Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease.

Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble α-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4 and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied.

Expression of lactate dehydrogenase A and B isoforms in the mouse kidney.

Cellular metabolic rates in the kidney are critical for maintaining normal renal function. In a hypoxic milieu, cells rely on glycolysis to meet energy needs, resulting in the generation of pyruvate and NADH. In the absence of oxidative phosphorylation, the continuation of glycolysis is dependent on the regeneration of NAD+ from NADH accompanied by the fermentation of pyruvate to lactate. This reaction is catalyzed by lactate dehydrogenase (LDH) isoform A (LDHA), whereas LDH isoform B (LDHB) catalyzes the opposite reaction. LDH is widely used as a potential injury marker as it is released from damaged cells into the urine and serum; however, the precise isoform-specific cellular localization of the enzyme along the nephron has not been characterized. By combining immunohistochemistry results and single-cell RNA-sequencing data on healthy mouse kidneys, we identified that LDHA is primarily expressed in proximal segments, whereas LDHB is expressed in the distal parts of the nephron. In vitro experiments in mouse and human renal proximal tubule cells showed an increase in LDHA following hypoxia with no change in LDHB. Using immunofluorescence, we observed that the overall expression of both LDHA and LDHB proteins decreased following renal ischemia-reperfusion injury as well as in the adenine-diet-induced model of chronic kidney disease. Single-nucleus RNA-sequencing analyses of kidneys following ischemia-reperfusion injury revealed a significant decline in the number of cells expressing detectable levels of Ldha and Ldhb; however, cells that were positive showed increased average expression postinjury, which subsided during the recovery phase. These data provide information on the cell-specific expression of LDHA and LDHB in the normal kidney as well as following acute and chronic kidney disease.NEW & NOTEWORTHY Cellular release of lactate dehydrogenase (LDH) is being used as an injury marker; however, the exact localization of LDH within the nephron remains unclear. We show that LDH isoform A is expressed proximally, whereas isoform B is expressed distally. Both subunit expressions were significantly altered in models of acute kidney injury and chronic kidney disease. Our study provides new insights into basal and postinjury renal lactate metabolism.

VEGFR3 tyrosine kinase inhibition aggravates cisplatin nephrotoxicity.

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity.NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

Regulation of renal NaDC1 expression and citrate excretion by NBCe1-A.

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

NBCe1-A Regulates Proximal Tubule Ammonia Metabolism under Basal Conditions and in Response to Metabolic Acidosis.

Renal ammonia metabolism is the primary mechanism through which the kidneys maintain acid-base homeostasis, but the molecular mechanisms regulating renal ammonia generation are unclear. In these studies, we evaluated the role of the proximal tubule basolateral plasma membrane electrogenic sodium bicarbonate cotransporter 1 variant A (NBCe1-A) in this process. Deletion of the NBCe1-A gene caused severe spontaneous metabolic acidosis in mice. Despite this metabolic acidosis, which normally causes a dramatic increase in ammonia excretion, absolute urinary ammonia concentration was unaltered. Additionally, NBCe1-A deletion almost completely blocked the ability to increase ammonia excretion after exogenous acid loading. Under basal conditions and during acid loading, urine pH was more acidic in mice with NBCe1-A deletion than in wild-type controls, indicating that the abnormal ammonia excretion was not caused by a primary failure of urine acidification. Instead, NBCe1-A deletion altered the expression levels of multiple enzymes involved in proximal tubule ammonia generation, including phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase, under basal conditions and after exogenous acid loading. Deletion of NBCe1-A did not impair expression of key proteins involved in collecting duct ammonia secretion. These studies demonstrate that the integral membrane protein NBCe1-A has a critical role in basal and acidosis-stimulated ammonia metabolism through the regulation of proximal tubule ammonia-metabolizing enzymes.

Differences in renal ammonia metabolism in male and female kidney.

Renal ammonia metabolism has a major role in the maintenance of acid-base homeostasis. Sex differences are well recognized as an important biological variable in many aspects of renal function, including fluid and electrolyte metabolism. However, sex differences in renal ammonia metabolism have not been previously reported. Therefore, the purpose of the current study was to investigate sex differences in renal ammonia metabolism. We studied 4-mo-old wild-type C57BL/6 mice fed a normal diet. Despite similar levels of food intake, and, thus, protein intake, which is the primary determinant of endogenous acid production, female mice excreted greater amounts of ammonia, but not titratable acids, than did male mice. This difference in ammonia metabolism was associated with fundamental structural differences between the female and male kidney. In the female mouse kidney, proximal tubules account for a lower percentage of the renal cortical parenchyma compared with the male kidney, whereas collecting ducts account for a greater percentage of the renal parenchyma than in male kidneys. To further investigate the mechanism(s) behind the greater ammonia excretion in female mice, we examined differences in the expression of proteins involved in renal ammonia metabolism and transport. Greater basal ammonia excretion in females was associated with greater expression of PEPCK, glutamine synthetase, NKCC2, Rhbg, and Rhcg than was observed in male mice. We conclude that there are sex differences in basal ammonia metabolism that involve both renal structural differences and differences in expression of proteins involved in ammonia metabolism.

