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. 2020 Oct;26(19-20):1091-1098.
doi: 10.1089/ten.TEA.2019.0294.

Metformin and Inhibition of Transforming Growth Factor-Beta Stimulate In Vitro Transport in Primary Renal Tubule Cells

Harold Love  1 , Rachel Evans  1 , Harvey David Humes  2 , Shuvo Roy  3 , Roy Zent  1   4 , Raymond Harris  1   4 , Matthew Wilson  1   4 , William Henry Fissell  1
Affiliations

Metformin and Inhibition of Transforming Growth Factor-Beta Stimulate In Vitro Transport in Primary Renal Tubule Cells

Harold Love et al. Tissue Eng Part A. 2020 Oct.

Abstract

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water. We previously identified transforming growth factor-beta as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-β receptor-1 led to active inhabitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5' AMP-activated kinase phosphorylation. The signals that undermine in vitro differentiation are complex, but susceptible to pharmacologic intervention. This achievement overcomes a major hurdle limiting the development of a bioreactor of cultured cells for renal replacement therapy that encompasses not only endocrine and metabolic functions but also transport and excretion. Impact statement Clinical tissue engineering requires functional fidelity of the cultured cell to its in vivo counterpart, but this has been elusive in renal tissue engineering. Typically, renal tubule cells in culture have a flattened morphology and do not express key transporters essential to their function. In this study, we build on our prior work by using small molecules to modulate pathways affected by substrate elasticity. In doing so, we are able to enhance differentiation of these cells on conventional noncompliant substrates and show transport. These results are fundamentally enabling a new generation of cell-based renal therapies.

Keywords: AMPK; NKCC2; TGF-beta; metformin; renal epithelial cells.

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Conflict of interest statement

W.H.F., H.D.H., and S.R. may have rights to certain intellectual property related to the subject matter of the article. W.H.F. and S.R. are founders of Silicon Kidney, LLC. H.D.L., R.E., M.W., R.Z., and R.H. have no disclosures.

Figures

FIG. 1.
FIG. 1.
Segmental origin of cultured cells. Immunofluorescent photomicrograph of cultured primary cells. Red, claudin-3, green, claudin-2. The vast majority of cells were claudin-3 positive and claudin-2 negative, consistent with origin in the thick ascending limb. Small islands of claudin-2-positive and claudin-3-negative cells were observed, suggesting a scarce population of proximal cells.
FIG. 2.
FIG. 2.
AMPK phosphorylation is increased by metformin in short-term (8 day) culture, but not long-term (6 week) culture. (A) Top, Western blot of lysates from primary human renal tubule cells grown on plastic substrates for 8 days; bottom, quantification of band density. Cells showed significantly increased AMPK phosphorylation when exposed to 200 μM metformin compared to controls (*p < 0.05). AICAR, an AMP mimic, showed a trend toward a dose-dependent effect at 0.5 and 1.0 mM concentration (p = 0.11). (B) Top, Western blot of lysates from primary human renal tubule cells grown on transwells for 6 weeks; quantification of band density. Exposure to SB431542 reduced AMPK phosphorylation with or without metformin (p = 0.050 metformin vs. combination and p < 0.02 control vs. combination). AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK, 5′ AMP-activated kinase.
FIG. 3.
FIG. 3.
(A) Protein expression of NKCC2 was increased when metformin, SB431542, or combination was added to media. C, control. M, metformin. SB, SB431542; MS, combination. Numbers denote replicates. The rightmost band (“MS3”) was run on a separate gel due to the limited number of lanes per gel. Despite strong denaturing conditions, we observed a very strong band at about 300 kDa, and a weaker band at about 150 kDa, both of which probed with antibody. We interpret the higher molecular weight bands as NKCC2 dimers as has been reported previously. (B) Quantification of Western blot protein expression data by scanning densitometry. N = 3 for all experiments. NKCC2 was significantly increased compared to controls in SB431542-treated and combination-treated cells, but not metformin-alone treated cells. *p < 0.05. NKCC2, sodium–potassium-two-chloride cotransporter 2.
FIG. 4.
FIG. 4.
Expression of NKCC2. Structured illumination microscopy three-dimensional reconstructions. Primary human renal epithelial cells stained with antibodies against NKCC (red) and ZO-1 (Green). Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). (A) Cross-section. NKCC2 is clearly expressed at the apical membrane surface. (B) Perspective rendering from above apical surface showing apical NKCC2 and ZO-1 at the apicolateral margin. (C) Perspective rendering from below the basal surface showing apical NKCC2 and ZO-1 at the apicolateral margin.
Fig. 5.
Fig. 5.
Transmission electron micrographs of renal tubule cells grown on permeable supports without (A) and with (“treated”) (B) SB431542 and metformin. Treated cells (B) were more cuboidal and less flattened, and had more dense mitochondrial networks (p = 0.044 for mitochondrial counts). Apical microvilli were sparse and appeared to be slightly more numerous at the periphery than the center, although the effect was subtle. Scale bar = 2 μm.
FIG. 6.
FIG. 6.
In vitro transport by primary renal tubule epithelial cells. Left cluster of columns: cells grown in standard hormonally defined media with added dimethyl sulfoxide transported very small fluid volumes (“Control,” pale gray bars). When metformin (medium gray bars) or SB431542, a TGF-βR1 inhibitor (dark gray bars), was added to media, transport increased (p < 0.001 and p < 0.02 vs. control, respectively.) When both metformin and SB431542 were added to media, transport increased further (p < 0.003 all comparisons, black bars). Middle cluster of columns: when mannitol was added to basolateral compartment of the transwells, transport increased slightly in control media, but was unchanged or decreased in cells that received metformin, SB431542, or both, indicating that paracellular apicobasal transport was minimal. Right cluster of columns: addition of ouabain (10 nM) significantly decreased transport in all groups. Active transport is seen in primary renal tubule cells allowed to differentiate on permeable supports for 8–10 weeks when metformin, SB431542, or both is added to media. Error bars are standard deviations. *p < 0.01 versus controls within each group; ‡p < 0.05 versus control within each group. TGF-βR1, transforming growth factor-beta receptor-1.
FIG. 7.
FIG. 7.
Diuretic response of cultured primary human renal tubule epithelial cells. Renal tubule epithelial cells were grown to confluence on permeable supports. Fourteen days postconfluence, metformin and SB431542, a TGF-βR1 inhibitor, and each diuretic were added to media. Transport volumes (about 140 μL/cm2/day) were normalized to volumes in control wells without diuretics. Furosemide abolished transport, while tenapanor and hydrochlorothiazide had no effect. Cultured cells showed specific and inhibitable active apicobasal transport. *p < 0.05.
FIG. 8.
FIG. 8.
Inulin leak rates before and after furosemide treatment. Fluorescein isothiocyanate-labeled inulin was added to the apical compartment of each transwell and incubated for ∼24 h. After 24 h, fluorescence in the apical and basal media were measured. Data were collected 24 h before incubation with furosemide, and then in the 24 h immediately after the furosemide experiment was complete. Typical ratios of basolateral to apical fluorescence are about 0.01–0.02. In this study, prefurosemide and postfurosemide leak rates are similar and differences did not reach statistical significance by two-tailed Student's t-test. The variation in fluorescence intensity between conditions reflects concentration of the apical media by transcellular transport. Furosemide did not cause paracellular leak.
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