Tubular CPT1A deletion minimally affects aging and chronic kidney injury.

Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal ß-oxidation when mitochondrial FAO is impaired.

MicroRNA-5010-5p ameliorates high-glucose induced inflammation in renal tubular epithelial cells by modulating the expression of PPP2R2D.

INTRODUCTION: We previously reported the significant upregulation of eight circulating exosomal microRNAs (miRNAs) in patients with diabetic kidney disease (DKD). However, their specific roles and molecular mechanisms in the kidney remain unknown. Among the eight miRNAs, we evaluated the effects of miR-5010-5p on renal tubular epithelial cells under diabetic conditions in this study. RESEARCH DESIGN AND METHODS: We transfected the renal tubular epithelial cell line, HK-2, with an miR-5010-5p mimic using recombinant plasmids. The target gene of hsa-miR-5010-5p was identified using a dual-luciferase assay. Cell viability was assessed via the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Moreover, mRNA and protein expression levels were determined via real-time PCR and western blotting, respectively. RESULTS: High glucose levels did not significantly affect the intracellular expression of miR-5010-5p in HK-2 cells. Transfection of the miR-5010-5p mimic caused no change in cell viability. However, miR-5010-5p-transfected HK-2 cells exhibited significantly decreased expression levels of inflammatory cytokines, such as the monocyte chemoattractant protein-1, interleukin-1ß, and tumor necrosis factor-ɑ, under high-glucose conditions. These changes were accompanied by the restored expression of phosphorylated AMP-activated protein kinase (AMPK) and decreased phosphorylation of nuclear factor-kappa B. Dual-luciferase assay revealed that miR-5010-5p targeted the gene, protein phosphatase 2 regulatory subunit B delta (PPP2R2D), a subunit of protein phosphatase 2A, which modulates AMPK phosphorylation. CONCLUSIONS: Our findings suggest that increased miR-5010-5p expression reduces high glucose-induced inflammatory responses in renal tubular epithelial cells via the regulation of the target gene, PPP2R2D, which modulates AMPK phosphorylation. Therefore, miR-5010-5p may be a promising therapeutic target for DKD.

The Ip6k1 and Ip6k2 Kinases Are Critical for Normal Renal Tubular Function.

SIGNIFICANCE STATEMENT: Kidneys are gatekeepers of systemic inorganic phosphate balance because they control urinary phosphate excretion. In yeast and plants, inositol hexakisphosphate kinases (IP6Ks) are central to regulate phosphate metabolism, whereas their role in mammalian phosphate homeostasis is mostly unknown. We demonstrate in a renal cell line and in mice that Ip6k1 and Ip6k2 are critical for normal expression and function of the major renal Na + /Pi transporters NaPi-IIa and NaPi-IIc. Moreover, Ip6k1/2-/- mice also show symptoms of more generalized kidney dysfunction. Thus, our results suggest that IP6Ks are essential for phosphate metabolism and proper kidney function in mammals. BACKGROUND: Inorganic phosphate is an essential mineral, and its plasma levels are tightly regulated. In mammals, kidneys are critical for maintaining phosphate homeostasis through mechanisms that ultimately regulate the expression of the Na + /Pi cotransporters NaPi-IIa and NaPi-IIc in proximal tubules. Inositol pyrophosphate 5-IP 7 , generated by IP6Ks, is a main regulator of phosphate metabolism in yeast and plants. IP6Ks are conserved in mammals, but their role in phosphate metabolism in vivo remains unexplored. METHODS: We used in vitro (opossum kidney cells) and in vivo (renal tubular-specific Ip6k1/2-/- mice) models to analyze the role of IP6K1/2 in phosphate homeostasis in mammals. RESULTS: In both systems, Ip6k1 and Ip6k2 are responsible for synthesis of 5-IP 7 . Depletion of Ip6k1/2 in vitro reduced phosphate transport and mRNA expression of Na + /Pi cotransporters, and it blunts phosphate transport adaptation to changes in ambient phosphate. Renal ablation of both kinases in mice also downregulates the expression of NaPi-IIa and NaPi-IIc and lowered the uptake of phosphate into proximal renal brush border membranes. In addition, the absence of Ip6k1 and Ip6k2 reduced the plasma concentration of fibroblast growth factor 23 and increased bone resorption, despite of which homozygous males develop hypophosphatemia. Ip6k1/2-/- mice also show increased diuresis, albuminuria, and hypercalciuria, although the morphology of glomeruli and proximal brush border membrane seemed unaffected. CONCLUSIONS: Depletion of renal Ip6k1/2 in mice not only altered phosphate homeostasis but also dysregulated other kidney functions.

