Intrarenal Expression Research Articles

HydroZitLa inhibits calcium oxalate stone formation in nephrolithic rats and promotes longevity in nematode Caenorhabditis elegans

Low fluid intake, low urinary citrate excretion, and high oxidative stress are main causative factors of calcium oxalate (CaOx) nephrolithiasis. HydroZitLa contains citrate and natural antioxidants and is developed to correct these three factors simultaneously. Antioxidants theoretically can prolong the lifespan of organisms. In this study, we preclinically investigated the antilithogenic, lifespan-extending and anti-aging effects of HydroZitLa in HK-2 cells, male Wistar rats, and Caenorhabditis elegans. HydroZitLa significantly inhibited CaOx crystal aggregation in vitro and reduced oxidative stress in HK-2 cells challenged with lithogenic factors. For experimental nephrolithiasis, rats were divided into four groups: ethylene glycol (EG), EG + HydroZitLa, EG + Uralyt-U, and untreated control. CaOx deposits in kidneys of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. Intrarenal expression of 4-hydroxynonenal in EG + HydroZitLa rats was significantly lower than that of EG rats. The urinary oxalate levels of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. The urinary citrate levels of EG + HydroZitLa and EG + Uralyt-U rats were restored to the level in normal control rats. In C. elegans, HydroZitLa supplementation significantly extended the median lifespan of nematodes up to 34% without altering feeding ability. Lipofuscin accumulation in HydroZitLa-supplemented nematodes was significantly lower than that of non-supplemented control. Additionally, HydroZitLa inhibited telomere shortening, p16 upregulation, and premature senescence in HK-2 cells exposed to lithogenic stressors. Conclusions, HydroZitLa inhibited oxidative stress and CaOx formation both in vitro and in vivo. HydroZitLa extended the lifespan and delayed the onset of aging in C. elegans and human kidney cells. This preclinical evidence suggests that HydroZitLa is beneficial for inhibiting CaOx stone formation, promoting longevity, and slowing down aging.

Urinary Galectin-3 as a Novel Biomarker for the Prediction of Renal Fibrosis and Kidney Disease Progression.

Plasma galectin-3 (Gal-3) is associated with organ fibrosis, but whether urinary Gal-3 is a potential biomarker of kidney disease progression has never been explored. Between 2018 and 2021, we prospectively enrolled 280 patients who underwent renal biopsy and were divided into three groups based on their urinary Gal-3 levels (<354.6, 354.6–510.7, and ≥510.8 pg/mL) to assess kidney disease progression (defined as ≥40% decline in the estimated glomerular filtration rate or end-stage renal disease) and renal histology findings. Patients in the highest urinary Gal-3 tertile had the lowest eGFRs and highest proteinuria levels. In multivariate Cox regression models, patients in the highest tertile had the highest risk of kidney disease progression (adjusted hazard ratio, 4.60; 95% confidence interval, 2.85–7.71) compared to those in the lowest tertile. Higher urinary Gal-3 levels were associated with more severe renal fibrosis. Intrarenal mRNA expression of LGALS3 (Gal-3-encoded gene) was most correlated with the renal stress biomarkers (IGFBP7 and TIMB2), renal function biomarkers (PTGDS) and fibrosis-associated genes (TGFB1). The urinary Gal-3 level may be useful for the identification of patients at high risk of kidney disease progression and renal fibrosis, and for the early initiation of treatments for these patients.

Renal mTORC1 activation is associated with disease activity and prognosis in lupus nephritis.

This study was initiated to evaluate mammalian target of rapamycin (mTOR) activation in renal tissue of LN patients. This retrospective study included 187 LN patients, 20 diabetic nephropathy (DN) patients, 10 minimal change disease (MCD) patients and 10 normal controls (NCs). Seven of 187 LN patients had repeated renal biopsies. mTORC1/2 activation was evaluated by immunohistochemistry and multiplexed immunofluorescence. The association of mTORC1/2 activation with the clinicopathologic indices and prognostic outcomes was analysed among 187 LN patients. Proteomics was performed in renal biopsies of 20 LN patients. Proteomics was employed to comprehensively evaluate the impact of mTOR activation on intrarenal gene expression. mTORC1/2 was significantly activated in podocytes, mesangial cells, endothelial cells and tubular epithelial cells of LN patients as compared with those with MCD or NC. The glomerular mTORC1 activation was higher in LN patients compared with DN patients. mTORC1, but not mTORC2, activation strongly correlated with serum albumin, complement C3, proteinuria and the following pathological biomarkers of LN: crescent formation, interstitial inflammation and fibrosis. Moreover, mTORC1 activation was identified as a prognostic marker in LN patients. Bioinformatic analyses of proteomics and immunohistochemical data unveiled increased complement activation, antigen presentation and phagocytosis in LN patients with mTORC1 activation. Renal mTORC1 activation could be a biomarker to reveal disease activity and predict clinical prognosis in LN patients.

