Kidney collecting duct-derived vasopressin is not essential for appropriate concentration or dilution of urine.

We have previously shown that kidney collecting ducts make vasopressin. However, the physiological role of collecting duct-derived vasopressin is uncertain. We hypothesized that collecting duct-derived vasopressin is required for the appropriate concentration of urine. We developed a vasopressin conditional knockout (KO) mouse model wherein Cre recombinase expression induces deletion of arginine vasopressin (Avp) exon 1 in the distal nephron. We then used age-matched 8- to 12-wk-old Avp fl/fl;Ksp-Cre(-) [wild type (WT)] and Avp fl/fl;Ksp-Cre(+) mice for all experiments. We collected urine, serum, and kidney lysates at baseline. We then challenged both WT and knockout (KO) mice with 24-h water restriction, water loading, and administration of the vasopressin type 2 receptor agonist desmopressin (1 µg/kg ip) followed by the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). We performed immunofluorescence and immunoblot analysis at baseline and confirmed vasopressin KO in the collecting duct. We found that urinary osmolality (UOsm), plasma Na+, K+, Cl-, blood urea nitrogen, and copeptin were similar in WT vs. KO mice at baseline. Immunoblots of the vasopressin-regulated proteins Na+-K+-2Cl- cotransporter, NaCl cotransporter, and water channel aquaporin-2 showed no difference in expression or phosphorylation at baseline. Following 24-h water restriction, WT and KO mice had no differences in UOsm, plasma Na+, K+, Cl-, blood urea nitrogen, or copeptin. In addition, there were no differences in the rate of urinary concentration or dilution as in WT and KO mice UOsm was nearly identical after desmopressin and OPC-31260 administration. We conclude that collecting duct-derived vasopressin is not essential to appropriately concentrate or dilute urine.NEW & NOTEWORTHY Hypothalamic vasopressin is required for appropriate urinary concentration. However, whether collecting duct-derived vasopressin is involved remains unknown. We developed a novel transgenic mouse model to induce tissue-specific deletion of vasopressin and showed that collecting duct-derived vasopressin is not required to concentrate or dilute urine.

Vasopressin, protein metabolism, and water conservation.

PURPOSE OF REVIEW: Highlight the mechanisms through which vasopressin and hypertonic stress regulate protein metabolism. RECENT FINDINGS: Mammals have an 'aestivation-like' response in which hypertonic stress increases muscle catabolism and urea productionVasopressin can directly regulate ureagenesis in the liver and the kidneyIn humans chronic hypertonic stress is associated with premature aging, diabetes, cardiovascular disease, and premature mortality. SUMMARY: There is an evolutionarily conserved 'aestivation-like' response in humans in which hypertonic stress results in activation of the vasopressin system, muscle catabolism, and ureagenesis in order to promote water conservation.

Age-related changes in hematological biomarkers in common marmosets.

Researchers and veterinarians often use hematology and clinical chemistry to evaluate animal health. These biomarkers are relatively easy to obtain, and understanding how they change across healthy aging is critical to clinical care and diagnostics for these animals. We aimed to evaluate how clinical biomarkers from a chemistry profile and complete blood count (CBC) change with age in common marmosets (Callithrix jacchus). We assessed blood samples collected during routine physical exams at the Southwest National Primate Research Center and the University of Texas Health San Antonio marmoset colonies from November 2020-November 2021. We found that chemistry and CBC profiles varied based on facility, sex, and age. Significant changes in albumin, phosphorus/creatinine ratio, albumin/globulin ratio, amylase, creatinine, lymphocyte percent, hematocrit, granulocytes percent, lymphocytes, hemoglobin, red cell distribution width, and platelet distribution width were all reported with advancing age. Aged individuals also demonstrated evidence for changes in liver, kidney, and immune system function compared with younger individuals. Our results suggest there may be regular changes associated with healthy aging in marmosets that are outside of the range typically considered as normal values for healthy young individuals, indicating the potential need for redefined healthy ranges for clinical biomarkers in aged animals. Identifying animals that exhibit values outside of this defined healthy aging reference will allow more accurate diagnostics and treatments for aging colonies.

Insulin-regulated aminopeptidase is required for water excretion in response to acute hypotonic stress.

