Claudin-2 and claudin-12 form independent, complementary pores required to maintain calcium homeostasis

Calcium (Ca2+) homeostasis is maintained through coordination between intestinal absorption, renal reabsorption, and bone remodeling. Intestinal and renal (re)absorption occurs via transcellular and paracellular pathways. The latter contributes the bulk of (re)absorption under conditions of adequate intake. Epithelial paracellular permeability is conferred by tight-junction proteins called claudins. However, the molecular identity of the paracellular Ca2+ pore remains to be delineated. Claudins (Cldn)-2 and -12 confer Ca2+ permeability, but deletion of either claudin does not result in a negative Ca2+ balance or increased calciotropic hormone levels, suggesting the existence of additional transport pathways or parallel roles for the two claudins. To test this, we generated a Cldn2/12 double knockout mouse (DKO). These animals have reduced intestinal Ca2+ absorption. Colonic Ca2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca2+ wasting than Cldn2 null animals. These perturbations lead to hypocalcemia and reduced bone mineral density, which was not observed in single-KO animals. Both claudins were localized to colonic epithelial crypts and renal proximal tubule cells, but they do not physically interact in vitro. Overexpression of either claudin increased Ca2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca2+ permeable pores in renal and colonic epithelia that enable paracellular Ca2+ (re)absorption in these segments, with either one sufficient to maintain Ca2+ balance.

Renal AT 2 Receptors Mediate Natriuresis via Protein Phosphatase PP2A

Background: How signals from activated angiotensin type-2 receptors (AT 2 R) mediate inhibition of sodium ion (Na+) reabsorption in renal proximal tubule cells (RPTCs) is currently unknown. Protein phosphatases including protein phosphatase 2A (PP2A) have been implicated in AT2R signaling in tissues other than kidney. We investigated whether inhibition of protein phosphatase PP2A reduced AT 2 R-mediated natriuresis and evaluated changes in PP2A activity and localization after renal AT 2 R activation in normal 4- and 10-week-old control Wistar-Kyoto rats (WKY) and 4-week-old pre-hypertensive and 10-week-old hypertensive spontaneously hypertensive rats (SHR). Methods and Results: In WKY, direct renal interstitial (RI) administration of selective AT 2 R non-peptide agonist Compound-21 (C-21) increased RI cyclic GMP (cGMP) levels, urine Na + excretion (U Na V), and simultaneously increased PP2A activity ≅ 2-fold in homogenates of renal cortical tubules. The cGMP and natriuretic responses were abolished by concurrent RI administration of protein phosphatase inhibitor calyculin A (CAL). In RPTCs in response to C-21, PP2A subunits A, B55α and C, but not B56γ, were recruited to apical plasma membranes together with AT 2 Rs. CAL treatment abolished C-21-induced translocation of both AT 2 R and PP2A regulatory subunit B55α to apical plasma membranes. Immunoprecipitation of AT 2 R solubilized from renal cortical homogenates demonstrated physical association of AT 2 R with PP2A A, B55α, and C but not B56γ subunits. In contrast, in SHR, administration of C-21 did not alter UNaV or PP2A activity and failed to translocate AT 2 Rs and PP2A subunits to apical plasma membranes. Conclusions: In RPTCs of WKY, PP2A is activated and PP2A subunits AB55αC are recruited to C-21-activated AT 2 Rs during induction of natriuresis. This response is defective in pre-hypertensive and hypertensive SHR, presenting a potential novel therapeutic target for treating renal Na+ retention and hypertension.