Renal Tissue miRNA Expression Profiles in ANCA-Associated Vasculitis—A Comparative Analysis

Anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) comprises autoimmune disease entities that cause target organ damage due to relapsing-remitting small vessel necrotizing vasculitis, and which affects various vascular beds. The pathogenesis of AAV is incompletely understood, which translates to considerable disease- and treatment-related morbidity and mortality. Recent advances have implicated microRNAs (miRNAs) in AAV; however, their accurate characterization in renal tissue is lacking. The goal of this study was to identify the intrarenal miRNA expression profile in AAV relative to healthy, non-inflammatory and inflammatory controls to identify candidate-specific miRNAs. Formalin-fixed, paraffin-embedded renal biopsy tissue samples from 85 patients were obtained. Comprehensive miRNA expression profiles were performed using panels with 752 miRNAs and revealed 17 miRNA that differentiated AAV from both controls. Identified miRNAs were annotated to characterize their involvement in pathways and to define their targets. A considerable subset of differentially expressed miRNAs was related to macrophage and lymphocyte polarization and cytokines previously deemed important in AAV pathogenesis, lending credence to the obtained results. Interestingly, several members of the miR-30 family were detected. However, a validation study of these differentially expressed miRNAs in an independent, larger sample cohort is needed to establish their potential diagnostic utility.

Protein-Bound Uremic Toxins Induce Reactive Oxygen Species-Dependent and Inflammasome-Mediated IL-1β Production in Kidney Proximal Tubule Cells.

Protein bound-uremic toxins (PBUTs) are not efficiently removed by hemodialysis in chronic kidney disease (CKD) patients and their accumulation leads to various co-morbidities via cellular dysfunction, inflammation and oxidative stress. Moreover, it has been shown that increased intrarenal expression of the NLRP3 receptor and IL-1β are associated with reduced kidney function, suggesting a critical role for the NLRP3 inflammasome in CKD progression. Here, we evaluated the effect of PBUTs on inflammasome-mediated IL-1β production in vitro and in vivo. Exposure of human conditionally immortalized proximal tubule epithelial cells to indoxyl sulfate (IS) and a mixture of anionic PBUTs (UT mix) increased expression levels of NLRP3, caspase-1 and IL-1β, accompanied by a significant increase in IL-1β secretion and caspase-1 activity. Furthermore, IS and UT mix induced the production of intracellular reactive oxygen species, and caspase-1 activity and IL-1β secretion were reduced in the presence of antioxidant N-acetylcysteine. IS and UT mix also induced NF-κB activation as evidenced by p65 nuclear translocation and IL-1β production, which was counteracted by an IKK inhibitor. In vivo, using subtotal nephrectomy CKD rats, a significant increase in total plasma levels of IS and the PBUTs, kynurenic acid and hippuric acid, was found, as well as enhanced urinary malondialdehyde levels. CKD kidney tissue showed an increasing trend in expression of NLRP3 inflammasome components, and a decreasing trend in superoxide dismutase-1 levels. In conclusion, we showed that PBUTs induce inflammasome-mediated IL-1β production in proximal tubule cells via oxidative stress and NF-κB signaling, suggesting their involvement in disease-associated inflammatory processes.

Abstract MP46: Mutagenesis Of PRR Cleavage Site In Mice Attenuates Angiotensin II-induced Hypertension And Suppresses Renin Expression