Proximal tubule glutamine synthetase expression is necessary for the normal response to dietary protein restriction.

Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion changes in parallel during changes in dietary protein intake. Dietary protein restriction decreases endogenous acid production and decreases urinary ammonia excretion, a major component of net acid excretion. Glutamine synthetase (GS) catalyzes the reaction of [Formula: see text] and glutamate, which regenerates the essential amino acid glutamine and decreases net ammonia generation. Because renal proximal tubule GS expression increases during dietary protein restriction, this could contribute to the decreased ammonia excretion. The purpose of the current study was to determine the role of proximal tubule GS in the renal response to protein restriction. We generated mice with proximal tubule-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Cre-negative (Control) and PT-GS-KO mice in metabolic cages were provided 20% protein diet for 2 days and were then changed to low-protein (6%) diet for the next 7 days. Additional PT-GS-KO mice were maintained on 20% protein diet. Dietary protein restriction caused a rapid decrease in urinary ammonia excretion in both genotypes, but PT-GS-KO blunted this adaptive response significantly. This occurred despite no significant genotype-dependent differences in urinary pH or in serum electrolytes. There were no significant differences between Control and PT-GS-KO mice in expression of multiple other proteins involved in renal ammonia handling. We conclude that proximal tubule GS expression is necessary for the appropriate decrease in ammonia excretion during dietary protein restriction.

Expression of sodium-dependent dicarboxylate transporter 1 (NaDC1/SLC13A2) in normal and neoplastic human kidney.

Regulated dicarboxylate transport is critical for acid-base homeostasis, prevention of calcium nephrolithiasis, regulation of collecting duct sodium chloride transport, and the regulation of blood pressure. Although luminal dicarboxylate reabsorption via NaDC1 (SLC13A2) is believed to be the primary mechanism regulating renal dicarboxylate transport, the specific localization of NaDC1 in the human kidney is currently unknown. This study's purpose was to determine NaDC1's expression in normal and neoplastic human kidneys. Immunoblot analysis demonstrated NaDC1 expression with an apparent molecular weight of ~61 kDa. Immunohistochemistry showed apical NaDC1 immunolabel in the proximal tubule of normal human kidney tissue; well-preserved proximal tubule brush border was clearly labeled. Apical NaDC1 expression was evident throughout the entire proximal tubule, including the initial proximal convoluted tubule, as identified by origination from the glomerular tuft, and extending through the terminal of the proximal tubule, the proximal straight tubule in the outer medulla. We confirmed proximal tubule localization by colocalization with the proximal tubule specific protein, NBCe1. NaDC1 immunolabel was not detected other than in the proximal tubule. In addition, NaDC1 immunolabel was not detected in tumors of presumed proximal tubule origin, clear cell and papillary renal cell carcinoma, or in tumors of nonproximal tubule origin, oncocytoma and chromophobe carcinoma. In summary, 1) in the human kidney, apical NaDC1 immunolabel is present throughout the entire proximal tubule, and is not detectable in other renal cells; and 2) NaDC1 immunolabel is not present in renal tumors. These studies provide important information regarding NaDC1's role in human dicarboxylate metabolism.

Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism.

Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na(+)-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression.

NBCe1 expression is required for normal renal ammonia metabolism.

The mechanisms regulating proximal tubule ammonia metabolism are incompletely understood. The present study addressed the role of the proximal tubule basolateral electrogenic Na(+)-coupled bicarbonate cotransporter (NBCe1; Slc4a4) in renal ammonia metabolism. We used mice with heterozygous and homozygous NBCe1 gene deletion and compared these mice with their wild-type littermates. Because homozygous NBCe1 gene deletion causes 100% mortality before day 25, we studied mice at day 8 (±1 day). Both heterozygous and homozygous gene deletion caused a gene dose-related decrease in serum bicarbonate. The ability to lower urinary pH was intact, and even accentuated, with NBCe1 deletion. However, in contrast to the well-known effect of metabolic acidosis to increase urinary ammonia excretion, NBCe1 deletion caused a gene dose-related decrease in ammonia excretion. There was no identifiable change in proximal tubule structure by light microscopy. Examination of proteins involved in renal ammonia metabolism showed decreased expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase, key enzymes in proximal tubule ammonia generation, and increased expression of glutamine synthetase, which recycles intrarenal ammonia and regenerates glutamine. Expression of key proteins involved in ammonia transport outside of the proximal tubule (rhesus B glycoprotein and rhesus C glycoprotein) was not significantly changed by NBCe1 deletion. We conclude from these findings that NBCe1 expression is necessary for normal proximal tubule ammonia metabolism.

