Biology subjects

Kleyman, T. R.

Publications and source records attributed to Kleyman, T. R..

10 recordsLinked to original sources

bioRxiv2026

Contribution of murine strain background to Na+ reabsorption in the kidney

Kidneys play an essential role in balancing fluid and electrolyte levels. Two mouse strains, C57Bl/6 and 129S2/SV, are routinely used to study renal physiology in laboratory settings, and prior observations suggest that significant differences in salt and water handling exist between them. This study aims to further establish the sources of these observed differences at both expressional and functional levels, in male and female mice. At baseline, male 129S2/SV mice displayed decreased Na+ and increased K+ plasma concentrations compared to C57Bl/6 males, while no statistical differences were observed between female mice. Interestingly, 129S2/SV male mice had lower glomerular density than C57Bl/6 males. Immunoblotting shows that 129S2/SV mice of both sexes had increased expression of NHE3 and NKCC2 compared to their C57Bl/6 counterparts. Both total and phosphorylated NCC were more abundant in female mice as compared to males, indicating sexual dimorphism. Furthermore, 129S2/SV females had higher expression of total and phosphorylated NCC compared to C57Bl/6 females. In contrast, the expression of SGLT2, ENaC subunits, and Na+/K+-ATPase were comparable between C57Bl/6 and 129S2/SV mice of both sexes. When challenged with diuretics intended to block NKCC2, NCC or ENaC, 129S2/SV male mice responded with a smaller diuresis and natriuresis than their C57Bl/6 counterparts. Taken together, our data suggest that differential expression of key Na+ transporters along the nephron contributes to differences in Na+/K+ homeostasis between these two mouse strains. NEW & NOTEWORTHYWe assessed the influence of genetic background on the expression of key Na+ transporters along the nephron in two commonly used inbred mouse strains, C57Bl/6 and 129S2/SV. We found that the kidney expression of NHE3, NKCC2, and NCC are strain dependent. Additionally, murine strain significantly contributes to the diuretic responses induced by hydrochlorothiazide, amiloride, and furosemide.

physiology↗

bioRxiv2026

An endoplasmic reticulum resident molecular chaperone, GRP170, prevents stress-induced glomerular injury

The glomerulus, a unique capillary network in the nephron, filters an entire blood volume approximately 300 times a day. Specialized epithelial cells known as podocytes form a critical component of the glomerular filtration barrier, and diseases linked to podocyte injury include minimal change disease, focal segmental glomerulosclerosis, and diabetic kidney disease. Because podocytes are terminally differentiated, their ability to respond to external stress is critical. The unfolded protein response (UPR), a cellular stress pathway, is associated with glomerular injury, although the role of the UPR in glomerular injury is undefined. The UPR is initially protective, leading to upregulation of molecular chaperones, a class of proteins that promote protein folding and are required to survive oxidative and ischemic injury. An unresolved UPR, however, leads to apoptosis. We previously found that one molecular chaperone, GRP170, provides protection against acute kidney injury since GRP170 depletion led to UPR induction and widespread kidney injury. Here we generated a new podocyte specific GRP170 knock out mouse (GRP170Pd-/-). Surprisingly, GRP170Pd-/- mice were born healthy, and podocyte development appeared normal. Within a month, however, the knockout mice exhibited profound glomerular injury manifesting as proteinuria, hypoalbuminemia, hyperlipidemia, and kidney injury. Concomitant with glomerular injury, we observed increased expression of the pro-apoptotic UPR target, CHOP, in podocytes. Together, our new model not only defines a protective role for GRP170 against glomerular injury but also provides a new model to test the therapeutic potential of small molecule UPR modulators to treat glomerular injury.

physiology↗

bioRxiv2024

Abnormal activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron contributes to fructose-induced salt-sensitive hypertension

