Negative Regulation of TGFβ Signaling by Stem Cell Antigen-1 Protects against Ischemic Acute Kidney Injury

<div><p>Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1), commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI), as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFβ receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFβ receptor—mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFβ-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.</p></div

hRPTEC and hMVEC in close-contact co-culture form a physiological PT tissue barrier.

<p>(A) The microfluidic channels overlap to create a renal epithelial filtrate channel in communication with an endothelial vascular channel. (B) A close-contact co-culture of hRPTEC and hMVEC were grown in channels on opposite sides of the membrane. hRPTEC were labeled with anti-ZO-1 (green) and hMVEC with anti-vWF (red). (C, D) Confocal slices of the co-cultured cells show a confluent hRPTEC tissue layer and hMVEC tissue layer with clear ZO-1 (green) and vWF (red) expression, respectively. Each tissue layer exists in the xy plane, but is separated in the z-axis by the membrane. Scale bars: 30 μm. (E) A collapsed xz view of the co-culture stack shows clear separation between the tissue layers and a thicker epithelial tissue layer vs. endothelial layer. (F) A z-profile plot illustrates the change in average intensity expression, normalized to respective blank channel intensity values, of ZO-1 and vWF signals through the 14 μm co-culture 3D tissue stack. The width of the peaks correspond to each cell layer thickness, indicating a cuboidal morphology of the hRPTEC tissue in and a squamous morphology of hMVEC tissue. At least 3 replicate samples were repeated over at least 3 batches of experiments.</p

Sca1^-/- kidneys display increased injury after IRI.

<p>(A) Histograms (mean<u>+</u>sd) comparing <i>Kim-1</i> and <i>Ngal</i> mRNA levels in uninjured NL and Sca1^<b>-/-</b> kidneys. p = 0.0258 and 0.0227, respectively. (B) Western blot detection of Kim-1 protein in uninjured kidneys and in kidneys 7 days post-IRI from three NL and Sca1^<b>-/-</b> animals. Kim-1 was significantly induced kidneys in uninjured kidneys (upper panel) and in IRI kidneys at day 7 (lower panel) post IRI in Sca1^<b>-/-</b> mice vs. NL mice (p = 0.0183 and p = 0.010, respectively). (C) Histograms (mean<u>+</u>sd) showing qPCR measurement of <i>Sca-1</i> expression in wild-type kidneys after IRI. Normal control animals: n = 6 (0h), 6 (24h), 4 (48h), 5 (72h), 5 (7d). Sca1^<b>-/-</b>: n = 6 (controls), 4 (24h), 3 (48h), 6 (72h), 7 (7d). (D) High power H&E stain of representative 6 μm sections from control (Cont.) and Sca1^<b>-/-</b> kidneys 7 days post IRI. Tubular casts (black arrows) and increased interstitial cellularity (yellow arrow) were frequently observed in Sca1^<b>-/-</b> kidneys. Kidney injury scores (mean<u>+</u>s.d.) 7 days post IRI were 3.0 and 2.75 for two controls and 4.5, 3.5 and 3.5 for three Sca1^<b>-/-</b> mice. Injury scores of zero were derived for kidneys from uninjured control and Sca1^<b>-/-</b> mice.</p

An in vitro 3D microfluidic model mimics the reabsorptive barrier of the proximal tubule.

<p>A) In vivo, water and solutes cross an epithelial-endothelial barrier in the reabsorption process from filtrate tubule to the peritubular capillaries. (B) The microfluidic channels overlap to create a filtrate channel (green) in communication with a vascular channel (purple). The cross-sectional architecture (inset) mimics in vivo epithelial-endothelial barrier and generates cell-mediated transport through the membrane. (C) A cross-sectional SEM of the device shows a semi-porous membrane, which serves as a scaffold for the epithelial and endothelial cells and separates the filtrate and vascular channels. (D) The membrane sub-micron ridge/groove topography influences tissue organization and function.</p

hMVEC presence enhances the hRPTEC layer.

<p>(A, B) TJ formation in 7-day cultures of hRPTEC without and with hMVEC in the microfluidic device, respectively. hRPTEC formed a more compact tissue layer with clear TJ formation under hRPTEC/hMVEC co-culture conditions. hRPTEC were labeled with anti-ZO-1 (green) and Hoechst (blue). At least 5 images for each tissue layer and at least three replicate samples were analyzed per group. (C) Average number of hRPTEC/mm² in co-culture conditions is more than double that of hRPTEC-only conditions after 7 days of culture. * P < 0.001. Results were verified for 3 independent experiments. (D) hRPTEC in co-culture conditions have increased mitochondrial activity compared to hRPTEC-only conditions, normalized to cell count. The mitochondrial activity of hMVEC cells in co-culture is negligible. * P = 0.002. Error bars represent standard error of the mean from 3 independent tissue samples.</p

Changes in renal injury markers in normal (NL) and Sca1^-/- animals after IRI.

