American journal of physiology. Renal physiology最新文献

Acute kidney injury (AKI) is a major clinical concern with limited therapeutic strategies, often leading to chronic kidney disease (CKD) and long-term morbidity. Mitochondrial dysfunction is a major causative factor for AKI onset and progression to CKD. Interventions that restore mitochondrial integrity and cellular energy represent promising therapeutic strategies. This study investigated the potential therapeutic role of MARY1, a novel, potent, and subtype-selective serotonin-2B receptor (5-HT_2BR) antagonist, following ischemia/reperfusion (I/R)-induced AKI in mice and rats. We previously demonstrated that MARY1 induces renal mitochondrial biogenesis (MB), the generation of new functional mitochondria, in vivo. MARY1 (0.3 mg/kg, i.p., daily) administration for 6 days following AKI improves renal function, restores mitochondrial homeostasis and renal vascular integrity, upregulates β-oxidation, and restores genes associated with proximal tubule repair. Moreover, daily treatment with MARY1 for 12 days following AKI restores mitochondrial homeostasis and increases autophagic activity in the renal cortex of mice. These findings establish MARY1-mediated 5-HT_2BR antagonism as a mitochondria-targeted therapeutic strategy that addresses multiple hallmarks of AKI, and as a potential intervention for mitochondrial dysfunction-associated renal diseases.NEW & NOTEWORTHY This study identifies MARY1, a subtype selective 5-HT_2B receptor antagonist, as a novel mitochondria-targeted therapeutic for AKI. MARY1 restores mitochondrial homeostasis, enhances renal vascular integrity, and promotes autophagy and β-oxidation following bilateral I/R injury-induced AKI, leading to improved renal recovery in vivo. These findings highlight a novel therapeutic strategy to mitigate AKI progression and mitochondrial dysfunction.

The distal convoluted tubule (DCT) plays a crucial role in potassium (K⁺) homeostasis, with electrogenic basolateral K⁺ flux well established as a regulator of its function. Although the involvement of electroneutral basolateral K⁺ transport has been hypothesized, its precise role remains unclear. The electroneutral potassium chloride (Cl^-) cotransporter, KCC3, is expressed in the kidney, but its role in DCT function has yet to be fully defined. To explore this, we generated a novel animal model with DCT-specific deletion of KCC3. Our results show that KCC3 deletion in DCT cells led to reduced levels of both total and phosphorylated sodium (Na⁺) Cl^- cotransporter (NCC), along with decreased NCC mRNA expression, indicating a regulatory role for KCC3 in NCC expression at the transcript level. Despite these changes, knockout animals maintained normal electrolyte balance under standard dietary conditions. In response to dietary K⁺ restriction, knockout mice showed no significant differences compared with controls-blood K⁺ levels, NCC phosphorylation, and with no lysine kinase (WNK) body formation in the DCT remained unchanged. These findings suggest that KCC3 is involved in the basal regulation of NCC expression but is not essential for DCT adaptation to K⁺ depletion or for overall K⁺ homeostasis.NEW & NOTEWORTHY Deletion of KCC3 specifically in distal convoluted tubule cells leads to decreased NCC mRNA transcript abundance as well as a reduction in both total and phosphorylated NCC protein levels. Despite these molecular changes, DCT-specific KCC3 deletion does not disrupt overall potassium homeostasis under either standard or low potassium dietary conditions. These findings suggest that other KCC isoforms, such as KCC4, may be involved in regulating the DCT response to reduced dietary potassium intake.

