Renal Brush Border Membrane Research Articles

Role of Leptin in Regulating Renal Phosphate Transport

Elevated plasma phosphate (Pi) levels, known as hyperphosphatemia, are associated with an increased risk of cardiovascular complications and mortality. Although studies have suggested a correlation between obesity and hyperphosphatemia/phosphate toxicity, the root cause of this observation has not been established. Since hyperleptinemia was also found to be associated with abnormalities in bone structure, we hypothesized that effects on renal Pi transport are responsible for this observation. To address this question, we studied hyperleptinemic mice (db/db, n=14) and compared them to wild-type mice (WT, n=10). Blood and urine samples, as well as kidney tissues, were collected and analyzed. Body weight was approximately twice as high in db/db compared to WT mice (59±3 vs. 30±2 g, P<0.05). Plasma Pi levels in db/db mice were significantly greater (~1.5-fold) compared to WT mice (2.6±0.1 vs. 1.7±0.1 mmol/L, P<0.05). However, urinary Pi /creatinine ratios were similar between genotypes. Plasma Ca2+ levels in db/db mice were significantly higher compared to WT mice (2.68±0.03 vs. 2.48±0.03 mmol/L, P<0.05) alongside urinary Ca2+/creatinine ratios being significantly higher in db/db mice compared to WT mice (1.6±0.4 vs. 0.3±0.1 mmol/mmol, P<0.05). Plasma parathyroid hormone (PTH, regulating Pi and Ca2+) levels were similar in both genotypes. The phosphaturic hormone fibroblast growth factor 23 (FGF23), which is produced and released from bone, was slightly but significantly elevated in db/db compared to WT mice (254±12 vs. 227±18 pg/mL, P<0.05). Bone formation markers (osteocalcin and procollagen type I N-propeptide) and bone resorption markers (tartrate-resistant acid phosphatase 5b and C-terminal telopeptide of type I collagen) were similar between genotypes, indicating that Pi and Ca2+ release from bone may not be causing these mineral differences. To determine the role of the kidney for the development of hyperphosphatemia, we performed 32P radiotracer studies in acutely isolated renal brush border membrane vesicles. Sodium-dependent 32P transport was not significantly different between genotypes. To determine the ambient in vivo contribution of the Na+-Pi cotransporter Npt2a, we employed acute pharmacological inhibition with PF-06869206 (Npt2a inhibitor, 30 mg/kg b.w, n=7) or vehicle (n=11) and compared plasma Pi before and 2 hours after administration in db/db mice. Vehicle treatment slightly increased plasma Pi levels compared to baseline (2.6±0.1 vs. 2.1±0.1 mmol/L, P<0.05); in contrast, the Npt2a inhibitor significantly reduced (~50%) plasma Pi levels compared to baseline (1.3±0.1 vs. 2.5±0.1 mmol/L, P<0.05). In summary, our study demonstrates that hyperphosphatemia in db/db mice is not caused by changes in renal Pi transport or bone turnover, suggesting that possibly intestinal mechanisms are responsible for increased Pi, and possibly Ca2+, absorption. Of note, Npt2a inhibition was effective in reducing hyperphosphatemia in db/db mice, indicating a potential therapeutic strategy. This work was supported by a VA Merit Review Award IBX004968A (to Dr. Rieg) and a Pilot Project from the USF Microbiomes Institute (to T.R. and J.D.R). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Ip6k1 and Ip6k2 Kinases Are Critical for Normal Renal Tubular Function.

Kidneys are gatekeepers of systemic inorganic phosphate balance because they control urinary phosphate excretion. In yeast and plants, inositol hexakisphosphate kinases (IP6Ks) are central to regulate phosphate metabolism, whereas their role in mammalian phosphate homeostasis is mostly unknown. We demonstrate in a renal cell line and in mice that Ip6k1 and Ip6k2 are critical for normal expression and function of the major renal Na + /Pi transporters NaPi-IIa and NaPi-IIc. Moreover, Ip6k1/2-/- mice also show symptoms of more generalized kidney dysfunction. Thus, our results suggest that IP6Ks are essential for phosphate metabolism and proper kidney function in mammals. Inorganic phosphate is an essential mineral, and its plasma levels are tightly regulated. In mammals, kidneys are critical for maintaining phosphate homeostasis through mechanisms that ultimately regulate the expression of the Na + /Pi cotransporters NaPi-IIa and NaPi-IIc in proximal tubules. Inositol pyrophosphate 5-IP 7 , generated by IP6Ks, is a main regulator of phosphate metabolism in yeast and plants. IP6Ks are conserved in mammals, but their role in phosphate metabolism in vivo remains unexplored. We used in vitro (opossum kidney cells) and in vivo (renal tubular-specific Ip6k1/2-/- mice) models to analyze the role of IP6K1/2 in phosphate homeostasis in mammals. In both systems, Ip6k1 and Ip6k2 are responsible for synthesis of 5-IP 7 . Depletion of Ip6k1/2 in vitro reduced phosphate transport and mRNA expression of Na + /Pi cotransporters, and it blunts phosphate transport adaptation to changes in ambient phosphate. Renal ablation of both kinases in mice also downregulates the expression of NaPi-IIa and NaPi-IIc and lowered the uptake of phosphate into proximal renal brush border membranes. In addition, the absence of Ip6k1 and Ip6k2 reduced the plasma concentration of fibroblast growth factor 23 and increased bone resorption, despite of which homozygous males develop hypophosphatemia. Ip6k1/2-/- mice also show increased diuresis, albuminuria, and hypercalciuria, although the morphology of glomeruli and proximal brush border membrane seemed unaffected. Depletion of renal Ip6k1/2 in mice not only altered phosphate homeostasis but also dysregulated other kidney functions.