Assessment of osteopontin as an early nephrotoxicity indicator in human renal proximal tubule cells and its application in evaluating lanthanum-induced nephrotoxicity.

Nephrotoxicity is a common adverse effect induced by various chemicals, necessitating the development of reliable toxicity screening models for nephrotoxicity assessment. In this study, we assessed a group of nephrotoxicity indicators derived from different toxicity pathways, including conventional endpoints and kidney tubular injury biomarkers such as clusterin (CLU), kidney injury molecule-I (KIM-1), osteopontin (OPN), and neutrophil gelatinase-associated lipocalin (NGAL), using HK-2 and induced pluripotent stem cells (iPSCs)-derived renal proximal tubular epithelial-like cells (PTLs). Among the biomarkers tested, OPN emerged as the most discerning and precise marker. The predictive potential of OPN was tested using a panel of 10 nephrotoxic and 5 non-nephrotoxic compounds. The results demonstrated that combining OPN with the half-maximal inhibitory concentration (IC50) enhanced the diagnostic accuracy in both cellular models. Additionally, PTLs cells showed superior predictive efficacy for nephrotoxicity compared to HK-2 cells in this investigation. The two cellular models were utilized to evaluate the nephrotoxicity of lanthanum. The findings indicated that lanthanum possesses nephrotoxic properties; however, the degree of nephrotoxicity was relatively low, consistent with the outcomes of in vivo experiments.

Corin protects against acute kidney injury in mice through anti-inflammatory effects.

Corin is a type II transmembrane serine protease mainly expressed in the heart. Recently, corin was detected in the kidney and was reported to be associated with multiple kidney diseases. To date, its effect on acute kidney injury (AKI) has not been clarified. Here, we found that corin was constitutively expressed in renal tubules, especially in proximal and distal tubular epithelial cells. The expression of corin was dramatically reduced in ischemia/reperfusion injury (IRI)-induced AKI mouse model and oxygen-glucose deprivation (OGD)-induced human proximal tubular epithelial (HK-2) cells injury model, suggesting a potential role of corin in AKI. Corin deficient mice exhibited aggravated renal injury in AKI, as indicated by higher elevation of serum creatinine (SCr) and blood urea nitrogen (BUN), more severe tubular damage, and increased cell death versus wild type mice, demonstrating a protective effect of corin on AKI. In vitro overexpression of corin didn't directly alleviate hypoxia-induced HK-2 cells death, revealing that the protective effect of corin against AKI is not due to direct protection of tubular epithelial cells but may be through indirect protection. Microarray analysis showed enhanced inflammatory chemokines signaling and leukocyte chemotaxis in corin-/- mice after AKI, identifying an important role of corin in halting leukocyte chemotaxis and inflammatory response. Consistently, corin-/- mice after AKI displayed increased tubulointerstitial neutrophils and macrophages infiltration, as well as higher inflammatory mediators in kidneys. Taken together, our study indicates that tubular corin exerts a protective effect against AKI through negative regulation of chemotaxis signaling and inflammation in the kidney.

Role of Angiotensin II Type 1a Receptor (AT1aR) of Renal Tubules in Regulating Inwardly Rectifying Potassium Channels 4.2 (Kir4.2), Kir4.1, and Epithelial Na+ Channel (ENaC).