The objective of this study was to understand the response of mice lacking insulin-regulated aminopeptidase (IRAP) to an acute water load. For mammals to respond appropriately to acute water loading, vasopressin activity needs to decrease. IRAP degrades vasopressin in vivo. Therefore, we hypothesized that mice lacking IRAP have an impaired ability to degrade vasopressin and, thus, have persistent urinary concentration. Age-matched 8- to 12-wk-old IRAP wild-type (WT) and knockout (KO) male mice were used for all experiments. Blood electrolytes and urine osmolality were measured before and 1 h after water load (∼2 mL sterile water via intraperitoneal injection). Urine was collected from IRAP WT and KO mice for urine osmolality measurements at baseline and after 1 h administration of the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). Immunofluorescence and immunoblot analysis were performed on kidneys at baseline and after 1 h acute water load. IRAP was expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting duct, and collecting duct. IRAP KO mice had elevated urine osmolality compared with WT mice due to higher membrane expression of aquaporin 2 (AQP2), which was restored to that of controls after administration of OPC-31260. IRAP KO mice developed hyponatremia after an acute water load because they were unable to increase free water excretion due to increased surface expression of AQP2. In conclusion, IRAP is required to increase water excretion in response to an acute water load due to persistent vasopressin stimulation of AQP2.NEW & NOTEWORTHY Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.

Activation of hypoxia-sensing pathways promotes renal ischemic preconditioning following myocardial infarction.

Ischemic heart disease is the leading cause of death worldwide and is frequently comorbid with chronic kidney disease. Physiological communication is known to occur between the heart and the kidney. Although primary dysfunction in either organ can induce dysfunction in the other, a clinical entity known as cardiorenal syndrome, mechanistic details are lacking. Here, we used a model of experimental myocardial infarction (MI) to test effects of chronic cardiac ischemia on acute and chronic kidney injury. Surprisingly, chronic cardiac damage protected animals from subsequent acute ischemic renal injury, an effect that was accompanied by evidence of chronic kidney hypoxia. The protection observed post-MI was similar to protection observed in a separate group of healthy animals housed in ambient hypoxic conditions prior to kidney injury, suggesting a common mechanism. There was evidence that chronic cardiac injury activates renal hypoxia-sensing pathways. Increased renal abundance of several glycolytic enzymes following MI suggested that a shift toward glycolysis may confer renal ischemic preconditioning. In contrast, effects on chronic renal injury followed a different pattern, with post-MI animals displaying worsened chronic renal injury and fibrosis. These data show that although chronic cardiac injury following MI protected against acute kidney injury via activation of hypoxia-sensing pathways, it worsened chronic kidney injury. The results further our understanding of cardiorenal signaling mechanisms and have implications for the treatment of heart failure patients with associated renal disease.NEW & NOTEWORTHY Experimental myocardial infarction (MI) protects from subsequent ischemic acute kidney injury but worsens chronic kidney injury. Observed protection from ischemic acute kidney injury after MI was accompanied by chronic kidney hypoxia and increased renal abundance of hypoxia-inducible transcripts. These data support the idea that MI confers protection from renal ischemic injury via chronic renal hypoxia and activation of downstream hypoxia-inducible signaling pathways.

Comparison of COVID-19 versus influenza on the incidence, features, and recovery from acute kidney injury in hospitalized United States Veterans.

Acute kidney injury is a common complication in patients hospitalized with SARSCoV-2 (COVID-19), with prior studies implicating multiple potential mechanisms of injury. Although COVID-19 is often compared to other respiratory viral illnesses, few formal comparisons of these viruses on kidney health exist. In this retrospective cohort study, we compared the incidence, features, and outcomes of acute kidney injury among Veterans hospitalized with COVID-19 or influenza and adjusted for baseline conditions using weighted comparisons. A total of 3402 hospitalizations for COVID-19 and 3680 hospitalizations for influenza admitted between October 1, 2019 and May 31, 2020 across 127 Veterans Administration hospitals nationally were studied using the electronic medical record. Acute kidney injury occurred more frequently among those with COVID-19 compared to those with influenza (40.9% versus 29.4%, weighted analysis) and was more severe. Patients with COVID-19 were more likely to require mechanical ventilation and vasopressors and experienced higher mortality. Proteinuria and hematuria were frequent in both groups but more common in COVID-19. Recovery of kidney function was less common in patients with COVID-19 and acute kidney injury but was similar among survivors. Thus, findings from this study confirm that acute kidney injury is more common and severe among patients hospitalized with COVID-19 compared to influenza, a finding that may be driven largely by illness severity. Hence, the combined impact of these two illnesses on kidney health may be significant and have important implications for resource allocation.

Phosphorylation by PKC and PKA regulate the kinase activity and downstream signaling of WNK4.

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation.

Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K(+) reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K(+) secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)--components of an E3 ubiquitin ligase complex that targets WNKs for degradation--cause constitutively increased renal salt reabsorption and impaired K(+) secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3(S433) phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K(+) secretion in the setting of volume depletion.

WNK3 abrogates the NEDD4-2-mediated inhibition of the renal Na+-Cl- cotransporter.

The serine/threonine kinase WNK3 and the ubiquitin-protein ligase NEDD4-2 are key regulators of the thiazide-sensitive Na+-Cl- cotransporter (NCC), WNK3 as an activator and NEDD2-4 as an inhibitor. Nedd4-2 was identified as an interacting partner of WNK3 through a glutathione-S-transferase pull-down assay using the N-terminal domain of WNK3, combined with LC-MS/MS analysis. This was validated by coimmunoprecipitation of WNK3 and NEDD4-2 expressed in HEK293 cells. Our data also revealed that the interaction between Nedd4-2 and WNK3 does not involve the PY-like motif found in WNK3. The level of WNK3 ubiquitylation did not change when NEDD4-2 was expressed in HEK293 cells. Moreover, in contrast to SGK1, WNK3 did not phosphorylate NEDD4-2 on S222 or S328. Coimmunoprecipitation assays showed that WNK3 does not regulate the interaction between NCC and NEDD4-2. Interestingly, in Xenopus laevis oocytes, WNK3 was able to recover the SGK1-resistant NEDD4-2 S222A/S328A-mediated inhibition of NCC and further activate NCC. Furthermore, elimination of the SPAK binding site in the kinase domain of WNK3 (WNK3-F242A, which lacks the capacity to bind the serine/threonine kinase SPAK) prevented the WNK3 NCC-activating effect, but not the Nedd4-2-inhibitory effect. Together, these results suggest that a novel role for WNK3 on NCC expression at the plasma membrane, an effect apparently independent of the SPAK kinase and the aldosterone-SGK1 pathway.

Low potassium activation of proximal mTOR/AKT signaling is mediated by Kir4.2.

The renal epithelium is sensitive to changes in blood potassium (K+). We identify the basolateral K+ channel, Kir4.2, as a mediator of the proximal tubule response to K+ deficiency. Mice lacking Kir4.2 have a compensated baseline phenotype whereby they increase their distal transport burden to maintain homeostasis. Upon dietary K+ depletion, knockout animals decompensate as evidenced by increased urinary K+ excretion and development of a proximal renal tubular acidosis. Potassium wasting is not proximal in origin but is caused by higher ENaC activity and depends upon increased distal sodium delivery. Three-dimensional imaging reveals Kir4.2 knockouts fail to undergo proximal tubule expansion, while the distal convoluted tubule response is exaggerated. AKT signaling mediates the dietary K+ response, which is blunted in Kir4.2 knockouts. Lastly, we demonstrate in isolated tubules that AKT phosphorylation in response to low K+ depends upon mTORC2 activation by secondary changes in Cl- transport. Data support a proximal role for cell Cl- which, as it does along the distal nephron, responds to K+ changes to activate kinase signaling.

Developing the Common Marmoset as a Translational Geroscience Model to Study the Microbiome and Healthy Aging.

Emerging data support associations between the depletion of the healthy gut microbiome and aging-related physiological decline and disease. In humans, fecal microbiota transplantation (FMT) has been used successfully to restore gut microbiome structure and function and to treat C. difficile infections, but its application to healthy aging has been scarcely investigated. The marmoset is an excellent model for evaluating microbiome-mediated changes with age and interventional treatments due to their relatively shorter lifespan and many social, behavioral, and physiological functions that mimic human aging. Prior work indicates that FMT is safe in marmosets and may successfully mediate gut microbiome function and host health. This narrative review (1) provides an overview of the rationale for FMT to support healthy aging using the marmoset as a translational geroscience model, (2) summarizes the prior use of FMT in marmosets, (3) outlines a protocol synthesized from prior literature for studying FMT in aging marmosets, and (4) describes limitations, knowledge gaps, and future research needs in this field.

Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

Prolonged obstruction of the ureter, which leads to injury of the kidney collecting ducts, results in permanent structural damage, while early reversal allows for repair. Cell structure is defined by the actin cytoskeleton, which is dynamically organized by small Rho guanosine triphosphatases (GTPases). In this study, we identified the Rho GTPase, Rac1, as a driver of postobstructive kidney collecting duct repair. After the relief of ureteric obstruction, Rac1 promoted actin cytoskeletal reconstitution, which was required to maintain normal mitotic morphology allowing for successful cell division. Mechanistically, Rac1 restricted excessive actomyosin activity that stabilized the negative mitotic entry kinase Wee1. This mechanism ensured mechanical G2-M checkpoint stability and prevented premature mitotic entry. The repair defects following injury could be rescued by direct myosin inhibition. Thus, Rac1-dependent control of the actin cytoskeleton integrates with the cell cycle to mediate kidney tubular repair by preventing dysmorphic cells from entering cell division.

Mitochondrial DNA diversity in two ethnic groups in southeastern Kenya: perspectives from the northeastern periphery of the Bantu expansion.

The Bantu languages are widely distributed throughout sub-Saharan Africa. Genetic research supports linguists and historians who argue that migration played an important role in the spread of this language family, but the genetic data also indicates a more complex process involving substantial gene flow with resident populations. In order to understand the Bantu expansion process in east Africa, mtDNA hypervariable region I variation in 352 individuals from the Taita and Mijikenda ethnic groups was analyzed, and we evaluated the interactions that took place between the Bantu- and non-Bantu-speaking populations in east Africa. The Taita and Mijikenda are Bantu-speaking agropastoralists from southeastern Kenya, at least some of whose ancestors probably migrated into the area as part of Bantu migrations that began around 3,000 BCE. Our analyses indicate that they show some distinctive differences that reflect their unique cultural histories. The Taita are genetically more diverse than the Mijikenda with larger estimates of genetic diversity. The Taita cluster with other east African groups, having high frequencies of haplogroups from that region, while the Mijikenda have high frequencies of central African haplogroups and cluster more closely with central African Bantu-speaking groups. The non-Bantu speakers who lived in southeastern Kenya before Bantu speaking groups arrived were at least partially incorporated into what are now Bantu-speaking Taita groups. In contrast, gene flow from non-Bantu speakers into the Mijikenda was more limited. These results suggest a more complex demographic history where the nature of Bantu and non-Bantu interactions varied throughout the area.

Rethinking Vasopressin: New Insights into Vasopressin Signaling and Its Implications.

Vasopressin is a highly conserved peptide hormone that has been traditionally associated with water homeostasis. There is accumulating evidence in both humans and animal models that vasopressin is implicated in the regulation of metabolism. This review focuses on the effects that vasopressin exerts on the regulation of glucose and fatty acids with a particular emphasis on the potential repercussions of metabolic dysregulation in kidney disease.

High sodium intake does not worsen low potassium-induced kidney damage.

High sodium and low potassium intake have both been linked to poor cardiovascular health outcomes and increased mortality rates. A combination of the two is thought to be particularly detrimental. While mechanisms are multiple, the kidney is an important target of harmful effects and low potassium influences on both proximal and distal nephron segments are especially potent. We recently reported that a combined high sodium/low potassium diet causes kidney injury and that low potassium in isolation can have similar effects. However, how sodium intake alters this process is not well-understood. Here we tested the hypothesis that a high sodium intake amplifies effects of low dietary potassium on kidney injury. We observed adding high sodium to low potassium caused an expected increase in blood pressure, but did not worsen markers of kidney injury, inflammation, and fibrosis. It also did not increase abundance or phosphorylation of the sodium chloride cotransporter or its regulatory kinases, SPAK and OxSR1, known renal targets of low potassium. Findings support the claim that dietary potassium deficiency, and not high sodium, is a dominant factor affecting kidney injury in animal models of high sodium/low potassium intake. This suggests further investigation is required to identify optimal ranges of sodium and potassium intake in both healthy populations and in those with kidney disease.

Aldosterone paradox: differential regulation of ion transport in distal nephron.

The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K(+) secretion remain poorly understood. The identification, localization, and physiological analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K(+) balance.

Advances in WNK signaling of salt and potassium metabolism: clinical implications.

Recent evidence due to the discovery of a family of kinases implicated in arterial hypertension now points to the underlying molecular mechanisms that dictate Na(+), K(+) and water handling in the nephron. These new key players need to be understood in order to fully comprehend the pathophysiology, manifestations, and treatment of common clinical entities such as hypovolemic shock, congestive heart failure, primary hyperaldosteronism, nephrotic syndrome and hypertension. It is through the analysis of the volume status and electrolyte abnormalities that commonly present with these diseases that we can begin to create a link between the abstract concept of a kinase regulation and how a patient will respond to a particular treatment. This review is an attempt to bridge that gap.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.