Uptake of Fluorescein via a pH-Dependent Monocarboxylate Transporter by Human Kidney 2 (HK-2) Cells.

Herein, we investigated whether a fluorescent probe for an organic anion transporter (OAT), fluorescein (FLS), could be accumulated by human kidney 2 (HK-2) cells derived from human kidney proximal tubular epithelia. HK-2 cells took up FLS in a pH-dependent and concentration-dependent manner. FLS accumulation by HK-2 cells was inhibited by monocarboxylic acids, ibuprofen, rosuvastatin, and indoleacetic acid but not by typical substrates for OATs. A typical protonophore, carbonyl cyanide p-trichloromethoxyphenylhydrazone completely abolished FLS accumulation by HK-2 cells. The FLS efflux process from the preloaded HK-2 cells exhibited substantial trans-stimulation by the excess amount of extracellular FLS transport inhibitable monocarboxylate compounds such as 2,4-dichloro phenoxyacetic acid, fluvastatin, ibuprofen, indoleacetic acid, salicylic acid and rosuvastatin, indicating that the FLS transporter can recognize and accumulate them into the cells in a pH-dependent manner. The involvement of the FLS transporter in the reabsorption of monocarboxylic compounds was indicated by demonstrating that the pH-dependent FLS uptake is inhibited by various monocarboxylates in rabbit renal brush border membrane vesicles. pH-dependent FLS uptake was trans-stimulated by the inhibitable monocarboxylates. Collectively, the present data indicate that the pH-dependent transporters expressed in HK-2 cells are involved in the reabsorption of monocarboxylates from the urinary fluid into the tubular epithelia.

Reducing kidney uptake of radiolabelled exendin-4 using variants of the renally cleavable linker MVK

BackgroundPeptidic radiotracers are preferentially excreted through the kidneys, which often results in high persistent renal retention of radioactivity, limiting or even preventing therapeutic clinical translation of these radiotracers. Exendin-4, which targets the glucagon-like-peptide 1 receptor (GLP-1R) overexpressed in insulinomas and in congenital hyperinsulinism, is an example thereof. The use of the tripeptide MVK, which is readily cleaved between methionine and valine by neprilysin at the renal brush border membrane, already showed promising results in reducing kidney uptake as reported in the literature. Based on our previous findings we were interested how linker variants with multiple copies of the MV-motive influence renal washout of radiolabelled exendin-4.ResultsThree exendin-4 derivatives, carrying either one MVK, a MV-MVK or a MVK-MVK linker were synthesized and compared to a reference compound lacking a cleavable linker. In vivo results of a biodistribution in GLP-1R overexpressing tumour bearing mice at 24 h post-injection demonstrated a significant reduction (at least 57%) of renal retention of all 111In-labeled exendin-4 compounds equipped with a cleavable linker compared to the reference compound. While the insertion of the single linker MVK led to a reduction in kidney uptake of 70%, the dual approach with the linker MV-MVK slightly, but not significantly enhanced this effect, with 77% reduction in kidney uptake compared to the reference. In vitro IC50 and cell uptake studies were conducted and demonstrated that though the cleavable linkers negatively influenced the affinity towards the GLP-1R, cell uptake remained largely unaffected, except for the MV-MVK cleavable linker conjugate, which displayed lower cell uptake than the other compounds. Importantly, the tumour uptake in the biodistribution study was not significantly affected with 2.9, 2.5, 3.2 and 1.5% iA/g for radiolabelled Ex4, MVK-Ex4, MV-MVK-Ex4 and MVK-MVK-Ex4, respectively.ConclusionCleavable linkers are highly efficient in reducing the radioactivity burden in the kidney. Though the dual linker approach using the instillation of MV-MVK or MVK-MVK between exendin-4 and the radiometal chelator did not significantly outperform the single cleavable linker MVK, further structural optimization or the combination of different cleavable linkers could be a stepping stone in reducing radiation-induced nephrotoxicity.