Abstract P238: Regulation Of G Protein-coupled Receptor Kinase 4 Expression By Serum In Breast Cancer And Renal Proximal Tubule Cells

G protein-coupled receptor kinase 4 (GRK4) is a member of the GRK family which play critical role in regulation of the function of G protein-coupled receptors. Our previous studies have shown that GRK4 not only plays a role in regulating sodium excretion in renal proximal tubule cells but also acts as a stimulator on proliferation of breast cancer cells. Uncontrolled proliferation is a characteristics of cancer cells and GRK4 is upregulated in breast cancer cells. We hypothesized that expression of GRK4 may be regulated differently in cancer and non-cancer cells. To test this hypothesis, expression of GRK4 in response to serum was compared in breast cancer cells and renal proximal tubule cells by Western analysis. In three breast cancer cell lines serum withdrawal caused rapid reduction in the levels of GRK4 which occurred as early as 15 min. GRK4 levels correlated with the concentrations of serum added to the culture media. To determine if growth factors were a critical element for maintaining GRK4 levels in the cells, EGF (10-20 ng/ml) was added to serum free medium for 24 h. There was no increase in GRK4 levels in the cells treated with EGF compared with the serum starvation control. Similarly, serum withdrawal (16 h) led to 40-80% decrease of GRK4 levels in renal proximal tubule cells even in the presence of EFG supplement. Serum feeding for 30 min after starvation dramatically increased the levels of GRK4 in both breast cancer cells and RPTC which exceeded the steady state levels. This rapid recovery of GRK4 protein do not need de novo protein synthesis because pretreatment of the cells with protein synthesis inhibitor, cycloheximide (10 μg/ml, 24 h), did not prevent this event. Expression of GRK2, another member of the GRK family, was not affected by serum starvation. Our results have shown that GRK4 is very sensitive to serum concentration in breast cancer cells as well as in RPTC. Preliminary studies suggest that rapid protein degradation rather than shutting down the protein synthesis plays a major role in this kind of GRK4 regulation. The biological significance of serum regulation of GRK4 in cancer and non-cancerous cells needs further investigation.

Abstract MP61: Sex Difference In The Response Of Human Renal Proximal Tubule Cells To A Nephrotoxic Agent.

Our previous work indicated that the renal dopamine D2 receptor (D2R) has a significant role in regulating renal inflammation and injury, as well as in blood pressure control. In mice, D2R has protective effects in the kidney by limiting the inflammatory and fibrotic reaction; impaired D2R function results in renal inflammation and damage. Some common single nucleotide polymorphisms (SNPs; rs 6276 and 6277) in the human DRD2 gene are associated with decreased D 2 R expression and function and high blood pressure. To determine the effects of the presence of SNPs in the response to the nephrotoxic aristolochic acid (AA, 5μg/ml, 24 h), we studied immortalized human renal proximal tubule cells isolated from normal tissue of nephrectomies and genotyped for DRD2 SNPs and DRD2 wild-type (WT). We also determined whether this response is sex dependent. D2R protein was higher in male than in female WT (135±5 vs 100±4%; n=3/group; P<0.04) and lower in males with SNPs (43±2%, P<0.05) and females with SNPs (23±2%,P<0.05), compared with their respective WT counterparts. In both male groups (WT and SNPs), AA increased D2R protein by 80-100% but had no effect in WT females and increased ~50% in females with SNPs. The TNFα mRNA was higher in males with WT and SNPs which was increased by AA 9-10-fold in WT males and females but only 2-3-fold in those with SNPs. The TGFβ mRNA was similar in WT males and females and increased to the same extent in both groups with SNPs and was not affected by AA in all groups. Col1a1 mRNA was higher (30%) in WT males and females than those with SNPs; AA decreased Col1a1 mRNA in all groups. FN1 mRNA was higher (30-40%) in males and females with SNPs than WT; AA increased FN1 mRNA only in males and females with SNPs. The mRNA expression of the cell proliferation marker Ki-67 was higher in WT females than WT males (1.5-2-fold) and higher with SNPs than WT in both groups; AA increased Ki-67 mRNA in both groups and to a greater extent in males than in females. Taken together our data indicate that the presence of DRD2 SNPs affects the baseline expression of inflammatory and fibrotic factors and the response to AA is dependent on both sex and the presence of DRD2 SNPs. These data may have potential clinical translation since rs6276/6277 is commonly expressed (42%/23%) in the human population.