Acute kidney injury (AKI) is a life-threatening condition with high morbidity and mortality, characterized by inflammation linked to organelle stress. Despite its clinical significance, effective therapies remain limited. Although organelle dysfunction is recognized as a driver of inflammation in AKI, the role of interorganelle communication in this process remains poorly understood. PDZ domain-containing 8 (PDZD8), a tethering protein on the endoplasmic reticulum (ER), facilitates ER-endolysosome contact that is essential for endolysosomal maturation. The mature endolysosome is a prerequisite for activating the DNA-sensing innate immune receptor, Toll-like receptor 9 (TLR9). Here, we investigated the role of PDZD8 in the TLR9-NF-κB pathway during AKI using Pdzd8 knockout (KO) mice and in vitro knockdown in human proximal tubular cells (PTCs). Pdzd8 KO mice showed reduced severity of cisplatin-induced AKI and reduced activation of the NF-κB pathway. Mechanistically, PDZD8 knockdown in PTCs impaired endolysosomal maturation and acidification. This functional disruption impeded the proper translocation of TLR9 to endolysosomes, thereby inhibiting the signaling cascade leading to NF-κB activation. Notably, PDZD8 knockdown did not alter mitochondrial morphology or the cytosolic leakage of mitochondrial DNA, an endogenous ligand for TLR9. These findings indicate that PDZD8 is crucial for maintaining endolysosomal homeostasis and regulating the TLR9-NF-κB pathway in cisplatin-induced tubular injury.NEW & NOTEWORTHY This study reveals the critical role of PDZD8 in maintaining endolysosomal homeostasis and regulating the TLR9-NF-κB inflammatory pathway in cisplatin-induced acute kidney injury (AKI). Loss of PDZD8 impaired endolysosomal function, suppressing TLR9 activation and downstream inflammation, leading to reduced tubular damage.

We previously reported that Na⁺-deprived mice lacking CAP1/Prss8 in kidney tubules maintained epithelial sodium channel-mediated sodium balance albeit persistent hypoaldosteronism, hence indicating an uncoupling from aldosterone production. This further suggested an implication of the serine protease CAP1/Prss8 (prostasin) in the cross talk of the kidney with the adrenal gland that does not express prostasin. When these knockout (Ko) mice were additionally exposed to a high K⁺ diet, plasma K⁺ levels and plasma aldosterone concentrations were normalized and no longer different from those of the control mice. The mRNA transcript expression of the adrenal aldosterone synthase Cyp11b2, which was lower in Na⁺-deprived CAP1/Prss8 Ko animals, was in the normal range. Plasma aldosterone levels were similar to control animals, indicating that K⁺ rescued the hypoaldosteronism in Na⁺-deprived CAP1/Prss8 Ko animals. These data suggest that CAP1/Prss8 (prostasin) is implicated in the regulation of aldosterone synthesis or production and that the consequences of CAP1/Prss8 deficiency can be compensated by high dietary K⁺ supplementation. Prostasin may therefore present a promising regulator of aldosterone production by affecting the adrenal steroidogenic pathway.NEW & NOTEWORTHY We explore the role of the serine protease CAP1/Prss8 in aldosterone synthesis. Described previously as a candidate gene for hypertension, the mechanism by which renal serine protease deficiency is implicated in aldosterone production is still largely unknown. Our findings underscore a role of prostasin in the regulation of aldosterone synthesis. In kidney-specific CAP1/Prss8 knockout mice, K⁺ supplementation is predominant over Na⁺ and restores normal aldosterone production proposing new pathways to treat hypo- or hypertension.

Under physiologic conditions, proximal tubules depend on basolateral fatty acid (FA) uptake for metabolism. In pathophysiologic conditions due to glomerular filtration barrier disruption, albumin-bound FA undergoes filtration and proximal tubule reabsorption, which leads to lipotoxicity and tubular atrophy. Apical proximal tubule albumin uptake is accomplished by the megalin/cubilin complex and receptor-mediated endocytosis, whereas apical proximal tubule FA uptake is primarily mediated by apical fatty acid transport protein-2 (FATP2). However, a commonly proposed (but untested) alternative model is that the intact albumin-FA complex is cotransported by megalin/cubilin-mediated endocytosis, similar to apolipoproteins. Microperfused mouse proximal tubules demonstrated divergent one- versus two-phase albumin and FA uptake kinetics, with significantly faster albumin compared with FA uptake. LLC-PK1, human proximal tubule cells (HPCT), and opossum kidney (OK) proximal tubule cell lines all expressed megalin, cubilin, and FATP2 mRNA, though in varying amounts. LLC-PK1 cells showed similar one-phase kinetics of dual fluorescently labeled albumin and FA uptake, whereas HPCT cells demonstrated one-phase albumin and two-phase FA uptake kinetics, with significantly faster albumin compared with FA uptake (similar to perfused proximal tubules). FATP2 inhibition blocked FA uptake, but had no effect on albumin uptake in LLC-PK1 and HPCT cells. Megalin and cubilin deletion in OK cells inhibited albumin uptake, but had no effect on FA uptake. We conclude that apical proximal tubule albumin and FA are transported by distinct mechanisms, implying that FAs dissociate from albumin within the proximal tubule lumen before uptake.NEW & NOTEWORTHY Reabsorption of aberrantly filtered albumin-bound fatty acids by the apical proximal tubule is important for chronic kidney disease progression. Whether fatty acids and albumin are taken up as intact complexes or dissociate within the lumen before uptake has been controversial. Data derived from in vitro and ex vivo models demonstrate separate albumin and fatty acid uptake kinetics, implying dissociation before uptake.