Reduction of the Renal Radioactivity of 111In-DOTA-Labeled Antibody Fragments with a Linkage Cleaved by the Renal Brush Border Membrane Enzymes.

The interposition of a cleavable linkage by enzymes on the renal brush border membrane constitutes a promising approach for reducing the renal radioactivity levels of radiolabeled low-molecular-weight antibody fragments and constructs (LMW Abs). Herein, we applied the molecular design to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-based reagents for radiotheranostic applications with trivalent radiometals. DOTA or a derivative thereof was conjugated to a Fab through an FGK linkage ([111In]In-DO3AiBu-Bn-FGK-Fab or [111In]In-DOTA-Bn-FGK-Fab). When injected into mice, both generated radiometabolites, [111In]In-DO3AiBu-Bn-F and [111In]In-DOTA-Bn-F, by the angiotensin-converting enzyme at similar rates. Both exhibited significantly lower renal radioactivity levels than a 111In-labeled Fab prepared by the conventional procedure ([111In]In-DOTA-Bn-SCN-Fab). The different elimination rates of each radiometabolite from the kidney significantly affected the renal radioactivity levels. [111In]In-DO3AiBu-Bn-FGK-Fab preferentially reduced the renal localization without impairing tumor accumulation. These findings would pave the way for developing a DOTA-based radiotheranostic platform for LMW Abs bearing cleavable linkers for renal brush border enzymes.

Enzymological Characterization of 64Cu-Labeled Neprilysin Substrates and Their Application for Modulating the Renal Clearance of Targeted Radiopharmaceuticals.

The applicability of radioligands for targeted endoradionuclide therapy is limited due to radiation-induced toxicity to healthy tissues, in particular to the kidneys as primary organs of elimination. The targeting of enzymes of the renal brush border membrane by cleavable linkers that permit the formation of fast eliminating radionuclide-carrying cleavage fragments gains increasing interest. Herein, we synthesized a small library of 64Cu-labeled cleavable linkers and quantified their substrate potentials toward neprilysin (NEP), a highly abundant peptidase at the renal brush border membrane. This allowed for the derivation of structure-activity relationships, and selected cleavable linkers were attached to the somatostatin receptor subtype 2 ligand [Tyr3]octreotate. Radiopharmacological characterization revealed that a substrate-based targeting of NEP in the kidneys with small peptides entails their premature cleavage in the blood circulation by soluble and endothelium-derived NEP. However, for a kidney-specific targeting of NEP, the additional targeting of albumin in the blood is highlighted.

Dual MVK cleavable linkers effectively reduce renal retention of 111In-fibronectin-binding peptides

BackgroundPreviously, we have exploited bacterial adhesins-derived fibronectin-binding peptides (FnBPs) for targeting mechanically altered fibronectin (Fn) fibrils within the cancer-associated extra-cellular matrix (ECM). However, despite the ability of FnBP probes to visualize pathological lesions, when labeled with metallic radionuclides and administered for targeted imaging, they exhibit high and persistent retention of radioactivity within the kidneys. Intending to overcome this issue towards a future translation of FnBPs to the clinic, the goal of the present study was to reduce the renal retention of 111In-labelled FnBPs employing dual renal brush border membrane (BBM) enzyme-sensitive Met-Val-Lys-based linkers, enabling a rapid washout of radioactivity from the kidneys. MethodsThree maleimide-activated NOTA-conjugated brush border-enzyme cleavable linkers equipped with either single or dual consecutive MVK-based cleavable moieties were designed and synthesized. Their respective NOTA-MVK-based FnBPA5.1 conjugates were obtained by means of maleimide-thiol mediated conjugation at the N-terminus of the Fn-binding sequence, radiolabeled with indium-111, and further evaluated in vitro and in vivo in comparison to the control [111In]In-FnBPA5.1. ResultsThe linker equipped with two MVK sites displayed a two-fold more effective cleavage rate than the single MVK featuring linker in vitro, as revealed by the quantification of the released Met-containing radiometabolites. SPECT/CT imaging and biodistribution studies of the series of FnBPA5.1 radioconjugates performed at 24 h post-injection (p.i.) confirmed the in vitro results, indicating that the renal retention of 111In-labelled FnBPs can be significantly lowered through the interposition of a single MVK-based sequence between the Fn-targeting moiety and the chelating unit (52.75 ± 9.79 vs 92.88 ± 4.85 % iA/g, P < 0.001), and even further reduced by the addition of a second one (down to 34.82 ± 6.04, P < 0.001), with minor influence on the biodistribution in other organs, such as tumors. ConclusionsIn summary, we report here promising 111In-labelled FnBP radiotracers equipped with dual MVK-based cleavable linkers leading to a more effective reduction of renal retention and improved tumor-to-kidney ratios compared to the single MVK-featuring derivative. Our dual MVK strategy is a crucial step towards the clinical translation of mechano-sensory FnBPs and might as well be adopted for other radiopharmaceuticals suffering from persistent renal retention of radioactivity.