Effect of dietary protein restriction on renal ammonia metabolism.

Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion change in parallel during protein restriction. Ammonia is the primary component of net acid excretion, and inappropriate ammonia excretion can lead to negative nitrogen balance. Accordingly, we examined ammonia excretion in response to protein restriction and then we determined the molecular mechanism of the changes observed. Wild-type C57Bl/6 mice fed a 20% protein diet and then changed to 6% protein developed an 85% reduction in ammonia excretion within 2 days, which persisted during a 10-day study. The expression of multiple proteins involved in renal ammonia metabolism was altered, including the ammonia-generating enzymes phosphate-dependent glutaminase (PDG) and phosphoenolpyruvate carboxykinase (PEPCK) and the ammonia-metabolizing enzyme glutamine synthetase. Rhbg, an ammonia transporter, increased in expression in the inner stripe of outer medullary collecting duct intercalated cell (OMCDis-IC). However, collecting duct-specific Rhbg deletion did not alter the response to protein restriction. Rhcg deletion did not alter ammonia excretion in response to dietary protein restriction. These results indicate 1) dietary protein restriction decreases renal ammonia excretion through coordinated regulation of multiple components of ammonia metabolism; 2) increased Rhbg expression in the OMCDis-IC may indicate a biological role in addition to ammonia transport; and 3) Rhcg expression is not necessary to decrease ammonia excretion during dietary protein restriction.

Expression of the ammonia transporter family member, Rh B Glycoprotein, in the human kidney.

The ammonia transporter family member, Rh B Glycoprotein (RhBG/Rhbg), is essential for ammonia transport by the rodent kidney, but in the human kidney mRNA but not protein expression has been reported. Because ammonia transport is fundamental for acid-base homeostasis, the current study addressed RhBG expression in the human kidney. Two distinct RhBG mRNA sequences have been reported, with different numbers of consecutive cytosines at nt1265 and thus encoding different carboxy-tails. Sequencing the region of difference in both human kidney and liver mRNA showed eight sequential cytosines, not seven as in some reports. Knowing the correct mRNA sequence for RhBG, we then assessed RhBG protein expression using antibodies against the correct amino acid sequence. Immunoblot analysis demonstrated RhBG protein expression in human kidney and immunohistochemistry identified basolateral RhBG in connecting segment (CNT) and the cortical and outer medullary collecting ducts. Colocalization of RhBG with multiple cell-specific markers demonstrated that that CNT cells and collecting duct type A intercalated cells express high levels of RhBG, and type B intercalated cells and principal cells do not express detectable RhBG. Thus, these studies identify the correct mRNA and thus protein sequence for human RhBG and show that the human kidney expresses basolateral RhBG protein in CNT, type A intercalated cells, and non-A, non-B cells. We conclude that RhBG can mediate an important role in human renal ammonia transport.

Effect of NBCe1 deletion on renal citrate and 2-oxoglutarate handling.

UNLABELLED: The bicarbonate transporter, NBCe1 (SLC4A4), is necessary for at least two components of the proximal tubule contribution to acid-base homeostasis, filtered bicarbonate reabsorption, and ammonia metabolism. This study's purpose was to determine NBCe1's role in a third component of acid-base homeostasis, organic anion metabolism, by studying mice with NBCe1 deletion. Because NBCe1 deletion causes metabolic acidosis, we also examined acid-loaded wild-type adult mice to determine if the effects of NBCe1 deletion were specific to NBCe1 deletion or were a non-specific effect of the associated metabolic acidosis. Both NBCe1 KO and acid-loading decreased citrate excretion, but in contrast to metabolic acidosis alone, NBCe1 KO decreased expression of the apical citrate transporter, NaDC-1. Thus, NBCe1 expression is necessary for normal NaDC-1 expression, and NBCe1 deletion induces a novel citrate reabsorptive pathway. Second, NBCe1 KO increased 2-oxoglutarate excretion. This could not be attributed to the metabolic acidosis as experimental acidosis decreased excretion. Increased 2-oxoglutarate excretion could not be explained by changes in plasma 2-oxoglutarate levels, the glutaminase I or the glutaminase II generation pathways, 2-oxoglutarate metabolism, its putative apical 2-oxoglutarate transporter, OAT10, or its basolateral transporter, NaDC-3. IN SUMMARY: (1) NBCe1 is necessary for normal proximal tubule NaDC-1 expression; (2) NBCe1 deletion results in stimulation of a novel citrate reabsorptive pathway; and (3) NBCe1 is necessary for normal 2-oxoglutarate metabolism through mechanisms independent of expression of known transport and metabolic pathways.