Abstract MP11: Decreased Expression Of Ace2 Is Associated Urine Derived Human Renal Proximal Tubule Cells From Inverse Salt Sensitivity Participants Cultured In Low Salt Conditions

Increased morbidity and mortality occurs in some individuals consuming low sodium diets. Inverse salt sensitivity (ISS) is the paradoxical increase in blood pressure of individuals to a low sodium diet. Our group previously reported decreased expression of dopamine type 2 receptor (D 2 R), increased expression Aminopeptidase N, and increased Ang II dependent sodium transport in human urine derived renal proximal tubule cells isolated from ISS participants. In an attempt to understand the increased Ang II sensitivity demonstrated in ISS cells, we examined angiotensin converting enzyme 2 (ACE2), a membrane associated enzyme involved in the metabolism of Ang II. Urine derived renal proximal tubule cells grown and immortalized from ISS participants were compared to cells from salt resistant (SR) participants cultured in iso-osmotic media with low salt (LS, 90 mM NaCl) normal salt (NS, 140 mM NaCl) and high salt (HS, 190 mM NaCl). Cells were incubated in LS, NS, and HS media with and without losartan (LOS,1 μM) overnight (18 hours) and ACE2 expression levels determined by in-cell western blot. A monoclonal antibody specific to an extracellular epitope of ACE2 was used as the primary antibody and an Alexa-647 anti-mouse secondary antibody. ACE2 expression was only reduced in ISS cells in LS condition (28.7±2.1 % reduction, ISS LS vs SR LS, N=4 per group, p<0.05). Addition of losartan completely blocked the decrease in ACE2 expression in low salt conditions in ISS in urine derived human renal proximal tubule cells. No other changes in ACE2 expression were found between ISS and SR in either NS or HS conditions and with or without losartan. In conclusion, a decreased expression of ACE2 in ISS urine proximal tubule cells could explain the previously reported increased sensitivity of ISS cells to Ang II by increasing the half-life of Ang II under low salt conditions.

Abstract MP29: A Cloned Anti-aminopeptidase N Scfv Antibody Blocks Enzymatic Activity And Rescues A Low Sodium Phenotype In Proximal Tubule Cells Isolated From Inverse Salt Sensitive Participants

Inverse Salt Sensitivity (ISS), defined as the paradoxical increase in blood pressure of individuals on a low sodium diet compared to a high sodium diet, may be associated with the increase in mortality and morbidity found in individuals on a low sodium diet. Our group has previously found that urine derived human renal proximal tubule cells (RPTC) isolated from ISS participants express higher Aminopeptidase N (APN) protein than cells isolated from salt resistant (SR) participants. An anti-Aminopeptidase N (APN) hybridoma was used construct a single chain variable fragment (scFv) into a bacterial expression system. The purified anti-APN scFv bound to live human renal proximal tubule cells, detected using an Alexa594 directly labelled anti-myc monoclonal antibody by immunofluorescent confocal microscopy. The anti-APN scFv was then tested to determine if it blocks APN enzymatic activity. We have previously shown that under low sodium conditions the amount of reduced-glutathione as determined by live cell fluorescence staining with the redox sensitive dye, monochlorobimane (mBCl), is decreased only in ISS cells under low sodium conditions at the two-hour time point. We hypothesized that the decreased mBCl signal may be due to the increased APN expression and activity altering the Ang II/Ang III ratio and reducing the Ang III activation of the AT 2 R. Low sodium reduced the mBCl signal in ISS (-29.2±4.3%, ISS vs SR, N=3 per group, p<0.05) and the addition of the anti-APN scFv at 1 ug/ml completely blocked the mBCl signal back to levels found in SR in normal salt conditions. In order to verify that the full rescue of this ISS specific response is due to enhanced Ang III – AT 2 R signaling, we next tried to block the effect of the anti-APN scFv by the addition of the AT 2 R antagonist, PD123319 (PD, 1 uM). Addition of PD alone to the ISS LS RPTCs did not significantly alter the mBCl signal, but when PD and anti-APN scFv are added to the LS treated ISS cells, there is complete reversal of the effect of anti-APN scFv alone (-38.1±3.9%, ISS anti-APN scFv + PD vs ISS anti-APN scFv, N=3 per group, p<0.05). The anti-APN scFv has potential therapeutic value by reducing APN enzymatic activity in ISS individuals and inducing the protective AT 2 R arm of the renin angiotensin system in low sodium conditions.