Does the composition of urinary extracellular vesicles reflect the abundance of renal Na+/phosphate transporters?

Studies addressing homeostasis of inorganic phosphate (Pi) are mostly restricted to murine models. Data provided by genetically modified mice suggest that renal Pi reabsorption is primarily mediated by the Na+/Pi cotransporter NaPi-IIa/Slc34a1, whereas the contribution of NaPi-IIc/Slc34a3 in adult animals seems negligible. However, mutations in both cotransporters associate with hypophosphatemic syndromes in humans, suggesting major inter-species heterogeneity. Urinary extracellular vesicles (UEV) have been proposed as an alternative source to analyse the intrinsic expression of renal proteins in vivo. Here, we analyse in rats whether the protein abundance of renal Pi transporters in UEV correlates with their renal content. For that, we compared the abundance of NaPi-IIa and NaPi-IIc in paired samples from kidneys and UEV from rats fed acutely and chronically on diets with low or high Pi. In renal brush border membranes (BBM) NaPi-IIa was detected as two fragments corresponding to the full-length protein and to a proteolytic product, whereas NaPi-IIc migrated as a single full-length band. The expression of NaPi-IIa (both fragments) in BBM adapted to acute as well to chronic changes of dietary Pi, whereas adaptation of NaPi-IIc was only detected in response to chronic administration. Both transporters were detected in UEV as well. UEV reflected the renal adaptation of the NaPi-IIa proteolytic fragment (but not the full-length protein) upon chronic but not acute dietary changes, while also reproducing the chronic regulation of NaPi-IIc. Thus, the composition of UEV reflects only partially changes in the expression of NaPi-IIa and NaPi-IIc at the BBM triggered by dietary Pi.

Changes in the Expression of Renal Brush Border Membrane N-Glycome in Model Rats with Chronic Kidney Diseases.

Chronic kidney disease (CKD) is defined by a reduced renal function i.e., glomerular filtration rate (GFR), and the presence of kidney damage is determined by measurement of proteinuria or albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush-border membranes (BBMs) on PT cells play an important role in maintaining the stability of PT functions. The PT BBM, a highly dynamic, organized, specialized membrane, contains a variety of glycoproteins required for the functions of PT. Since protein glycosylation regulates many protein functions, the alteration of glycosylation due to the glycan changes has attracted more interests for a variety of disease studies recently. In this work, liquid chromatography-tandem mass spectrometry was utilized to analyze the abundances of permethylated glycans from rats under control to mild CKD, severe CKD, and diabetic conditions. The most significant differences were observed in sialylation level with the highest present in the severe CKD and diabetic groups. Moreover, high mannose N-glycans was enriched in the CKD BBMs. Characterization of all the BBM N-glycan changes supports that these changes are likely to impact the functional properties of the dynamic PT BBM. Further, these changes may lead to the potential discovery of glycan biomarkers for improved CKD diagnosis and new avenues for therapeutic treatments.

Altered O-glycomes of Renal Brush-Border Membrane in Model Rats with Chronic Kidney Diseases.

Chronic kidney disease (CKD) is defined as a decrease in renal function or glomerular filtration rate (GFR), and proteinuria is often present. Proteinuria increases with age and can be caused by glomerular and/or proximal tubule (PT) alterations. PT cells have an apical brush border membrane (BBM), which is a highly dynamic, organized, and specialized membrane region containing multiple glycoproteins required for its functions including regulating uptake, secretion, and signaling dependent upon the physiologic state. PT disorders contribute to the dysfunction observed in CKD. Many glycoprotein functions have been attributed to their N- and O-glycans, which are highly regulated and complex. In this study, the O-glycans present in rat BBMs from animals with different levels of kidney disease and proteinuria were characterized and analyzed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A principal component analysis (PCA) documented that each group has distinct O-glycan distributions. Higher fucosylation levels were observed in the CKD and diabetic groups, which may contribute to PT dysfunction by altering physiologic glycoprotein interactions. Fucosylated O-glycans such as 1-1-1-0 exhibited higher abundance in the severe proteinuric groups. These glycomic results revealed that differential O-glycan expressions in CKD progressions has the potential to define the mechanism of proteinuria in kidney disease and to identify potential therapeutic interventions.