BACKGROUND: Kir4.2 and Kir4.1 play a role in regulating membrane transport in the proximal tubule (PT) and in the distal-convoluted-tubule (DCT), respectively. METHODS: We generated kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) mice to examine whether renal AT1aR regulates Kir4.2 and Kir4.1. RESULTS: Ks-AT1aR-KO mice had a lower systolic blood pressure than Agtr1aflox/flox (control) mice. Ks-AT1aR-KO mice had a lower expression of NHE3 (Na+/H+-exchanger 3) and Kir4.2, a major Kir-channel in PT, than Agtr1aflox/flox mice. Whole-cell recording also demonstrated that the membrane potential in PT of Ks-AT1aR-KO mice was lesser negative than Agtr1aflox/flox mice. The expression of Kir4.1 and Kir5.1, Kir4.1/Kir5.1-mediated K+ currents of DCT and DCT membrane potential in Ks-AT1aR-KO mice, were similar to Agtr1aflox/flox mice. However, angiotensin II perfusion for 7 days hyperpolarized the membrane potential in PT and DCT of the control mice but not in Ks-AT1aR-KO mice, while angiotensin II perfusion did not change the expression of Kir4.1, Kir4.2, and Kir5.1. Deletion of AT1aR did not significantly affect the expression of αENaC (epithelial Na+ channel) and ßENaC but increased cleaved γENaC expression. Patch-clamp experiments demonstrated that deletion of AT1aR increased amiloride-sensitive Na+-currents in the cortical-collecting duct but not in late-DCT. However, tertiapin-Q sensitive renal outer medullary potassium channel currents were similar in both genotypes. CONCLUSIONS: AT1aR determines the baseline membrane potential of PT by controlling Kir4.2 expression/activity but AT1aR is not required for determining the baseline membrane potential of the DCT and Kir4.1/Kir5.1 activity/expression. However, AT1aR is required for angiotensin II-induced hyperpolarization of basolateral membrane of PT and DCT. Deletion of AT1aR had no effect on baseline renal outer medullary potassium channel activity but increased ENaC activity in the CCD.

Mitochondrial Dysfunction in Kidney Tubulopathies.

Mitochondria play a key role in kidney physiology and pathology. They produce ATP to fuel energy-demanding water and solute reabsorption processes along the nephron. Moreover, mitochondria contribute to cellular health by the regulation of autophagy, (oxidative) stress responses, and apoptosis. Mitochondrial abundance is particularly high in cortical segments, including proximal and distal convoluted tubules. Dysfunction of the mitochondria has been described for tubulopathies such as Fanconi, Gitelman, and Bartter-like syndromes and renal tubular acidosis. In addition, mitochondrial cytopathies often affect renal (tubular) tissues, such as in Kearns-Sayre and Leigh syndromes. Nevertheless, the mechanisms by which mitochondrial dysfunction results in renal tubular diseases are only scarcely being explored. This review provides an overview of mitochondrial dysfunction in the development and progression of kidney tubulopathies. Furthermore, it emphasizes the need for further mechanistic investigations to identify links between mitochondrial function and renal electrolyte reabsorption.

ACSS2 gene variants determine kidney disease risk by controlling de novo lipogenesis in kidney tubules.

Worldwide, over 800 million people are affected by kidney disease, yet its pathogenesis remains elusive, hindering the development of novel therapeutics. In this study, we used kidney-specific expression of quantitative traits and single-nucleus open chromatin analysis to show that genetic variants linked to kidney dysfunction on chromosome 20 target the acyl-CoA synthetase short-chain family 2 (ACSS2). By generating ACSS2-KO mice, we demonstrated their protection from kidney fibrosis in multiple disease models. Our analysis of primary tubular cells revealed that ACSS2 regulated de novo lipogenesis (DNL), causing NADPH depletion and increasing ROS levels, ultimately leading to NLRP3-dependent pyroptosis. Additionally, we discovered that pharmacological inhibition or genetic ablation of fatty acid synthase safeguarded kidney cells against profibrotic gene expression and prevented kidney disease in mice. Lipid accumulation and the expression of genes related to DNL were elevated in the kidneys of patients with fibrosis. Our findings pinpoint ACSS2 as a critical kidney disease gene and reveal the role of DNL in kidney disease.