Vasopressin (VP)-induced cellular trafficking of the water channel aquaporin-2 (AQP2) to maintain systemic water balance is regulated in large part by the actin cytoskeleton in collecting duct principal cells (PCs). Actin depolymerization/polymerization is involved in both constitutive AQP2 recycling and the VP-stimulated pathway; in many cells, focal adhesion kinase (FAK) modulates the actin cytoskeleton by inhibiting small GTPases. To understand the contribution of FAK to this process, we examined its involvement in AQP2 trafficking. We first showed that FAK inhibition using the drug VS-4718 caused membrane accumulation of AQP2 in LLC-AQP2 epithelial cells in culture and PC in situ (kidney slices), by immunofluorescence staining and biotinylation. This was associated with significantly reduced endocytosis of AQP2 via the clathrin-mediated pathway, along with a reduction in RhoA activation and F-actin depolymerization. Importantly, AQP2 membrane accumulation induced by VS-4718 also occurred in cells expressing the dephosphorylation mutant of AQP2, S256A. Unlike VP, FAK inhibition did not increase cellular cAMP, nor AQP2 S256 phosphorylation. As expected, treatment with the FAK inhibitor VS-4718 suppressed FAK phosphorylation at Tyr397 in both cultured cells and kidney tissue. However, VP stimulation induced a redistribution of phospho-FAK (Tyr397) from basolateral toward the apical region of collecting duct principal cells in tissue slices, concomitant with AQP2 accumulation, but no overall change in the level of FAK phosphorylation was detectable after VP treatment. These data, therefore, identify FAK signaling as a distinct pathway that could provide a novel therapeutic avenue for regulating AQP2 trafficking in water balance disorders.NEW & NOTEWORTHY Focal adhesion kinase (FAK) signaling plays a previously unrecognized role in regulating AQP2 trafficking by modulating the actin cytoskeleton and inhibiting the small GTPase RhoA in renal epithelial cells. Our findings demonstrate that FAK inhibition reduces AQP2 endocytosis via a cAMP- and serine 256 phosphorylation-independent mechanism, leading to its membrane accumulation. This study identifies FAK as a potential therapeutic target for water balance disorders by revealing its role in actin-mediated AQP2 regulation through RhoA inhibition.

Crystalline nephropathies are associated with kidney injury. Uromodulin (Umod), a glycoprotein produced in the kidneys, regulates salt transport, protecting against urinary tract infections, kidney stones, and kidney injury, contributing to innate immunity. After cleavage by the protease hepsin, Umod is secreted into the tubular lumen. We hypothesize that exogenous Umod may reduce injury associated with crystalline nephropathy. Both in vivo and in vitro models were used. Eight-week-old C57BL/6J male mice were treated with sodium oxalate (NaOx, 9 mg/100 g body wt) and/or Umod (5 µg/animal) and compared with controls. The ST-1 cell line (mouse thick ascending limb of loop of Henle) was treated with calcium oxalate (CaOx; 200 µg/mL) for 6 or 24 h and compared with controls. NaOx treatment caused tubular injury and upregulated proinflammatory and profibrotic factors. Exogenous Umod attenuated NaOx-induced kidney injury. In vitro CaOx treatment decreased Umod expression and induced apoptosis in ST-1 cells, confirmed by elevated caspase-8 immunostaining, whereas Umod reduced the apoptotic response. This study demonstrates that Umod cotreatment attenuated several aspects of NaOx-induced kidney injury. These findings suggest that the multifunctional nature of Umod may have clinical relevance and support the potential utility of urinary Umod as a biomarker of kidney health.NEW & NOTEWORTHY This study sheds light on the potential role of exogenous uromodulin in modulating tubular responses to oxalate-induced kidney injury. By exploring its influence on epithelial stress, inflammation, and protein trafficking, these findings provide a more comprehensive understanding of uromodulin's function beyond its known structural and antimicrobial properties. These insights may inform future strategies for preserving tubular integrity in crystal-related renal disorders.