Copper-64-Labeled Antibody Fragments for Immuno-PET/Radioimmunotherapy with Low Renal Radioactivity Levels and Amplified Tumor-Kidney Ratios.

Copper-64 (64Cu)-labeled antibody fragments such as Fab are useful for molecular imaging (immuno-PET) and radioimmunotherapy. However, these fragments cause high and persistent localization of radioactivity in the kidneys after injection. To solve this problem, this study assessed the applicability of a molecular design to 64Cu, which reduces renal radioactivity levels by liberating a urinary excretory radiometabolite from antibody fragments at the renal brush border membrane (BBM). Since 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) forms a stable complex with Cu, NOTA-conjugated Met-Val-Lys-maleimide (NOTA-MVK-Mal), which is a radio-gallium labeling agent for antibody fragments, was evaluated for applicability to 64Cu. The MVK linkage was recognized by the BBM enzymes to liberate [64Cu]Cu-NOTA-Met although the recognition of the MVK sequence for the [64Cu]Cu-NOTA-MVK derivative was reduced compared with that of its [67Ga]Ga-counterpart, probably due to the difference in the charge of the metal-NOTA complexes. When injected into mice, [64Cu]Cu-NOTA-MVK-Fab resulted in similar renal radioactivity levels to the 67Ga-labeled counterpart. In addition, [64Cu]Cu-NOTA-MVK-Fab resulted in lower renal radioactivity levels than those from 64Cu-labeled Fab using a conventional method, without a reduction in the tumor radioactivity levels. These findings indicate that our approach to reducing renal radioactivity levels by liberating a radiolabeled compound from antibody fragments at the renal BBM for urinary excretion is applicable to 64Cu-labeled antibody fragments and useful for immuno-PET and radioimmunotherapy.

Sub-fractions from Carica Papaya Seed Extracts Can Prevent Potassium Bromate- induced Changes in Activities of Renal Brush Border Membrane Enzymes and Some Enzymes of Carbohydrate Metabolism in the Kidney of Rats

Aim: To investigate the effect of the chromatographic fractions of Carica papaya seed on KBrO3 –induced reduction in the activities of renal brush border membrane (BBM) marker enzymes and the changes in activities of some enzymes of carbohydrate metabolism in the kidney of rats.&#x0D; Study Design: twenty male Wistar rats were divided into four groups, five rats per group; normal control, KBrO3 control, papaya fraction control and KBrO3 group administered with 126mg/kg body weight of the most active fraction of partially purified methanol extract of C. papaya for 48 hours.&#x0D; Place and Duration of Study: Department of Biochemistry Laboratory, Faculty of Basic Medical Sciences, Bayero University Kano, Nigeria.&#x0D; Methodology: The activities of renal BBM marker enzymes: γ-glutamyl transferase, alkaline phosphatase, maltase and leucine aminopeptidase were assayed in homogenates of renal cortex and medulla, and in brush border membrane vesicle (BBMV) isolated from cortex using standard methods. Furthermore, activities of the following enzymes representing different pathways of carbohydrate metabolism were determined in renal homogenates: hexokinase (HK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose 6-phosphatase (G6P), fructose 1,6-bisphosphatase (FBP), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME).&#x0D; Results: KBrO3 administration significantly (P&lt;0.05) decreases the activities of all the BBM marker enzymes in renal homogenates and BBMV. It also decreases the activities of MDH, G6P, FBP and G6PD, and significantly increases (P&lt;0.05) that of HK, LDH and ME in renal homogenates however co-administration of most active fraction of C. papaya seed prevented all the KBrO3 -induced changes in these biochemical parameters.&#x0D; Conclusion: Chromatographic fractions of C. papaya seed extract possesses potent phytochemicals that could prevent KBrO3 –induced reduction in activities of renal BBM marker enzymes and the changes in enzymes of carbohydrate metabolism studied and therefore could be analyzed further to isolate the bioactive compounds.

Renal Handling of 99mTc-Labeled Antibody Fab Fragments with a Linkage Cleavable by Enzymes on Brush Border Membrane.