Proenkephalin-A secreted by renal proximal tubules functions as a brake in kidney regeneration.

Organ regeneration necessitates precise coordination of accelerators and brakes to restore organ function. However, the mechanisms underlying this intricate molecular crosstalk remain elusive. In this study, the level of proenkephalin-A (PENK-A), expressed by renal proximal tubular epithelial cells, decreases significantly with the loss of renal proximal tubules and increased at the termination phase of zebrafish kidney regeneration. Notably, this change contrasts with the role of hydrogen peroxide (H2O2), which acts as an accelerator in kidney regeneration. Through experiments with penka mutants and pharmaceutical treatments, we demonstrate that PENK-A inhibits H2O2 production in a dose-dependent manner, suggesting its involvement in regulating the rate and termination of regeneration. Furthermore, H2O2 influences the expression of tcf21, a vital factor in the formation of renal progenitor cell aggregates, by remodeling H3K4me3 in renal cells. Overall, our findings highlight the regulatory role of PENK-A as a brake in kidney regeneration.

Fibrocystin/Polyductin releases a C-terminal fragment that translocates into mitochondria and suppresses cystogenesis.

Fibrocystin/Polyductin (FPC), encoded by PKHD1, is associated with autosomal recessive polycystic kidney disease (ARPKD), yet its precise role in cystogenesis remains unclear. Here we show that FPC undergoes complex proteolytic processing in developing kidneys, generating three soluble C-terminal fragments (ICDs). Notably, ICD15, contains a novel mitochondrial targeting sequence at its N-terminus, facilitating its translocation into mitochondria. This enhances mitochondrial respiration in renal epithelial cells, partially restoring impaired mitochondrial function caused by FPC loss. FPC inactivation leads to abnormal ultrastructural morphology of mitochondria in kidney tubules without cyst formation. Moreover, FPC inactivation significantly exacerbates renal cystogenesis and triggers severe pancreatic cystogenesis in a Pkd1 mouse mutant Pkd1V/V in which cleavage of Pkd1-encoded Polycystin-1 at the GPCR Proteolysis Site is blocked. Deleting ICD15 enhances renal cystogenesis without inducing pancreatic cysts in Pkd1V/V mice. These findings reveal a direct link between FPC and a mitochondrial pathway through ICD15 cleavage, crucial for cystogenesis mechanisms.

Key enzyme in charge of ketone reabsorption of renal tubular SMCT1 may be a new target in diabetic kidney disease.

OBJECTIVE: A ketogenic diet or mildly increased ketone body levels are beneficial for diabetic kidney disease (DKD) patients. Our previous study has found that sodium-coupled monocarboxylate transporter 1 (SMCT1), a key enzyme in charge of ketone reabsorption, possesses beneficial effects on the function of renal tubular epithelial cells (TECs) in energy crisis. Our present study is to investigate whether SMCT1 is important in maintaining the physiological function of renal tubular and plays a role in DKD. METHODS: We tested the expression of SMCT1 in kidney tissues from DKD patients receiving kidney biopsy as well as diabetes mice. We compared the difference of ß-hydroxybutyrate (ß-HB) levels in serum, urine and kidney tissues between diabetic mice and control. Using recombinant adeno-associated viral vector containing SMCT1 (encoded by Slc5a8 gene), we tested the effect of SMCT1 upregulation on microalbuminuria as well as its effects on mitochondrial energy metabolism in diabetic mice. Then we investigated the role of SMCT1 and its ß-HB reabsorption function in maintaining the physiological function of renal tubular using renal tubule-specific Slc5a8 gene knockout mice. Transcriptomes and proteomics analysis were used to explore the underlying mechanism. RESULTS: SMCT1 downregulation was found in DKD patients as well as in diabetic mice. Moreover, diabetic mice had a decreased renal ß-HB level compared with control, and SMCT1 upregulation could improve microalbuminuria and mitochondrial energy metabolism. In renal tubule-specific Slc5a8 gene knockout mice, microalbuminuria occurred early at 24 weeks of age, accompanied by ATP shortage and metabolic reprogramming in the kidney; however, supplementation with ß-HB precursor substance 1,3-butanediol in food alleviated kidney damage as well as energy metabolic reprogramming. CONCLUSIONS: Decreased SMCT1 expression and its ketone reabsorption function play an important role in the occurrence of DKD. SMCT1 may be a new promising target in treating DKD.