Fructose high-salt (FHS) diets increase blood pressure (BP) in an angiotensin II (Ang II)-dependent manner. Ang II stimulates aldosterone release, which, by acting on the mineralocorticoid receptor (MR), regulates Na+ reabsorption by the aldosterone-sensitive distal nephron (ASDN). The MR can be transactivated by glucocorticoids, including those locally produced by 11{beta}-HSD1. The epithelial sodium channel (ENaC) is a key transporter regulated by MRs. We hypothesized that fructose-induced salt-sensitive hypertension depends in part on abnormal activation of MRs in the ASDN with consequent increases in ENaC expression. We found that aldosterone-upregulated genes in mice ASDN, significantly overlapped with 74 genes upregulated by FHS in the rat kidney cortex (13/74; p[≤]1x10-8), and that these 74 genes are prominently expressed in rat ASDN cells. Additionally, the average z-score expression of mice-aldosterone-upregulated genes is highly correlated with FHS compared to glucose high-salt (GHS) in the rat kidney cortex (Pearson correlation; r=0.66; p[≤]0.005). There were no significant differences in plasma aldosterone concentrations between the FHS and GHS. However, 11{beta}-HSD1 transcripts were upregulated by FHS (log2FC=0.26, p[≤]0.02). FHS increased BP by 23{+/-}6 mmHg compared to GHS, and blocking MRs with eplerenone prevented this increase. Additionally, inhibiting ENaC with amiloride significantly reduced BP in FHS from 148{+/-}6 to 134{+/-}5 mmHg (p[≤]0.019). Compared to GHS, FHS increased total and cleaved ENaC protein by 89{+/-}14 % (p[≤]0.03) and 47{+/-}16 % (p[≤]0.01) respectively. FHS did not change {beta}- or {gamma}-subunit expression. These results suggest that fructose-induced salt-sensitive hypertension depends, in part, on abnormal Na+ retention by ENaC, resulting from the activation of MRs by glucocorticoids.

physiology↗

bioRxiv2024

Myeloid Cell Glucocorticoid, Not Mineralocorticoid Receptor Signaling, Contributes to Salt-Sensitive Hypertension in Humans via Cortisol

BACKGROUNDSalt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular morbidity and mortality, yet the etiology is poorly understood. We previously found that serum/glucocorticoid-regulated kinase 1 (SGK1) and epoxyeicosatrienoic acids (EETs) regulate epithelial sodium channel (ENaC)-dependent sodium entry into monocyte-derived antigen-presenting cells (APCs) and activation of NADPH oxidase, leading to the formation of isolevuglandins (IsoLGs) in SSBP. Whereas aldosterone via the mineralocorticoid receptor (MR) activates SGK1 leading to hypertension, our past findings indicate that levels of plasma aldosterone do not correlate with SSBP, and there is little to no MR expression in APCs. Thus, we hypothesized that cortisol acting via the glucocorticoid receptor (GR), not the MR in APCs mediates SGK1 actions to induce SSBP. METHODSWe performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) analysis on peripheral blood mononuclear cells of humans rigorously phenotyped for SSBP using an inpatient salt loading/depletion protocol to determine expression of MR, GR, and SGK1 in immune cells. In additional experiments, we performed bulk transcriptomic analysis on isolated human monocytes following in vitro treatment with high salt from a separate cohort. We then measured urine and plasma cortisol, cortisone, renin, and aldosterone. Subsequently, we measured the association of these hormones with changes in systolic, diastolic, mean arterial pressure and pulse pressure as well as immune cell activation via IsoLG formation. RESULTSWe found that myeloid APCs predominantly express the GR and SGK1 with no expression of the MR. Expression of the GR in APCs increased after salt loading and decreased with salt depletion in salt-sensitive but not salt-resistant people and was associated with increased expression of SGK1. Moreover, we found that plasma and urine cortisol/cortisone but not aldosterone/renin correlated with SSBP and APCs activation via IsoLGs. We also found that cortisol negatively correlates with EETs. CONCLUSIONOur findings suggest that renal cortisol signaling via the GR but not the MR in APCs contributes to SSBP via cortisol. Urine and plasma cortisol may provide an important currently unavailable feasible diagnostic tool for SSBP. Moreover, cortisol-GR-SGK1-ENaC signaling pathway may provide treatment options for SSBP. Novelty and RelevanceO_ST_ABSWhat Is New?C_ST_ABSO_LIAlthough salt sensitivity is a major risk factor for cardiovascular morbidity and mortality, the mechanisms underlying the salt sensitivity of blood pressure (SSBP) are poorly understood. C_LIO_LIHigh salt modifies glucocorticoid-receptor expression in antigen-presenting cells (APCs), suggesting a critical role of glucocorticoids in SSBP. C_LIO_LIElevated glucocorticoid receptor (GR) expression compared to mineralocorticoid receptor (MR) expression in APCs provides evidence for a GR-dependent pathway to SSBP. Isolevuglandins (IsoLGs) increased in APCs in vitro after hydrocortisone treatment compared to aldosterone treatment, indicating that cortisol was the predominant driver of IsoLG production in these cells. C_LIO_LIOur studies suggest a mechanism for SGK1 expression through GR activation by cortisol that differs from the currently accepted mechanism for SSBP pathogenesis. C_LI What Is Relevant?O_LIAlthough aldosterone has been used to study SSBP, there has been no consideration of cortisol as a major driver of the condition. C_LIO_LIUnderstanding alternative inflammatory pathways that affect SSBP may provide insights into the mechanism of SSBP and suggest a range of therapeutic targets. C_LIO_LIOur studies may provide a practical approach to understanding and treating salt-sensitive hypertension. C_LI Clinical/Pathophysiological Implications?O_LIOur findings firmly support a GR-dependent signaling pathway for activating SSBP via SGK1 expression. A cortisol-driven mechanism could provide a practical approach for targeted treatments for salt-sensitive hypertension. Moreover, it could pave the way for a diagnostic approach. C_LI