The high and persistent renal radioactivity levels after injection of radiolabeled low-molecular-weight polypeptides constitute a significant problem for their diagnostic and therapeutic applications, especially when they are labeled with metallic radionuclides. To improve the renal radioactivity levels of technetium-99m (99mTc)-labeled Fab fragments, a mercaptoacetyltriglycine (MAG3)-based new bifunctional chelating agent with a cleavable glycyl-phenylalanyl-lysine (GFK) linkage, MAG3-GFK-suc-TFP, was designed, synthesized, and evaluated. 99mTc-labeled Fab was obtained by reacting MAG3-GFK-Fab conjugate with 99mTc-glucarate. The GFK linkage remained stable in plasma but was cleaved by enzymes on the renal brush border membrane. The comparative biodistribution studies with indium-111 (111In)-labeled Fab using SCN-CHX-A″-DTPA showed that while both radiolabeled Fabs exhibited similar elimination rates from the blood, [99mTc]Tc-MAG3-GFK-Fab registered much lower renal radioactivity levels from 30 min post-injection onward due to the release and subsequent urinary excretion of [99mTc]Tc-MAG3-Gly. However, [99mTc]Tc-MAG3-GFK-Fab showed an increase in the intestinal radioactivity levels with the time that was not observed with 111In-labeled Fab. The analysis of the intestinal contents suggested the redistribution of [99mTc]Tc-MAG3-Gly to the intestine. The retrospective comparison of [99mTc]Tc-MAG3-GFK-Fab with the radiolabeled Fabs so far prepared under the identical concept suggested that some portion of [99mTc]Tc-MAG3-Gly was generated after the coated vesicle formation and they were excreted into the blood, and subsequently redistributed in the intestine via hepatobiliary excretion. In conclusion, MAG3-GFK-suc-TFP provided 99mTc-labeled Fabs that exhibit low renal radioactivity shortly after injection by the post-labeling procedure. The present study indicated that, contrary to our earlier proposal, the generation of the radiometabolites would proceed not only during the internalization process of the parental antibody fragments but also after coated vesicle formation. This study also showed that the intracellular behaviors of radiometabolites played crucial roles in the elimination rates and the routes of the radioactivity from the kidney.

1,25-Dihydroxyvitamin D Maintains Brush Border Membrane NaPi2a and Attenuates Phosphaturia in Hyp Mice.

Phosphate homeostasis is critical for many cellular processes and is tightly regulated. The sodium-dependent phosphate cotransporter, NaPi2a, is the major regulator of urinary phosphate reabsorption in the renal proximal tubule. Its activity is dependent upon its brush border localization that is regulated by fibroblast growth factor 23 (FGF23) and PTH. High levels of FGF23, as are seen in the Hyp mouse model of human X-linked hypophosphatemia, lead to renal phosphate wasting. Long-term treatment of Hyp mice with 1,25-dihydroxyvitamin D (1,25D) or 1,25D analogues has been shown to improve renal phosphate wasting in the setting of increased FGF23 mRNA expression. Studies were undertaken to define the cellular and molecular basis for this apparent FGF23 resistance. 1,25D increased FGF23 protein levels in the cortical bone and circulation of Hyp mice but did not impair FGF23 cleavage. 1,25D attenuated urinary phosphate wasting as early as one hour postadministration, without suppressing FGF23 receptor/coreceptor expression. Although 1,25D treatment induced expression of early growth response 1, an early FGF23 responsive gene required for its phosphaturic effects, it paradoxically enhanced renal phosphate reabsorption and NaPi2a protein expression in renal brush border membranes (BBMs) within one hour. The Na-H+ exchange regulatory factor 1 (NHERF1) is a scaffolding protein thought to anchor NaPi2a to the BBM. Although 1,25D did not alter NHERF1 protein levels acutely, it enhanced NHERF1-NaPi2a interactions in Hyp mice. 1,25D also prevented the decrease in NHERF1/NaPi2a interactions in PTH-treated wild-type mice. Thus, these investigations identify a novel role for 1,25D in the hormonal regulation of renal phosphate handling.

Dose-Dependent Chemopreventive Effect of Methanol Extract of Carica papaya Seed on Potassium Bromate- Induced Nephrotoxicity in Rats

Aim: To investigate the effect of Carica papaya seed extract on KBrO3 - induced nephrotoxicity in rats. Renal toxicity was induced by a single oral dose of 100 mg/kg body weight of KBrO3.&#x0D; Study Design: Thirty (30) male albino rats were divided into six groups, five rats per group; normal control, KBrO3 control, papaya control and KBrO3 group administered with methanol seed extract of 200 mg/kg, 400 mg/kg and 600 mg/kg body weight for 48 hours.&#x0D; Place and Duration of Study: Department of Biochemistry Laboratory, Faculty of Basic Medical Sciences, Bayero University Kano, Nigeria, from April 2018 to August 2018.&#x0D; Methodology: Serum urea, creatinine, uric acid and electrolytes were determined using kits from randox laboratories. Furthermore, activities of renal brush border membrane marker enzymes namely γ-glutamyltransferase (GGT), alkaline phosphatase (ALP), maltase (Mal) and leucine aminopeptidase (LAP) and some parameters of oxidative stress including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH) and malondialdehyde (MDA) were determined in homogenates prepared from renal cortex and medulla of the kidney of rats using colorimetric methods.&#x0D; Results: Administration of KBrO3 significantly (P&lt;0.05) increases the serum levels of urea, creatinine, uric acid and all electrolytes studied in a dose-dependent fashion from 200mg/kg to 400mg/kg and 600mg/kg in that order. Furthermore, the activities of GGT, ALP, Mal and LAP decrease in renal homogenates with KBrO3 administration. Also the activities of CAT, SOD, GPx and level of GSH decreases while the level of MDA significantly (P&lt;0.05) increases however concurrent administration of Carica papaya seed extract prevented all the KBrO3- induced changes in the biochemical parameters studied .&#x0D; Conclusion: It was suggested that methanol seed extract of Carica papaya possess nephroprotective effect against KBrO3 –induced renal toxicity and oxidative stress, and the most effective dose was 600 mg/kg body weight.