CD8 T Cell-Derived Exosomal miR-186-5p Elicits Renal Inflammation via Activating Tubular TLR7/8 Signal Axis.

T cells play an important role in the development of focal segmental glomerulosclerosis (FSGS). The mechanism underlying such T cell-based kidney disease, however, remains elusive. Here the authors report that activated CD8 T cells elicit renal inflammation and tissue injury via releasing miR-186-5p-enriched exosomes. Continuing the cohort study identifying the correlation of plasma level of miR-186-5p with proteinuria in FSGS patients, it is demonstrated that circulating miR-186-5p is mainly derived from activated CD8 T cell exosomes. Renal miR-186-5p, which is markedly increased in FSGS patients and mice with adriamycin-induced renal injury, is mainly delivered by CD8 T cell exosomes. Depleting miR-186-5p strongly attenuates adriamycin-induced mouse renal injury. Supporting the function of exosomal miR-186-5p as a key circulating pathogenic factor, intravenous injection of miR-186-5p or miR-186-5p-containing T cell exosomes results in mouse renal inflammation and tissue injury. Tracing the injected T cell exosomes shows their preferential distribution in mouse renal tubules, not glomerulus. Mechanistically, miR-186-5p directly activates renal tubular TLR7/8 signal and initiates tubular cell apoptosis. Mutating the TLR7-binding sequence on miR-186-5p or deleting mouse TLR7 largely abolishes renal tubular injuries induced by miR-186-5p or adriamycin. These findings reveal a causative role of exosomal miR-186-5p in T cell-mediated renal dysfunction.

MiR-132-3p activation aggravates renal ischemia-reperfusion injury by targeting Sirt1/PGC1alpha axis.

The pathogenesis of renal ischemic diseases remains unclear. In this study, we demonstrate the induction of microRNA-132-3p (miR-132-3p) in ischemic acute kidney injury (AKI) and cultured renal tubular cells under oxidative stress. miR-132-3p mimic increased apoptosis in renal tubular cells and enhanced ischemic AKI in mice, whereas miR-132-3p inhibition offered protective effects. We analyzed miR-132-3p target genes through bioinformatic analysis and Sirt1 was predicted as the target gene of miR-132-3p. Luciferase microRNA target reporter assay further verified Sirt1 as a direct target of miR-132-3p. In cultured tubular cells and mouse kidneys, IRI and H2O2 treatment repressed Sirt1 and PGC-1α/NRF2/HO-1 expression, whereas anti-miR-132-3p preserved Sirt1 and PGC-1α/NRF2/HO-1 expression. In renal tubular, Sirt1 inhibitor suppressed PGC1-1α/NRF2/HO-1 expression and aggravated tubular apoptosis. Together, the results suggest that miR-132-3p induction aggravates ischemic AKI and oxidative stress by repressing Sirt1 expression, and miR-132-3p inhibition offers renal protection and may be a potential therapeutic target.

A renal YY1-KIM1-DR5 axis regulates the progression of acute kidney injury.

Acute kidney injury (AKI) exhibits high morbidity and mortality. Kidney injury molecule-1 (KIM1) is dramatically upregulated in renal tubules upon injury, and acts as a biomarker for various renal diseases. However, the exact role and underlying mechanism of KIM1 in the progression of AKI remain elusive. Herein, we report that renal tubular specific knockout of Kim1 attenuates cisplatin- or ischemia/reperfusion-induced AKI in male mice. Mechanistically, transcription factor Yin Yang 1 (YY1), which is downregulated upon AKI, binds to the promoter of KIM1 and represses its expression. Injury-induced KIM1 binds to the ECD domain of death receptor 5 (DR5), which activates DR5 and the following caspase cascade by promoting its multimerization, thus induces renal cell apoptosis and exacerbates AKI. Blocking the KIM1-DR5 interaction with rationally designed peptides exhibit reno-protective effects against AKI. Here, we reveal a YY1-KIM1-DR5 axis in the progression of AKI, which warrants future exploration as therapeutic targets.