Abstract P212: Peroxiredoxin-4 And Dopamine D5 Receptor Interact To Reduce Oxidative Stress And Inflammation In The Kidney

Peroxiredoxin-4 (PRDX4), an endoplasmic reticulum peroxiredoxin protein, plays a protective role against oxidative stress and inflammation by reducing hydrogen peroxide to water. The dopamine D5 receptor (D 5 R) is also important in protecting against oxidative stress, but the interaction between PRDX4 and D 5 R in regulating oxidative stress in the kidney is not known. In D 5 R-HEK 293 cells, fenoldopam (FEN, 25 nM/12 hr, n=4), a D1-likereceptor agonist, increased PRDX4 protein expression (1.92±0.12-fold over basal level, n=4), mainly in non-lipid rafts (LRs: 24.9±11.4%, non-LRs: 75.1±11.4%, baseline; LRs: 30.9±13.9%, non-LRs: 174.1±16.7%, FEN). FEN also increased the co-immunoprecipitation of D 5 R and PRDX4 and their colocalization, particularly in the endoplasmic reticulum. In human renal proximal tubule cells (hRPTCs), FEN (25 nM/12 hr, n=3) increased PRDX4 and D 5 R interaction in non-LRs, also. Si-RNA silencing of PRDX4 increased reactive oxygen species (ROS) production and impaired the inhibitory effect of FEN on ROS production (scrambled siRNA: 100.0±11.6% and 65.4±5.6% for Vehicle (Veh) and FEN, respectively; PRDX4 siRNA: 147.7±11.8% and 134.8±11.2% for Veh and FEN, respectively, n=4/group) detected by Amplex Red. In addition in both D 5 R-HEK 293 and hRPTCs, siRNA silencing of PRDX4 increased the production of interleukin-1β (26.88±3.8 and 46.40±4.2 pg/mL [n=3, D 5 R-HEK 293]; 15.87±1.2 and 37.9±1.4 pg/mL [n=3 in hRPTCs]), tumor necrosis factor (131.7±6.5 and 271.2±18.1 pg/mL [n=4, D 5 R-HEK 293]; 108.8±11.8 and 240.1±13.7 pg/mL [n=4 in hRPTCs]), and caspase-12 (15.21±3.8 and 40.78±4.3 ng/mL [n=4,n D 5 R-HEK 293]; 8.8±1.1 and 27.9±2.0 ng/mL [n=4, hRPTCs]). Furthermore, the protein expression of D 5 R was decreased in PRDX4 siRNA-treated D 5 R-HEK293 (~41.2%, n=3) and hRPTCs (~39.6%, n=3). PRDX4 protein was also reduced in the kidney homogenates from D 5 R -/- mice (WT: 1.00±0.18, n=5; D 5 R -/- : 0.686±0.14, n=4; P<0.05). Taken together, PRDX4 interacts with D 5 R to decrease oxidative stress and inflammation in the kidney.

Non-invasive assessment of exfoliated kidney cells extracted from urine using multispectral autofluorescence features

Optimally preserved urinary exfoliated renal proximal tubule cells were assessed by multispectral imaging of cell autofluorescence. We demonstrated different multispectral autofluorescence signals in such cells extracted from the urine of patients with healthy or diseased kidneys. Using up to 10 features, we were able to differentiate cells from individuals with heathy kidneys and impaired renal function (indicated by estimated glomerular filtration rate (eGFR) values) with the receiver operating characteristic area under the curve (AUC) of 0.99. Using the same method, we were also able to discriminate such urine cells from patients with and without renal fibrosis on biopsy, where significant differences in multispectral autofluorescence signals (AUC = 0.90) were demonstrated between healthy and diseased patients (p < 0.05). These findings show that multispectral assessment of the cell autofluorescence in urine exfoliated proximal tubule kidney cells has the potential to be developed as a sensitive, non-invasive diagnostic method for CKD.