Transport of 2,4-dichloro phenoxyacetic acid by human Na+-coupled monocarboxylate transporter 1 (hSMCT1, SLC5A8)

Using X. laevis oocyte expression system, we investigated whether human Na+-coupled monocarboxylate transporter 1 (SLC5A8, hSMCT1) is involved in 2,4-dichlorophenoxyacetate (2,4-D) uptake by the renal tubular epithelial cells. 2,4-D is a herbicide that causes nephrotoxicity. Heterologous expression of hSMCT1 in X. laevis oocytes conferred the ability to take up 2,4-D; the induced uptake process was Na+-dependent and electrogenic. The Na+-dependent uptake of 2,4-D was inhibited not only by known hSMCT1 substrates, but also by many structural analogs of 2,4-D. The currents induced by 2,4-D, 4-chlorophenoxyacetate (4-CPA) and 2-methyl-4-chlorophenoxyacetate (MCPA) were saturable: the rank order of the maximal induced current and the affinity for hSMCT1was 2,4-D > 4-CPA > MCPA. The relationship between the structures of the derivatives and their transport activity implied specific structural features in a compound for recognition as a substrate by hSMCT1. Furthermore, we have demonstrated using purified rabbit renal brush-border membrane vesicles that 2,4-D potently inhibited the Na+-dependent uptake of pyroglutamate, a typical substrate for Smct1, and that 2,4-D uptake process was Na+-dependent, saturable and inhibitable by a potent blocker, ibuprofen. We conclude that hSMCT1 is involved partially in the renal reabsorption of 2,4-D and its derivatives and their nephrotoxicity.

A Gallium-67/68-Labeled Antibody Fragment for Immuno-SPECT/PET Shows Low Renal Radioactivity Without Loss of Tumor Uptake.

Purpose: This study was undertaken to evaluate the renal radioactivity levels of a newly designed 67Ga-labeled antibody fragment with a linkage cleaved by enzymes present on the brush border membrane (BBM) lining the lumen of the renal tubule.Experimental Design:67Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) was conjugated with an antibody Fab fragment through a Met-Val-Lys linkage (67Ga-NOTA-MVK-Fab) considering that a Met-Val sequence is a substrate of enzymes on the renal BBM and 67Ga-NOTA-Met is excreted from the kidney into the urine. The enzymatic recognition of the linkage was evaluated with a low-molecular-weight 67Ga-NOTA-Met-Val-Lys derivative. Biodistribution of radioactivity after injection of 67Ga-NOTA-MVK-Fab into mice was compared with 67Ga-NOTA-conjugated Fab fragments through a Met-Ile linkage that liberates 67Ga-NOTA-Met (67Ga-NOTA-MI-Fab) or a conventional thiourea linkage (67Ga-NOTA-Fab).Results: The MVK linkage remained stable in plasma and was recognized by enzymes on renal BBM to liberate 67Ga-NOTA-Met. When injected into mice, all three 67Ga-labeled Fab exhibited similar blood clearance rates and tumor accumulation. Significant differences were observed in the kidney where 67Ga-NOTA-MVK-Fab registered the lowest renal radioactivity levels from early postinjection time (P < 0.05), followed by 67Ga-NOTA-MI-Fab, which was well reflected in the SPECT/CT images.Conclusions: These findings indicated that our proposal of liberating a radiolabeled compound to urinary excretion from antibody fragments at the renal BBM to reduce the renal radioactivity levels was applicable to 67/68Ga-labeled antibody fragments. Because antibody fragments and constructs share similar metabolic fates in the kidney, the present labeling procedure would also apply to a variety of antibody fragments and constructs of interest. Clin Cancer Res; 24(14); 3309-16. ©2018 AACR.

In vivo evidence for an interplay of FGF23/Klotho/PTH axis on the phosphate handling in renal proximal tubules.