Markers of Kidney Tubule Dysfunction and Major Adverse Kidney Events.

BACKGROUND: Serum creatinine and albuminuria are primary markers of glomerular function and injury, respectively. Tubular secretion, acid-base homeostasis, protein reabsorption, among other tubular functions, are largely ignored. This mini-review aimed to discuss how two tubular functions, secretion, and acid-base homeostasis are associated with major adverse kidney events (MAKEs). SUMMARY: Proximal tubular secretion is an essential function that allows the elimination of endogenous substances and drugs. Recently discovered endogenous markers in urine and plasma allow a noninvasive way of assessing tubular secretion markers. Several studies have found an association between these markers and a higher risk of chronic kidney disease (CKD) progression and mortality. In a study we recently performed among patients with CKD and at risk of cardiovascular events, lower tubular secretion was associated with an increased risk of acute kidney injury and metabolic acidosis, independent of baseline eGFR and albuminuria. The kidney tubules also play a crucial role in acid-base homeostasis. Although the standard clinical assessment of acidosis consists of measuring serum bicarbonate, urinary ammonium excretion decreases before over metabolic acidosis. Urinary ammonium excretion is associated with CKD progression, a higher risk of kidney failure, and an increased mortality risk, independent of baseline eGFR and albuminuria. KEY MESSAGES: Novel biomarkers of kidney tubular health consistently associate with MAKEs, above and beyond baseline eGFR, albuminuria, and other CKD risk factors. Tubular markers may provide new opportunities to improve kidney prognosis, drug dosing, and monitoring for adverse events.

Rap1 regulates lumen continuity via Afadin in renal epithelia.

The continuity of a lumen within an epithelial tubule is critical for its function. We previously found that the F-actin binding protein Afadin is required for timely lumen formation and continuity in renal tubules formed from the nephrogenic mesenchyme in mice. Afadin is a known effector and interactor of the small GTPase Rap1, and in the current study, we examine the role of Rap1 in nephron tubulogenesis. Here, we demonstrate that Rap1 is required for nascent lumen formation and continuity in cultured 3D epithelial spheroids and in vivo in murine renal epithelial tubules derived from the nephrogenic mesenchyme, where its absence ultimately leads to severe morphogenetic defects in the tubules. By contrast, Rap1 is not required for lumen continuity or morphogenesis in renal tubules derived from the ureteric epithelium, which differ in that they form by extension from a pre-existing tubule. We further demonstrate that Rap1 is required for correct localization of Afadin to adherens junctions both in vitro and in vivo. Together, these results suggest a model in which Rap1 localizes Afadin to junctional complexes, which in turn regulates nascent lumen formation and positioning to ensure continuous tubulogenesis.

Lower Circulating Cytotoxic T-Cell Frequency and Higher Intragraft Granzyme-B Expression Are Associated with Inflammatory Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Recipients.