physiology↗

bioRxiv2024

Proteolytic Cleavage of the ENaC gamma Subunit: Impact Upon Na+ and K+ Handling

The ENaC gamma subunit is essential for homeostasis of Na+, K+, and body fluid. Dual subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146 in mouse). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (Q4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, Q4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in Q4/Q4 mice was not altered. Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.

physiology↗

bioRxiv2024

Excess dietary sodium partially restores salt and water homeostasis caused by loss of the endoplasmic reticulum molecular chaperone, GRP170, in the mouse nephron

The maintenance of fluid and electrolyte homeostasis by the kidney requires proper folding and trafficking of ion channels and transporters in kidney epithelia. Each of these processes requires a specific subset of a diverse class of proteins termed molecular chaperones. One such chaperone is GRP170, which is an Hsp70-like, endoplasmic reticulum (ER)-localized chaperone that plays roles in protein quality control and protein folding in the ER. We previously determined that loss of GRP170 in the mouse nephron leads to hypovolemia, electrolyte imbalance, and rapid weight loss. In addition, GRP170-deficient mice develop an AKI-like phenotype, typified by tubular injury, elevation of clinical kidney injury markers, and induction of the unfolded protein response (UPR). By using an inducible GRP170 knockout cellular model, we confirmed that GRP170 depletion induces the UPR, triggers an apoptotic response, and disrupts protein homeostasis. Based on these data, we hypothesized that UPR induction underlies hyponatremia and volume depletion in rodents, but that these and other phenotypes might be rectified by supplementation with high salt. To test this hypothesis, control and GRP170 tubule-specific knockout mice were provided with a diet containing 8% sodium chloride. We discovered that sodium supplementation improved electrolyte imbalance and reduced clinical kidney injury markers, but was unable to restore weight or tubule integrity. These results are consistent with UPR induction contributing to the kidney injury phenotype in the nephron-specific GR170 knockout model, and that the role of GRP170 in kidney epithelia is essential to both maintain electrolyte balance and cellular protein homeostasis.