It is well demonstrated that activation of renal (pro)renin receptor (PRR) contributes to AngII-induced hypertension. Relatively, less is known about involvement of soluble PRR (sPRR), the extracellular domain of PRR, primarily generated by site-1 protease(S1P) and furin. Moreover, the relationship between PRR/sPRR and the renin-angiotensin system (RAS) has been debated. In the present study, we used CRISPR/Cas9 strategy to generate mice with mutagenesis of the overlapping cleavage site for both proteases in PRR (termed as PRR R279V/L282V ) to examine the phenotype during angiotensin II (AngII) infusion with particular emphasis on circulating and intrarenal renin status. PRR R279V/L282V mice exhibited a reduction of sPRR level in plasma by ~53% and in the kidney by ~82%, were fertile, and had no gross developmental abnormalities. At basal condition, PRR R279V/L282V mice had drastically suppressed renin levels from plasma (plasma total prorenin/renin content [ng/ml]: 9.3 ± 0.7 in PRR R279V/L282V mice vs. 12.8 ± 0.9 in WT mice, n = 5, p &lt; 0.05), urine (urinary total prorenin/renin excretion [ng/24h]: 0.54 ± 0.11 in PRR R279V/L282V mice vs. 1.05 ± 0.15 in WT mice, n = 5, p &lt; 0.05), and the kidney as compared to wild-type controls (WT). By telemetry, the hypertensive response of PRR R279V/L282V to AngII infusion (300 ng/min/kg) was blunted (MAP [mmHg] in Day 10: 125.9 ± 4.6 in PRR R279V/L282V mice vs. 138.5 ± 2.8 in WT mice, n = 8 for each group, p &lt; 0.05) in parallel with attenuated response of intrarenal renin and renal medullary α-ENaC expression. We further examined the direct role of sPRR in renin regulation in As4.1 cells, a renin-expressing cell line isolated from mouse renal tumor and M-1 cells, a mouse cell line derived from the collecting duct. The exposure to sPRR-His in both cell types consistently elevated renin activity, and renin expression at both protein and mRNA levels, all of which were sensitive to inhibition by ICG-001, a β-catenin signaling inhibitor. Together, these results represent strong evidence favoring sPRR as a mediator of AngII-induced hypertension and a master regulator of renin expression at systemic and local levels. Therefore, PRR should be considered as an integrative member of the RAS.

Tetrahydrocurcumin Ameliorates Kidney Injury and High Systolic Blood Pressure in High-Fat Diet-Induced Type 2 Diabetic Mice.

BackgroundActivation of the intrarenal renin-angiotensin system (RAS) is implicated in the pathogenesis of kidney injury and hypertension. We aimed to investigate the protective effect of tetrahydrocurcumin (THU) on intrarenal RAS expression, kidney injury, and systolic blood pressure (SBP) in high-fat diet (HFD)-induced type 2 diabetic mice.MethodsEight-week-old male mice were fed a regular diet (RD) or HFD for 12 weeks, and THU (50 or 100 mg/kg/day) was intragastrically administered with HFD. Physiological and metabolic changes were monitored and the expression of RAS components and markers of kidney injury were assessed.ResultsHFD-fed mice exhibited hyperglycemia, insulin resistance, and dyslipidemia compared to those in the RD group (P<0.05). Kidney injury in these mice was indicated by an increase in the ratio of albumin to creatinine, glomerular hypertrophy, and the effacement of podocyte foot processes. Expression of intrarenal angiotensin-converting enzyme, angiotensin II type I receptor, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4, and monocyte chemoattractant protein-1 was also markedly increased in HFD-fed mice. HFD-fed mice exhibited elevated SBP that was accompanied by an increase in the wall thickness and vascular cross-sectional area (P<0.05), 12 weeks post-HFD consumption. Treatment with THU (100 mg/kg/day) suppressed intrarenal RAS activation, improved insulin sensitivity, and reduced SBP, thus, attenuating kidney injury in these mice.ConclusionTHU alleviated kidney injury in mice with HFD-induced type 2 diabetes, possibly by blunting the activation of the intrarenal RAS/nicotinamide adenine dinucleotide phosphate oxidase IV (NOX4)/monocyte chemoattractant protein 1 (MCP-1) axis and by lowering the high SBP.

Connexin mRNA distribution in adult mouse kidneys

Kidneys are thought to express eight different connexin isoforms (i.e., Cx 26, 30, 32, 37, 40, 43, 45, and 46), which form either hemichannels or gap junctions serving to intercellular communication and functional synchronization. Proper function of connexins has already been shown to be crucial for regulation of renal hemodynamics and renin secretion, and there is also growing evidence for connexins to play a role in pathologic conditions such as renal fibrosis or diabetic nephropathy. Therefore, exact intrarenal localization of the different connexin isoforms gains particular interest. Until now intrarenal expression of connexins has mainly been examined by immunohistochemistry, which in part generated conflicting results depending on antibodies and fixation protocols used. In this work, we used fluorescent RNAscope as an alternative technical approach to localize renal connexin mRNAs in healthy mouse kidneys. Addition of RNAscope probes for cell type specific mRNAs was used to assign connexin mRNA signals to specific cell types. We hereby found Cx26 mRNA strongly expressed in proximal tubules, Cx30 mRNA was selectively detected in the urothelium, and Cx32 mRNA was found in proximal tubules and to a lesser extent also in collecting ducts. Cx37 mRNA was mainly associated with vascular endothelium, Cx40 mRNA was largely found in glomerular mesangial and less in vascular endothelial cells, Cx43 mRNA was sparsely expressed by interstitial cells of all kidney zones, and Cx45 mRNA was predominantly found in smooth muscle cell layers of both blood vessels and ureter as well as in mesangial and interstitial (fibroblastic) cells. Cx46 mRNA could not be detected. In summary our results essentially confirm previous data on connexin expression in the renal vasculature and in glomeruli. In addition, they demonstrate strong connexin gene expression in proximal tubules, and they suggest significant connexin expression in resident tubulointerstitial cells.