Background and Objectives: Inflammatory interstitial fibrosis and tubular atrophy (i-IFTA) is an inflammation in the area of tubular atrophy and fibrosis. i-IFTA is poorly associated with graft outcome and associated with infiltration of inflammatory mononuclear cells. A cytotoxic T cell is a granzyme B+CD8+CD3+ T cell, mainly secret granzyme B. Granzyme B is a serine protease that may mediate allograft injury and inflammatory interstitial fibrosis and tubular atrophy (i-IFTA). However, there is no report identifying the association of granzyme B with i-IFTA after a long post-transplant interval. Material and Methods: In this study, we have measured the cytotoxic T-cell frequency with flow cytometry, serum and PBMCs culture supernatants granzyme-B levels with ELISA and intragraft granzyme-B mRNA transcript expression with the RT-PCR in RTRs in 30 patients with biopsy-proven i-IFTA and 10 patients with stable graft function. Result: The frequency of cytotoxic T cells (CD3+CD8+ granzyme B+) in SGF vs. i-IFTA was (27.96 ± 4.86 vs. 23.19 ± 3.85%, p = 0.011), the serum granzyme-B level was (100.82 ± 22.41 vs. 130.32 ± 46.60, p = 0.038 pg/mL) and the intragraft granzyme-B mRNA transcript expression was (1.01 ± 0.048 vs. 2.10 ± 1.02, p < 0.001 fold). The frequency of CD3+ T cells in SGF vs. i-IFTA was (66.08 ± 6.8 vs. 65.18 ± 9.35%; p = 0.68) and that of CD3+CD8+ T cells was (37.29 ± 4.11 vs. 34.68 ± 5.43%; p = 0.28), which were similar between the 2 groups. CTLc frequency was negatively correlated with urine proteinuria (r = -0.51, p < 0.001), serum creatinine (r = -0.28, p = 0.007) and eGFR (r = -0.28, p = 0.037). Similarly, the PBMC culture supernatants granzyme-B level was negatively correlated with urine proteinuria (r = -0.37, p < 0.001) and serum creatinine (r = -0.31, p = 0.002), while the serum granzyme-B level (r = 0.343, p = 0.001) and intragraft granzyme-B mRNA transcript expression (r = 0.38, p < 0.001) were positively correlated with proteinuria. Conclusions: A decrease in the CTLc frequency in circulation and an increased serum granzyme-B level and intragraft granzyme-B mRNA expression shows that cytotoxic T cells may mediate the allograft injury in RTRs with i-IFTA by releasing granzyme B in serum and intragraft tissue.

Localization of Diacylglycerol Kinase ζ in the Kidney of Adult Rats: Its Dominant Expression in Collecting Tubules and its Selective Lack in Proximal Tubules / Localización de Diacilglicerol Quinasa ζ en el Riñón de Ratas Adultas: su Expresión Dominante en Túbulos Colectores y su Carencia Selectiva en Túbulos Proximales

SUMMARY: Diacylglycerol kinase (DGK) exerts balancing the intracellular level between two-second messengers, diacylglycerol and phosphatidic acid, by its phosphorylation activity. DGK ζ is often localized in cell nuclei, suggesting its involvement in the regulation of intranuclear activities, including mitosis and apoptosis. The present immunohistochemical study of rat kidneys first revealed no detection levels of DGK ζ -immunoreactivity in nuclei of most proximal tubule epithelia in contrast to its distinct occurrence in cell nuclei of collecting and distal tubules with the former more dominant. This finding suggests that DGK ζ is a key factor regulating vulnerability to acute kidney injury in various renal tubules: its low expression represents the high vulnerability of proximal tubule cells, and its distinct expression does the resistance of collecting and distal tubule cells. In addition, this isozyme was more or less localized in nuclei of cells forming glomeruli as well as in endothelial nuclei of peritubular capillaries and other intrarenal blood vessels, and epithelial nuclei of glomerular capsules (Bowman's capsules) and renal calyces, including intrarenal interstitial cells.

La diacilglicerol quinasa (DGK) ejerce el equilibrio del nivel intracelular entre dos segundos mensajeros, diacilglicerol y ácido fosfatídico, por su actividad de fosforilación. La DGK ζ a menudo se localiza en los núcleos celulares, lo que sugiere su participación en la regulación de las actividades intranucleares, incluidas la mitosis y la apoptosis. El presente estudio inmunohistoquímico en riñones de rata no reveló niveles de detección de inmunorreactividad de DGK ζ en los núcleos de la mayoría de los epitelios de los túbulos proximales, en contraste a la detección en los núcleos celulares de los túbulos colectores y distales, siendo el primero más dominante. Este hallazgo sugiere que DGK ζ es un factor clave que regula la vulnerabilidad a la lesión renal aguda en varios túbulos renales: su baja expresión representa la alta vulnerabilidad de las células del túbulo proximal, y su expresión distinta hace a la resistencia de las células del túbulo colector y distal. Además, esta isoenzima estaba más o menos localizada en los núcleos de las células que forman los glomérulos, así como en los núcleos endoteliales de los capilares peritubulares y otros vasos sanguíneos intrarrenales, y en los núcleos epiteliales de las cápsulas glomerulares (cápsulas de Bowman) y los cálices renales, incluidas las células intersticiales intrarrenales.

