and streptavidin-Cy2 (1:400; Amersham) were used in double-immunofluorescence studies. An average of 200 to 400 cells per mouse were counted in a blinded manner.

TEM and Morphometry Analysis

Kidneys were fixed in situ with 2% glutaraldehyde in PBS, followed by immersion fixation for 24 h of renal tissue slices. Blocks of tissue dissected around the inner stripe of the outer medulla were submitted to standard technique of TEM. On toluidine blue–stained semithin sections, only transverse sections of inner stripe collecting ducts were selected and studied on thin sections. Ten collecting ducts per mouse (n = 3 in control and acidotic groups) were photographed at ×2500 magnification so as to include a complete collecting duct section in a picture. For calibration, a reference grid was photographed at the same magnification.

Using ImageJ program and a macro written by one of us (D.H., available upon request), image analysis allowed measurement of the following parameters in collecting duct TEM photographs: Numbers of PC and IC, surface areas of individual PC and IC and of the tubular lumen, and number of cells per section.

Microdissection and Reverse Transcription–PCR

The various segments of nephron were dissected from collagenase-treated kidney slices under RNase-free conditions, as described previously,39 and tubular length was determined by image analysis (Visilog, Noesis, France).

Total RNA were extracted from pools of 40 to 60 nephron segments using an adaptation of the procedure of Chomczynski and Sacchi.40 Reverse transcription was performed using first-strand cDNA synthesis kit for reverse transcription–PCR (Roche Diagnostics, Meylan, France), according to the manufacturer's protocol.

Real-time PCR was performed using a cDNA quantity corresponding to 0.1 mm of nephron on a LightCycler (Roche Diagnostics) with LightCycler 480 SYBR Green I Master qPCR kit (Roche Diagnostics) according to the manufacturer's protocol, except that the reaction volume was reduced to 10 μl. We verified that no amplification product was produced when reverse transcriptase was omitted. Results (arbitrary units per millimeter of tubule length) are expressed as means ± SEM from several mice. Specific primers (Supplemental Table) were designed using ProbeDesign (Roche Diagnostics).

DISCLOSURES

None.

Acknowledgments

We thank Dr. Se-Jin Lee (Johns Hopkins University, Baltimore, MD) for kindly providing GDF15−/− mice.

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

  • J.P.D.V.H. and L.C. contributed equally to this work.

  • Supplemental information for this article is available online at http://www.jasn.org/.

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GDF15 Triggers Homeostatic Proliferation of Acid-Secreting Collecting Duct Cells
Jean Paul Duong Van Huyen, Lydie Cheval, May Bloch-Faure, Marie France Belair, Didier Heudes, Patrick Bruneval, Alain Doucet
JASN Oct 2008, 19 (10) 1965-1974; DOI: 10.1681/ASN.2007070781
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