Tuberous sclerosis complex (TSC) promotes renal cyst formation and chronic kidney disease through mechanistic target of rapamycin complex 1 (mTORC1) dysregulation, yet effective treatments remain limited. Using mouse models with Tsc1 deletion in nephron progenitor cells and CRISPR-edited human kidney cells, we assessed the role of the endocannabinoid system in TSC-associated kidney disease. Tsc1 deletion led to significant alterations in endocannabinoid levels and the expression of metabolizing enzymes. These molecular changes were accompanied by receptor dysregulation, characterized by CB1R upregulation and CB2R downregulation in cyst-lining epithelial cells. A similar receptor imbalance was observed in TSC1-deficient human kidney cells, suggesting a conserved pathogenic mechanism. Treatment with the peripheral CB1R antagonist JD5037 significantly reduced mTORC1 activity and c-Myc expression in cultured cells and ex vivo kidney organ cultures. These findings identified CB1R as a potential therapeutic target, linking endocannabinoid dysregulation to TSC kidney pathology.NEW & NOTEWORTHY This study reveals for the first time that TSC-associated kidney disease involves significant dysregulation of the endocannabinoid system in both murine models and human kidneys, characterized by altered endocannabinoid levels, enzyme expression changes, CB1R upregulation, and CB2R downregulation in cyst-lining epithelial cells. Treatment with the peripheral CB1R antagonist effectively suppressed mTORC1 hyperactivation and c-Myc expression, identifying CB1R as a novel therapeutic target for TSC-associated renal pathology.

Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease representing the most frequent genetic cause of end-stage kidney failure in children and adolescents. NPH is caused by genetic variants in >20 NPHP genes. Although nearly all NPHP genes encode ciliary proteins, classifying NPH as a renal ciliopathy, there is evidence for a pathogenic role of a compromised DNA damage response (DDR). Here, we present a novel Nphp7/Glis2-deficient mouse model with an early stop codon using CRISPR/Cas9-mediated genome editing (Glis2^Y122X). Homozygous mice displayed dilated kidney tubules progressing to cystic kidney disease with significant fibrosis at a higher age. Interestingly, the kidneys of these animals exhibited an accumulation of DNA damage (DD) early on, even before any functional impairment of the kidneys became apparent. Interactome analysis for GLIS2 revealed an array of DDR-related proteins within the GLIS2 protein complex. Consistent with the in vivo data, the knockdown of Glis2 in kidney epithelial cells led to increased DNA damage. Moreover, supporting the role of GLIS2 in the DDR, we demonstrate that a substantial proportion of GLIS2 is present within the chromatin fraction of cells, which is further increased upon UV-induced DD. Live-cell imaging revealed the rapid recruitment of green fluorescent protein (GFP)-tagged GLIS2 to sites of laser-induced DD, a response diminished in Glis2^Y122X and a variant of Glis2 resembling a known patient mutation. Overall, our data provide compelling evidence for the direct involvement of GLIS2 in the DDR, highlighting the loss of genome stability as an important factor contributing to the pathogenesis of renal ciliopathies.NEW & NOTEWORTHY Nephronophthisis (NPH) is a pediatric cystic kidney disease and ciliopathy. We present a novel Glis2/Nphp7-deficient mouse model that shows early accumulation of DNA damage before detectable kidney dysfunction. The GLIS2 protein complex includes DNA damage response factors. GLIS2 localizes to chromatin and rapidly relocates to sites of DNA damage. These findings position GLIS2 as a direct player in genome stability, highlighting impaired DDR as a key contributor to NPH pathogenesis.