Renal denervation does not affect hypertension or the renin-angiotensin system in a rodent model of juvenile-onset polycystic kidney disease: clinical implications

We examined the effect of total and afferent renal denervation (RDN) on hypertension and the renin-angiotensin system (RAS) in a rodent model of juvenile-onset polycystic kidney disease (PKD). Lewis Polycystic Kidney (LPK) and control rats received total, afferent or sham RDN by periaxonal application of phenol, capsaicin or normal saline, respectively, and were monitored for 4-weeks. Afferent RDN did not affect systolic blood pressure (SBP) determined by radiotelemetry in either strain (n = 19) while total RDN significantly reduced SBP in Lewis rats 4-weeks post-denervation (total vs. sham, 122 ± 1 vs. 130 ± 2 mmHg, P = 0.002, n = 25). Plasma and kidney renin content determined by radioimmunoassay were significantly lower in LPK vs. Lewis (plasma: 278.2 ± 6.7 vs. 376.5 ± 11.9 ng Ang I/ml/h; kidney: 260.1 ± 6.3 vs. 753.2 ± 37.9 ng Ang I/mg/h, P < 0.001, n = 26). These parameters were not affected by RDN. Intrarenal mRNA expression levels of renin, angiotensinogen, angiotensin-converting enzyme (ACE)2, and angiotensin II receptor type 1a were significantly lower, whereas ACE1 expression was significantly higher in the LPK vs. Lewis (all P < 0.05, n = 26). This pattern of intrarenal RAS expression was not changed by RDN. In conclusion, RDN does not affect hypertension or the RAS in the LPK model and indicates RDN might not be a suitable antihypertensive strategy for individuals with juvenile-onset PKD.

MO328ASYMPTOMATIC HYPERURICEMIA ACTS AS ANTIOXIDANT DURING ACUTE KIDNEY INJURY AND DISEASE*

Abstract Background and Aims Acute kidney injury (AKI) and disease (AKD) are major causes of morbidity and mortality worldwide. Hyperuricemia (HU) is common in patients with impaired kidney function. While there is no doubt that crystalline uric acid (UA) causes acute and chronic UA nephropathy, urolithiasis and kidney stone disease, the pathogenesis of asymptomatic HU in AKI/AKD is incompletely understood. In animal studies, elevated serum UA levels may lead to endothelial dysfunction, renin-angiotensin system activation and oxidative stress. However, such models do not mimic human HU. To overcome this issue, we established a model of AKI/AKD with clinically relevant serum UA levels and hypothesized that asymptomatic HU improves the outcomes after AKI/AKD by restoring metabolic activity and mitochondrial biogenesis in macrophages and tubular epithelial cells. Method Alb-creERT2;Glut9lox/lox and Glut9lox/lox control mice were injected with tamoxifen and placed on a chow diet enriched with inosine. Hyperuricemic mice (serum UA ≥7 mg/dL) and mice without HU (serum UA 4-5 mg/dL) underwent uninephrectomy followed by unilateral ischemia-reperfusion (IR) to induce AKI/AKD. Serum and kidneys were collected on day 3 and 14 after AKI/AKD, and kidney function, tubular injury, inflammation, mitochondrial dysfunction, metabolic activity (fatty acid oxidation) and macrophage infiltration were quantified using GFR measurement, immunohistochemistry, colorimetric assays, electron microscopy, RT-PCR and flow cytometry. Results We observed an increase in serum UA levels from 7 to 10 mg/dL in hyperuricemic mice on day 3 after IR-induced AKI/AKD that returned to 7 mg/dL after 14 days (Figure left). While there was no difference in GFR between hyperuricemic and mice without HU with AKI/AKD on day 3, we found an improved kidney function in hyperuricemic mice on day 14 (Figure middle). This was associated with significantly less tubular injury and inflammation as well as an increase in the number of infiltrating anti-inflammatory M2-like macrophages as compared to mice without HU. Intrarenal mRNA expression level of the pro-oxidant heme-oxygenase-1 was reduced in hyperuricemic mice. However, the expression of anti-oxidant enzymes (Nrf-1 and Sod) and metabolic genes associated with fatty acid oxidation (Cpt1, Pparg, and Pgc1b) significantly increased as compared to mice without HU 14 days after AKI/AKD. In addition, HU increased the number of phospho-Histone-3 and intact proximal tubules and restored tubular mitochondrial morphology as indicated by an increased mitochondrial aspect ratio (Figure right). Conclusion Our data imply that asymptomatic HU improves kidney outcomes after IR-induced AKI/AKD because HU attenuates tubular injury and inflammation. In addition, we found that HU enhances the metabolic activity and anti-inflammatory M2-like macrophage polarization as well as restores mitochondrial biogenesis in tubular epithelial cells, suggesting that HU acts as antioxidant by improving kidney recovery after AKI/AKD.