TRPV4 expression in the renal tubule is necessary for maintaining whole body K+ homeostasis.

The Ca2+-permeable transient receptor potential vanilloid type 4 (TRPV4) channel serves as the sensor of tubular flow, thus being well suited to govern mechanosensitive K+ transport in the distal renal tubule. Here, we directly tested whether the TRPV4 function is significant in affecting K+ balance. We used balance metabolic cage experiments and systemic measurements with different K+ feeding regimens [high (5% K+), regular (0.9% K+), and low (<0.01% K+)] in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4fl/fl-Pax8Cre) and their littermate controls (TRPV4fl/fl). Deletion was verified by the absence of TRPV4 protein expression and lack of TRPV4-dependent Ca2+ influx. There were no differences in plasma electrolytes, urinary volume, and K+ levels at baseline. In contrast, plasma K+ levels were significantly elevated in TRPV4fl/fl-Pax8Cre mice on high K+ intake. K+-loaded knockout mice exhibited lower urinary K+ levels than TRPV4fl/fl mice, which was accompanied by higher aldosterone levels by day 7. Moreover, TRPV4fl/fl-Pax8Cre mice had more efficient renal K+ conservation and higher plasma K+ levels in the state of dietary K+ deficiency. H+-K+-ATPase levels were significantly increased in TRPV4fl/fl-Pax8Cre mice on a regular diet and especially on a low-K+ diet, pointing to augmented K+ reabsorption in the collecting duct. Consistently, we found a significantly faster intracellular pH recovery after intracellular acidification, as an index of H+-K+-ATPase activity, in split-opened collecting ducts from TRPV4fl/fl-Pax8Cre mice. In summary, our results demonstrate an indispensable prokaliuretic role of TRPV4 in the renal tubule in controlling K+ balance and urinary K+ excretion during variations in dietary K+ intake. NEW & NOTEWORTHY The mechanoactivated transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in distal tubule segments, where it controls flow-dependent K+ transport. Global TRPV4 deficiency causes impaired adaptation to variations in dietary K+ intake. Here, we demonstrate that renal tubule-specific TRPV4 deletion is sufficient to recapitulate the phenotype by causing antikaliuresis and higher plasma K+ levels in both states of K+ load and deficiency.

Chilling injury in human kidney tubule cells after subzero storage is not mitigated by antifreeze protein addition.

By preventing freezing, antifreeze proteins (AFPs) can permit cells and organs to be stored at subzero temperatures. As metabolic rates decrease with decreasing temperature, subzero static cold storage (SZ-SCS) could provide more time for tissue matching and potentially lead to fewer discarded organs. Human kidneys are generally stored for under 24 h and the tubule epithelium is known to be particularly sensitive to static cold storage (SCS). Here, telomerase-immortalized proximal-tubule epithelial cells from humans, which closely resemble their progenitors, were used as a proxy to assess the potential benefit of SZ-SCS for kidneys. The effects of hyperactive AFPs from a beetle and Cryostasis Storage Solution were compared to University of Wisconsin Solution at standard SCS temperatures (4 °C) and at -6 °C for up to six days. Although the AFPs helped guard against freezing, lower storage temperatures under these conditions were not beneficial. Compared to cells at 4 °C, those stored at -6 °C showed decreased viability as well as increased lactate dehydrogenase release and apoptosis. This suggests that this kidney cell type might be prone to chilling injury and that the addition of AFPs to enable SZ-SCS may not be effective for increasing storage times.