cell biology↗

bioRxiv2023

Genome mining yields new disease-associated ROMK variants with distinct defects

Bartter syndrome is a group of rare genetic disorders that compromise kidney function by impairing electrolyte reabsorption. Left untreated, the resulting hyponatremia, hypokalemia, and dehydration can be fatal. Although there is no cure for this disease, specific genes that lead to different Bartter syndrome subtypes have been identified. Bartter syndrome type II specifically arises from mutations in the KCNJ1 gene, which encodes the renal outer medullary potassium channel, ROMK. To date, over 40 Bartter syndrome-associated mutations in KCNJ1 have been identified. Yet, their molecular defects are mostly uncharacterized. Nevertheless, a subset of disease-linked mutations compromise ROMK folding in the endoplasmic reticulum (ER), which in turn results in premature degradation via the ER associated degradation (ERAD) pathway. To identify uncharacterized human variants that might similarly lead to premature degradation and thus disease, we mined three genomic databases. First, phenotypic data in the UK Biobank were analyzed using a recently developed computational platform to identify individuals carrying KCNJ1 variants with clinical features consistent with Bartter syndrome type II. In parallel, we examined ROMK genomic data in both the NIH TOPMed and ClinVar databases with the aid of a computational algorithm that predicts protein misfolding and disease severity. Subsequent phenotypic studies using a high throughput yeast screen to assess ROMK function--and analyses of ROMK biogenesis in yeast and human cells--identified four previously uncharacterized mutations. Among these, one mutation uncovered from the two parallel approaches (G228E) destabilized ROMK and targeted it for ERAD, resulting in reduced protein expression at the cell surface. Another ERAD-targeted ROMK mutant (L320P) was found in only one of the screens. In contrast, another mutation (T300R) was ERAD-resistant, but defects in ROMK activity were apparent after expression and two-electrode voltage clamp measurements in Xenopus oocytes. Together, our results outline a new computational and experimental pipeline that can be applied to identify disease-associated alleles linked to a range of other potassium channels, and further our understanding of the ROMK structure-function relationship that may aid future therapeutic strategies. Author SummaryBartter syndrome is a rare genetic disorder characterized by defective renal electrolyte handing, leading to debilitating symptoms and, in some patients, death in infancy. Currently, there is no cure for this disease. Bartter syndrome is divided into five types based on the causative gene. Bartter syndrome type II results from genetic variants in the gene encoding the ROMK protein, which is expressed in the kidney and assists in regulating sodium, potassium, and water homeostasis. Prior work established that some disease-associated ROMK mutants misfold and are destroyed soon after their synthesis in the endoplasmic reticulum (ER). Because a growing number of drugs have been identified that correct defective protein folding, we wished to identify an expanded cohort of similarly misshapen and unstable disease-associated ROMK variants. To this end, we developed a pipeline that employs computational analyses of human genome databases with genetic and biochemical assays. Next, we both confirmed the identity of known variants and uncovered previously uncharacterized ROMK variants associated with Bartter syndrome type II. Further analyses indicated that select mutants are targeted for ER-associated degradation, while another mutant compromises ROMK function. This work sets-the-stage for continued mining for ROMK loss of function alleles as well as other potassium channels, and positions select Bartter syndrome mutations for correction using emerging pharmaceuticals.

genetics↗

bioRxiv2022

Influence of MUC1 on trafficking of TRPV5 and TRPV6 and in vivo Ca2+ homeostasis.

Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5 in cultured unpolarized cells. Using biotinylation of cell-surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+.

physiology↗

bioRxiv2021

L-WNK1 is required for BK channel activation in intercalated cells

BK channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high K+ diet. In cell culture, the long isoform of the kinase WNK1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high K+ diet. Mice with an IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high K+ (5% K+ as KCl) diet for at least 10 days. IC-L-WNK1-KO mice exhibited higher blood K+ concentrations ([K+]) than controls. BK channel-dependent whole-cell currents in ICs from cortical collecting ducts of high K+ fed IC-L-WNK1-KO mice were reduced compared to controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice have higher blood [K+] and reduced IC BK channel currents are consistent with impaired urinary K+ secretion, and suggest that IC L-WNK1 has a role in the renal adaptation to a high K+ diet.

physiology↗

bioRxiv2021

Water and Electrolyte Homeostasis in a Mouse Model with Reduced ENaC Gamma Subunit Expression

The epithelial Na+ channel (ENaC) promotes the absorption of Na+ in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding ENaCs subunits results in early post-natal mortality. We present initial characterization of a mouse with dramatically suppressed expression of the {gamma} subunit. We use this hypomorphic ({gamma}mt) allele to explore the importance of ENaCs {gamma} subunit in homeostasis of electrolytes and body fluid volume. At baseline, {gamma} subunit expression in {gamma}mt/mt mice is markedly suppressed in kidney and lung, while electrolytes resemble those of littermate controls. Challenge with a high K+ diet does not cause significant differences in blood K+, but provokes higher aldosterone in {gamma}mt/mt mice than controls. Quantitative magnetic resonance (QMR) measurement of body composition reveals similar baseline body water, lean tissue mass, and fat tissue mass in {gamma}mt/mt mice and controls. Surprisingly, euvolemia is sustained without significant changes in aldosterone or atrial natriuretic peptide. {gamma}mt/mt mice exhibit a more rapid decline in body water and lean tissue mass in response to a low Na+ diet than controls. Replacement of drinking water with 2% saline induces dramatic increases in body fat in both genotypes, and a selective transient increase in body water and lean tissue mass in {gamma}mt/mt mice. While ENaC in renal tubules and colon work to prevent extracellular fluid volume depletion, our observations suggest that ENaC in non-epithelial tissues may have a role in preventing extracellular fluid volume overload.

physiology↗