Interaction between Renal Sodium‐Glucose Cotransporter 2 and Renin‐Angiotensin System in a PCOS Rat Model

Introduction : Polycystic Ovary Syndrome (PCOS) is characterized by androgen excess, oligo/anovulation, and polycystic ovaries. PCOS is co-prevalent with insulin resistance (IR), increased blood pressure (BP), and a dysfunctional renin-angiotensin system (RAS). The RAS has both a vasoconstrictive arm with angiotensin-converting enzyme (ACE) and a vasodilatory arm with ACE2. While PCOS patients require multiple medications to control their comorbidities, it is unknown how these medications interact with each other. ACE inhibitors (ACEi) and sodium-glucose cotransporter 2 inhibitors (SGLT2i) seem to influence each other in male rodents. However, how androgens influence these interactions in females is unknown. We have a well-established rat model that mimics PCOS, including IR, increased BP, and overactive RAS. Using this model, we tested the hypothesis that SGLT2i would attenuate the RAS and that ACEi would decrease renal SGLT2 expression in a rat model of PCOS. Methods : In Experiment 1, four-week old female Sprague Dawley (SD) rats implanted with DHT pellets (7.5mg/90 days) or control were randomized to drinking water with and without the SGLT2i empagliflozin at a dose of 10 mg/kg/day during the last 3 weeks of the study (n=10/group). At 3 months of DHT, fasting plasma was collected for the homeostatic model assessment of IR (HOMA-IR). BP was measured by radiotelemetry. Renal cortex and medulla were collected for quantitative real-time polymerase chain reaction (qRT-PCR) and western blot for ACE and ACE2. In Experiment 2, four-week old female SD rats implanted with DHT pellets (7.5mg/90 days) or control were randomized to drinking water with and without the ACEi enalapril at a dose of 250 mg/mL throughout the study (n=10/group). At 3 months of DHT, kidneys were collected for qRT-PCR for SGLT2. Results : PCOS rats had increased IR, BP, and renal expression of ACE and ACE2 compared to controls. SGLT2i had no impact on IR while causing a small reduction in BP in both groups. SGLT2i decreased cortical ACE mRNA and protein (1.03 ± 0.08 vs 1.37 ± 0.04, P<0.05) in PCOS, and had no impact in controls. SGLT2i also decreased cortical ACE2 protein (1.02 ± 0.05 vs 1.18 ± 0.03, P<0.05) in PCOS, but not in controls. SGLT2i decreased mRNA and protein of medullar ACE and ACE2 in both groups. In Experiment 2, cortical SGLT2 mRNA was increased in PCOS compared to controls. ACEi increased SGLT2 mRNA in controls (30.32 ± 8.03 vs 1.00 ± 0.32, P<0.01) with no effect in PCOS. Conclusions In summary, PCOS rats had increased IR, BP, and intrarenal ACE. Without impacting IR, SGLT2i attenuated both intrarenal ACE and ACE2 expression in PCOS. This suggests ACEi could work synergistically with SGLT2i to reduce BP. PCOS rats have increased SGLT2 mRNA, suggesting a differential response to SGLT2i between PCOS rats and controls, such as shown with cortical ACE. ACEi also increased cortical SGLT2 mRNA in controls, but androgen excess appeared to blunt this effect. Overall, this suggests that there could be a differential response in hyperandrogenemic women taking both SGLT2i and ACEi compared to a woman with normal androgen levels.