<p>(A) Serum creatinine measurements in normal (NL) and Sca1^<b>-/-</b> animals prior to (0h) and at different times following IRI. Significant differences were found between animals at 0h (uninjured)(p = 0.0019), and at day 7 post injury (p = 0.002). (B and C) Comparisons of fold induction of <i>Kim-1</i> and <i>Ngal</i> mRNA as measured by quantitative PCR (qPCR) in kidneys from Sca1^<b>-/-</b> vs. NL animals. Kim-1 (B): p = 0.0036 (48h), p = 0.0013(72h), p = 0.0071(7d); Ngal (C): p = 0.0128 (24h), p = 0.0056 (48h)). *, p<0.05; **, p<0.005; ***, p<0.0005. Normal control animals: n = 6 (0h), 6 (24h), 4 (48h), 5 (72h), 5 (7d). Sca1^<b>-/-</b>: n = 6 (controls), 4 (24h), 3 (48h), 6 (72h), 7 (7d).</p

The microfluidic PT model altered sodium-dependent reabsorption of glucose analog in response to ouabain.

<p>(A) hRPTEC expressed polarized transport proteins Na⁺/K⁺ ATPase and SGLT2 under co-culture conditions. (B) A schematic representation of experimental conditions. Under all conditions, the transport of a fluorescent glucose analog, 2-NBDG, from the filtrate channel (top) into the vascular channel (bottom) was observed using confocal z-stack and time-lapse microscopy to quantify intensity in the vascular channel. (C) 2-NBDG intensity versus time for one device subjected to all conditions sequentially shows the reduction in active transport due to ouabain administration. (D) The bar graph indicates the mean 2-NBDG intensity in the vascular channel at each condition, measured over ~15 minutes for 3 devices. Data are from 3 replicates of the experiment. Error bars represent standard error of the main effects as computed from the error term in the analysis of covariance model. Introducing ouabain to the vascular channel blocked 2-NBDG transport. The 2-NBDG transport recovered when ouabain was rinsed from the system. A second administration of ouabain again blocked 2-NBDG transport. The tissue recovery and repeat effect of ouabain demonstrates dynamic reabsorptive cell-mediated reabsorption function. * P < 0.0005. Experimental results were verified for 3 independent experiments.</p

Sca-1 regulates TGFβ signaling in proximal renal tubule cells.

<p>(A) Western blots from a representative experiment, one of three conducted, showing phospho-Smad3 (p-Smad3) in untreated cell lysates from serum-starved control and Sca-1 silenced D5 and C8 cells and after exposure to TGFβ₁ for 15 or 30 min. Smad2/3 detection served as a loading control. (B) Blocking TβRI activity abrogates ectopic Smad3 activation in Sca-1 silenced cells. TKPTS or Sca-1 silenced cells (D5) were serum starved and pretreated with SB431542 for 30 minutes prior to addition of TGFβ₁. Cell lysates were analyzed by Western blot for detection of p-Smad3, with Smad2/3 used to control for protein loading. (C) Histograms (mean<u>+</u>sd, n = 3) showing <i>Pai-1</i> mRNA levels in TKPTS, D5, and C8 cells at baseline and following treatment with TGFβ₁ and/or SB431542.</p

Sca-1 interacts with the TGFβ signaling pathway in mouse proximal tubule epithelial (TKPTS) cells.

<p>(A) Immunoprecipitation of TβRI and TβRII from lysates of serum-starved normal TKPTS cells by anti-Sca-1 antibody. Both TRI and TRII were immunoprecipitated with anti-Sca-1 in the absence of TGFβ_1, but only TβRI co-precipitated with Sca-1 in the presence of TGFβ₁. <i>Lower panel</i>, reciprocal immunoprecipitations displayed the same trend. A representative experiment, one of three, is shown. Equal loading of samples was reflected in observed levels of Sca-1 (<i>upper panel</i>) and TβRI (<i>lower panel</i>). (B) Co-localization of Sca-1 with TβRI and TβRII in TKPTS cells. Cells were stained with antibodies against Sca-1 (green), TβRI or TβRII (red). Colocalization of Sca-1 and TR can be seen in focal surface membrane regions as well as intracellularly (yellow staining in merged images). (C) Western blots showing Smad3 phosphorylation (p-Smad3) in normal TKPTS and the Sca-1 silenced cells D5 and C8. Smad2/3 expression was used as loading control. Scale bar = 20μm. The differences in p-Smad3 in control cells perhaps reflect stochastic baseline variations in replicate confluency. Averages of p-Smad/total Smad ratios of duplicate samples from TKPTS, D5 and C8 were 0.33, 1.55 and 1.76, respectively. (D) Loss of Sca-1 expression increased Smad2/3 nuclear localization in C8 cells. Immunostaining of control TKPTS and C8 (Sca-1 KD) cells showing Smad2/3 localization (green), with actin detected with Alexa555 phalloidin (red), and nuclei labeled with DAPI (blue). Scale bar = 20μm. (E) A representative experiment, one of two, of a Western blot of TRII immunoprecipitates from serum-starved TKPTS, D5, and C8 cells, detected with anti-TRI antibody. Silencing Sca-1 in D5 and C8 cells resulted in constitutive TRI/TRII complex formation in the absence of ligand.</p

FLIM lifetimes of the Alexa488 donor fluorescence in the presence and absence of the acceptor FM.

<p>A) Representative Alexa488 fluorescence intensity and lifetime images. The panel shows examples of data collected in the absence (left, WT 488) and presence of the acceptor FM (right, WT 488+FM). The pseudocolor scale is shown at the bottom. B, histogram of measured Alexa488 lifetimes generated by integrating lifetime measurements for individual cells. The histogram bars show the number of cells in 50 ps bins for Alexa488 in the absence (blue) and presence (red) of the acceptor FM. The average and standard deviation were generated by fitting the histogram to a single Gaussian function (shown as a solid line).</p