American journal of physiology. Renal physiology最新文献

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.

Chronic kidney disease (CKD) is associated with heightened cardiovascular disease (CVD) risk, partly due to impaired peripheral vascular function. Symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA) are emerging biomarkers implicated in nitric oxide (NO) regulation and vascular health. Although ADMA is a well-established inhibitor of NO synthesis, recent evidence suggests that SDMA may also play a critical role in vascular health, especially in CKD before end-stage. Thus, in 23 stages 3 and 4 patients with CKD (66 ± 9 yr) and 32 age-matched controls (64 ± 8 yr), we compared serum SDMA and ADMA levels and examined their associations with vascular function, including flow-mediated dilation (FMD), peak blood velocity to reactive hyperemia, and carotid-femoral pulse wave velocity (cfPWV). SDMA was significantly elevated in patients with CKD (163 ± 37 vs. 100 ± 15 ng/mL, P < 0.0001), while ADMA did not differ significantly between groups (111 ± 22 vs. 103 ± 12 ng/mL, P = 0.083). Patients with CKD had lower FMD (3.66 ± 2.45 vs. 4.47 ± 2.45%, P = 0.048) and peak blood velocity (47.43 ± 16.67 vs. 60.18 ± 16.88 cm/s, P = 0.009), but higher cfPWV (8.82 ± 1.53 vs. 7.69 ± 1.35 m/s, P = 0.004) than controls. Pooled analysis revealed that SDMA correlated inversely with eGFR (r = -0.86, P < 0.0001), FMD (r_s = -0.28, P = 0.039), and peak blood velocity (r_s = -0.40, P = 0.001) but not cfPWV (r = 0.14, P = 0.338). ADMA correlated inversely with peak blood velocity (r_s = -0.28, P = 0.042) but not eGFR (r = -0.25, P = 0.063), FMD (r_s = -0.06, P = 0.664), or cfPWV (r = 0.21, P = 0.146). Collectively, these findings suggest that SDMA, relative to ADMA, may be a stronger marker of vascular dysfunction in stages 3 and 4 CKD. However, the predictive value of SDMA for vascular function was modest, which may limit its overall potential as a biomarker for vascular function in CKD.NEW & NOTEWORTHY The associations between symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA) and measures of vascular function were investigated in patients with stage 3-4 chronic kidney disease (CKD). We found that SDMA exhibited stronger relationships with vascular function than ADMA. However, the strength of associations was modest, potentially limiting their role as standalone predictors of vascular dysfunction. Nonetheless, these data support emerging evidence of a differential impact of SDMA and ADMA in patients with CKD.

Extracellular cold-inducible RNA-binding protein (eCIRP) was discovered as a potent damage-associated molecular pattern (DAMP). It has been shown that eCIRP is linked to various types of programmed cell death and acute inflammation. However, the role of eCIRP in chronic inflammation and renal fibrosis has not been elucidated. Accumulating evidence indicates that renal tubular epithelial cells (RTECs) play a significant role in renal fibrosis. C23, a small molecular peptide inhibitor of eCIRP, has been implicated as a therapeutic agent in the context of acute inflammation and tissue injury. PANoptosis or synchronized cell death is observed as simultaneous triggering of apoptosis, pyroptosis, and necroptosis. However, its role in renal fibrosis is not known. We therefore hypothesize that eCIRP induced-chronic inflammation and injury in RTECs are mediated by PANoptosis and that inhibition of eCIRP by C23 decreases RTEC PANoptosis and attenuates renal injury and fibrosis in a mouse model of unilateral ureter obstruction (UUO) injury. By using primary RTECs, we demonstrated that eCIRP induces inflammatory cytokines, Z-DNA-binding protein-1, and other PANoptosome markers and markers of apoptosis, pyroptosis, and necroptosis. We then substantiated that C23 downregulated proinflammatory cytokines and inhibited PANoptosis in the RTECs. Using the UUO mouse model, we demonstrated renal cell PANoptosis and renal fibrosis 7 days after UUO. Importantly, treatment with C23 effectively inhibited PANoptosis and concurrently ameliorated renal fibrosis. Taken together, eCIRP induces inflammation and PANoptosis in RTECs, whereas C23 inhibits PANoptosis in these cells and attenuates renal fibrosis in UUO mice.NEW & NOTEWORTHY Renal fibrosis is a common pathological manifestation of chronic kidney disease (CKD). Extracellular cold-inducible RNA-binding protein (eCIRP) was discovered as a potent damage-associated molecular pattern (DAMP). eCIRP is linked to various types of programmed cell death. PANoptosis or synchronized cell death is observed as simultaneous triggering of apoptosis, pyroptosis, and necroptosis. Inhibiting eCIRP by C23, a small molecular peptide inhibitor of eCIRP, attenuated PANoptosis and renal fibrosis in CKD.

Ischemia-reperfusion injury (IRI) remains a critical challenge to the survival of kidney transplantation (KTX) graft, with no effective prevention or treatment strategies currently available. Neuronal nitric oxide synthase 1β (NOS1β), the predominant splice variant of NOS1 and the main source of NO in the macula densa (MD), mediates tubuloglomerular feedback and regulates glomerular filtration rates. NOS1β activity in the MD is influenced by renal pH; however, the role of pH-dependent regulation of NOS1β in mitigating IRI and protecting transplanted kidney graft function remains unclear. To explore this, C57BL/6J mice were given oral sodium bicarbonate (NaHCO₃) or NaCl for 2 wk before KTX. Blood and urine pH, NOS1β expression, NO levels, and transplant outcomes were evaluated. MD-specific NOS1 knockout (MD-NOS1KO) mice were used to assess the direct role of NOS1β. NOS1β expression decreased by approximately 60% 3 days after KTX. MD-NOS1β deletion exacerbated graft injury. NOS1β activities showed a strong tubular pH dependence, with maximal activity near pH 8.0. Bicarbonate treatment increased NOS1β expression in the MD by 65% and significantly improved graft outcomes, lowering plasma creatinine by ∼30% relative to NaCl-treated group. These protective effects were absent in MD-NOS1KO mice. Proteomic analysis revealed 718 differentially expressed proteins, with several showing enrichment in NO signaling, tissue repair, and inflammatory response pathways. In summary, MD-NOS1β downregulation after transplantation contributes to graft injury. Raising renal pH with bicarbonate enhances NOS1β activity and protects graft function, suggesting a potential therapeutic strategy to reduce IRI in kidney transplants.NEW & NOTEWORTHY This study reveals that raising renal tubular pH with oral bicarbonate enhances macula densa-specific NOS1β activity, protecting against ischemia-reperfusion injury in kidney transplants. These benefits are lost in macula densa NOS1β knockout mice, confirming its key role in graft protection. The findings suggest that modulating renal pH is a promising, noninvasive strategy to improve transplant outcomes by targeting macula densa-NOS1β.