Genetic Background but Not Intestinal Microbiota After Co-Housing Determines Hyperoxaluria-Related Nephrocalcinosis in Common Inbred Mouse Strains.

Calcium oxalate (CaOx) crystal formation, aggregation and growth is a common cause of kidney stone disease and nephrocalcinosis-related chronic kidney disease (CKD). Genetically modified mouse strains are frequently used as an experimental tool in this context but observed phenotypes may also relate to the genetic background or intestinal microbiota. We hypothesized that the genetic background or intestinal microbiota of mice determine CaOx crystal deposition and thus the outcome of nephrocalcinosis. Indeed, Casp1 -/-, Cybb -/- or Casp1 -/-/Cybb -/- knockout mice on a 129/C57BL/6J (B6J) background that were fed an oxalate-rich diet for 14 days did neither encounter intrarenal CaOx crystal deposits nor nephrocalcinosis-related CKD. To test our assumption, we fed C57BL/6N (B6N), 129, B6J and Balb/c mice an oxalate-rich diet for 14 days. Only B6N mice displayed CaOx crystal deposits and developed CKD associated with tubular injury, inflammation and interstitial fibrosis. Intrarenal mRNA expression profiling of 64 known nephrocalcinosis-related genes revealed that healthy B6N mice had lower mRNA levels of uromodulin (Umod) compared to the other three strains. Feeding an oxalate-rich diet caused an increase in uromodulin protein expression and CaOx crystal deposition in the kidney as well as in urinary uromodulin excretion in B6N mice but not 129, B6J and Balb/c mice. However, backcrossing 129 mice on a B6N background resulted in a gradual increase in CaOx crystal deposits from F2 to F7, of which all B6N/129 mice from the 7th generation developed CaOx-related nephropathy similar to B6N mice. Co-housing experiments tested for a putative role of the intestinal microbiota but B6N co-housed with 129 mice or B6N/129 (3rd and 6th generation) mice did not affect nephrocalcinosis. In summary, genetic background but not the intestinal microbiome account for strain-specific crystal formation and, the levels of uromodulin secretion may contribute to this phenomenon. Our results imply that only littermate controls of the identical genetic background strain are appropriate when performing knockout mouse studies in this context, while co-housing is optional.

Angiotensin II up-regulates sodium-glucose co-transporter 2 expression and SGLT2 inhibitor attenuates Ang II-induced hypertensive renal injury in mice.

Clinical trials indicate that sodium/glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) improve kidney function, yet, the molecular regulation of SGLT2 expression is incompletely understood. Here, we investigated the role of the intrarenal renin-angiotensin system (RAS) on SGLT2 expression. In adult non-diabetic participants in the Nephrotic Syndrome Study Network (NEPTUNE, n=163), multivariable linear regression analysis showed SGLT2 mRNA was significantly associated with angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE) mRNA levels (P<0.001). In vitro, angiotensin II (Ang II) dose-dependently stimulated SGLT2 expression in HK-2, human immortalized renal proximal tubular cells (RPTCs); losartan and antioxidants inhibited it. Sglt2 expression was increased in transgenic (Tg) mice specifically overexpressing Agt in their RPTCs, as well as in WT mice with a single subcutaneous injection of Ang II (1.44 mg/kg). Moreover, Ang II (1000 ng/kg/min) infusion via osmotic mini-pump in WT mice for 4 weeks increased systolic blood pressure (SBP), glomerulosclerosis, tubulointerstitial fibrosis, and albuminuria; canaglifozin (Cana, 15 mg/kg/day) reversed these changes, with the exception of SBP. Fractional glucose excretion (FeGlu) was higher in Ang II+Cana than WT+Cana, whereas Sglt2 expression was similar. Our data demonstrate a link between intrarenal RAS and SGLT2 expression and that SGLT2i ameliorates Ang II-induced renal injury independent of SBP.

CD107a+ (LAMP-1) Cytotoxic CD8+ T-Cells in Lupus Nephritis Patients.