Phosphate homeostasis is primarily maintained in the renal proximal tubules, where the expression of sodium/phosphate cotransporters (Npt2a and Npt2c) is modified by the endocrine actions of both fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). However, the specific contribution of each regulatory pathway in the proximal tubules has not been fully elucidated in vivo. We have previously demonstrated that proximal tubule-specific deletion of the FGF23 coreceptor Klotho results in mild hyperphosphatemia with little to no change in serum levels of FGF23, 1,25(OH)2D3, and PTH. In the present study, we characterized mice in which the PTH receptor PTH1R was specifically deleted from the proximal tubules, either alone or in combination with Klotho ( PT-PTH1R-/- and PT-PTH1R/KL-/-, respectively). PT-PTH1R-/- mice showed significant increases in serum FGF23 and PTH levels, whereas serum phosphate levels were maintained in the normal range, and Npt2a and Npt2c expression in brush border membrane (BBM) did not change compared with control mice. In contrast, PT-PTH1R/KL-/- mice displayed hyperphosphatemia and an increased abundance of Npt2a and Npt2c in the renal BBM, along with increased circulating FGF23 levels. While serum calcium was normal, 1,25(OH)2D3 levels were significantly decreased, leading to extremely high levels of PTH. Collectively, mice with a deletion of PTH1R alone in proximal tubules results in only minor changes in phosphate regulation, whereas deletion of both PTH1R and Klotho leads to a severe disturbance, including hyperphosphatemia with increased sodium/phosphate cotransporter expression in BBM. These results suggest an important interplay between the PTH/PTH1R and FGF23/Klotho pathways to affect renal phosphate handling in the proximal tubules.

Preferential Cleavage of a Tripeptide Linkage by Enzymes on Renal Brush Border Membrane To Reduce Renal Radioactivity Levels of Radiolabeled Antibody Fragments.

The obstructive renal radioactivity after injection of antibody fragments/constructs labeled with metallic radionuclides would be improved by liberating a radiometal chelate of urinary excretion from the antibody molecules by enzymes on the renal brush border membrane (BBM). A tripeptide GFK sequence was newly evaluated as an enzyme-cleavable linkage and conjugated to a 99mTc chelate of an isonicotinic acid derivative of 2-picolylglycine (99mTc-IPG). 99mTc-IPG-glycine was liberated from 99mTc-IPG-GFK by the enzymes, while 99mTc-IPG-GK (where the tripeptide GFK was substituted with a dipeptide GK) did not. When injected into mice, 99mTc-IPG-GFK-conjugated Fab exhibited lower renal radioactivity levels than directly radioiodinated Fab shortly after injection without reducing the tumor radioactivity levels, due to a release and excretion of 99mTc-IPG-glycine by enzymes present on the renal BBM. These findings would provide insights to develop antibody fragments/constructs labeled with metallic radionuclides of the clinical relevance for improved renal radioactivity levels.

Ubiquitin COOH-terminal hydrolase L1 deletion is associated with urinary α-klotho deficiency and perturbed phosphate homeostasis.

Loss of ubiquitin COOH-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme required for neuronal function, led to hyperphosphatemia accompanied by phosphaturia in mice, while calcium homeostasis remained intact. We therefore investigated the mechanisms underlying the phosphate imbalance in Uchl1-/- mice. Interestingly, phosphaturia was not a result of lower renal brush border membrane sodium-phosphate cotransporter expression as sodium-phosphate cotransporter 2a and 2c expression levels was similar to wild-type levels. Plasma parathyroid hormone and fibroblast growth factor 23 levels were not different; however, fibroblast growth factor 23 mRNA levels were significantly increased in femur homogenates from Uchl1-/- mice. Full-length and soluble α-klotho levels were comparable in kidneys from wild-type and Uchl1-/- mice; however, soluble α-klotho was reduced in Uchl1-/- mice urine. Consistent with unchanged components of 1,25(OH)2D3 metabolism (i.e., CYP27B1 and CYP24A1), sodium-phosphate cotransporter 2b protein levels were not different in ileum brush borders from Uchl1-/- mice, suggesting that the intestine is not the source of hyperphosphatemia. Nonetheless, when Uchl1-/- mice were fed a low-phosphate diet, plasma phosphate, urinary phosphate, and fractional excretion of phosphate were significantly attenuated and comparable to levels of low-phosphate diet-fed wild-type mice. Our findings demonstrate that Uchl1-deleted mice exhibit perturbed phosphate homeostasis, likely consequent to decreased urinary soluble α-klotho, which can be rescued with a low-phosphate diet. Uchl1-/- mice may provide a useful mouse model to study mild perturbations in phosphate homeostasis.