Cytotoxic CD8+ T-cells play a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). The aim of this study was to investigate the role of CD107a (LAMP-1) on cytotoxic CD8+ T-cells in SLE-patients in particular with lupus nephritis. Peripheral blood of SLE-patients (n = 31) and healthy controls (n = 21) was analyzed for the expression of CD314 and CD107a by flow cytometry. Kidney biopsies of lupus nephritis patients were investigated for the presence of CD8+ and C107a+ cells by immunohistochemistry and immunofluorescence staining. The percentages of CD107a+ on CD8+ T-cells were significantly decreased in SLE-patients as compared to healthy controls (40.2 ± 18.5% vs. 47.9 ± 15.0%, p = 0.02). This was even more significant in SLE-patients with inactive disease. There was a significant correlation between the percentages of CD107a+CD8+ T-cells and SLEDAI. The evaluation of lupus nephritis biopsies showed a significant number of CD107a+CD8+ T-cells mainly located in the peritubular infiltrates. The intrarenal expression of CD107a+ was significantly correlated with proteinuria. These results demonstrate that CD8+ T-cells of patients with systemic lupus erythematosus have an altered expression of CD107a which seems to be associated with disease activity. The proof of intrarenal CD107a+CD8+ suggests a role in the pathogenesis of lupus nephritis.

Urinary soluble CD90 predicts renal prognosis in patients with diabetic kidney disease.

BackgroundDiabetic kidney disease (DKD), the major cause of chronic kidney disease, is associated with progressive renal fibrosis. The expression of CD90 correlated with fibrogenesis. However, the association between urinary soluble CD90 and renal disease severity, and whether it predicts outcomes in patients with DKD are still unclear.MethodsUrinary sCD90 was measured in 285 patients with DKD in a longitudinal cohort. The composite endpoint was defined as end-stage renal disease (ESRD) or 40% reduction of estimated glomerular filtration rate (eGFR). The associations between urinary sCD90/Cr and clinical parameters, as well as renal outcomes were evaluated. Moreover, we detected the intrarenal CD90 expression, and demonstrated the correlation of intrarenal CD90 with clinico-pathological parameters.ResultsThe urinary sCD90 level of DKD patients is significantly higher than diabetes patients without kidney injuries and healthy controls. We further showed urinary sCD90/Cr had significantly correlations with eGFR (r=−0.373, P<0.001), uACR (r=0.303, P<0.001), serum creatinine (r=0.344, P<0.001), and the eGFR slope (r=−0.27, P<0.001). Elevated urinary sCD90/Cr was an independent risk factor for the composite endpoint, adjustment for potential confounders in DKD patients (HR 1.20, 95% CI: 1.04–1.38, P=0.015). However, the CD90 expression in the renal tubulointerstitial compartment in DKD patients was significantly lower than healthy controls, and showed significant negative correlations with the interstitial fibrosis and tubular atrophy score (IFTA) (r=−0.3, P=0.047), and urinary sCD90/Cr (r=−0.399, P=0.029).ConclusionsThis study provided evidence that urinary sCD90 could reflect the disease severity and serve as a valuable factor for renal outcome prediction in patients with DKD.

Reno-protective effect of IL-34 inhibition on cisplatin-induced nephrotoxicity in mice.

Interleukin-34 (IL-34) shares a receptor (cFMS) with colony stimulating factor-1 (CSF-1), and these two ligands mediate macrophage proliferation. However, in contrast to CSF-1, the influence of IL-34 on tubular epithelial cells (TECs) injury remains unclear. We investigated the physiological effects of IL-34 on TEC damage caused by cisplatin nephrotoxicity (CP-N). Mice were administered anti-mouse IL-34 antibody (anti-IL-34 Ab; 400 ng/kg) or vehicle from 1 day before and up to 2 days after CP-N induction. In vitro, mouse renal proximal TECs (MRPTEpiC) were cultured to analyze the inhibitory effects of IL-34 on CP-induced TEC apoptosis. Compared to vehicle treatment, anti-IL-34 Ab treatment significantly suppressed the intra-renal expression of IL-34 and its two receptors, cFMS and PTP-ζ, and significantly improved renal function, ameliorated tubulointerstitial injury, suppressed macrophage infiltration, and reduced apoptotic cell numbers in CP-N mice. It also significantly reduced the renal transcript levels of Kim-1, MIP-1/CCL3, TNF-α, and Bax in CP-N mice. Furthermore, anti-IL-34 Ab-treated CP-N mice showed less renal infiltration of F4/80+TNF-α+ cells. In vitro, stimulation with CP induced the expression of IL-34 and its two receptors in MRPTEpiC. Anti-IL-34 Ab treatment significantly suppressed CP-induced Bax expression with the degradation of ERK1/2 phosphorylation in damaged MRPTEpiC. IL-34 secreted from damaged TECs appeared to be involved in the progression of CP-N. Inhibition of IL-34 with neutralizing antibody directly prevented CP-induced TEC apoptosis by inhibiting the phosphorylation of ERK 1/2. Blocking of IL-34 appears to suppress the proliferation of cytotoxic macrophages, which indirectly attenuates CP-N. Thus, IL-34 represents a potential therapeutic target for TEC injury, and the inhibition of IL-34 might have a reno-protective effect.

Kidney microRNA-21 Expression and Kidney Function in IgA Nephropathy

Rationale & ObjectivePrevious studies have suggested that microRNA-21 (miR-21) plays an important role in kidney fibrosis. We examined the relationship between intrarenal miR-21 level and rate of kidney function loss in immunoglobulin A nephropathy (IgAN).Study DesignProspective cohort study.Setting & Participants40 patients with IgAN and 10 with hypertensive nephrosclerosis as controls.PredictorsmiR-21 levels in kidney biopsy specimen and urinary sediment, quantified as ratio to the housekeeping gene.OutcomesKidney event–free survival and rate of kidney function decline.Analytic ApproachTime-to-event and correlation analysis.ResultsThe IgAN group had significantly higher intrarenal miR-21 expression compared with the hypertensive nephrosclerosis group (1.71 [IQR, 0.99-2.77] vs 0.31 [IQR, 0.25-1.32]; P < 0.0001), but urinary miR-21 levels were similar. Intrarenal miR-21 expression had significant but modest correlation with severity of glomerulosclerosis (r = 0.293; P = 0.05) and tubulointerstitial fibrosis (r = 0.341; P = 0.03). Patients with high intrarenal miR-21 expression had significantly higher risk for developing kidney end points compared with those with low expression (log-rank test, P = 0.017). Univariate Cox analysis showed that intrarenal miR-21 expression significantly predicted the development of kidney end points (unadjusted HR, 1.586; 95% CI, 1.179-2.134; P = 0.002). However, the result was just short of statistical significance after adjusting for the severity of histologic damage (P = 0.06). There was also a significant correlation between intrarenal miR-21 expression and the slope of kidney function decline by univariate analysis (r = −0.399; P = 0.02).LimitationsSmall sample size; uncertain cellular origin of miR-21.ConclusionsWe found that intrarenal miR-21 expression is increased in patients with IgAN, modestly correlated with the severity of histologic damage, and predictive of subsequent kidney function loss.

MicroRNA-133a-Dependent Inhibition of Proximal Tubule Angiotensinogen by Renal TNF (Tumor Necrosis Factor).

We showed that intrarenal suppression of TNF (tumor necrosis factor) production under low salt (LS) conditions increases renal cortical AGT (angiotensinogen) mRNA and protein expression. Intrarenal injection of murine recombinant TNF attenuated increases of AGT in mice ingesting LS. Moreover, AGT mRNA and protein expression increased ≈6-fold and 2-fold, respectively, in mice ingesting LS that also received an intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6-TNF-ex4). Silencing of TNF under normal salt and high salt (HS) conditions also resulted in increased AGT expression. Since renal TNF production decreases in response to LS and increases in response to HS, the data suggest that alterations in TNF production under these conditions modulate the degree of AGT expression. We also tested the hypothesis that TNF inhibits intrarenal AGT expression by a mechanism involving miR-133a. Expression of miR-133a decreased in mice given LS and increased in response to HS for 7 days. Intrarenal silencing of TNF reversed the effects of HS on miR-133a-dependent AGT expression. In contrast, intrarenal TNF administration increased miR-133a expression in the kidney. Collectively, the data suggest that miR-133a is a salt-sensitive microRNA that inhibits AGT in the kidney and is increased by TNF. The HS-induced increase in blood pressure observed following silencing of TNF was markedly reduced upon intrarenal administration of miR-133a suggesting that intrinsic effects of TNF in the kidney to limit the blood pressure response to HS include an increase in miR-133a, which suppresses AGT expression.

B Cell Activating Factor (BAFF) Is Required for the Development of Intra-Renal Tertiary Lymphoid Organs in Experimental Kidney Transplantation in Rats.

Intra-renal tertiary lymphoid organs (TLOs) are associated with worsened outcome in kidney transplantation (Ktx). We used an anti-BAFF (B cell activating factor) intervention to investigate whether BAFF is required for TLO formation in a full MHC-mismatch Ktx model in rats. Rats received either therapeutic immunosuppression (no rejection, NR) or subtherapeutic immunosuppression (chronic rejection, CR) and were sacrificed on d56. One group additionally received an anti-BAFF antibody (CR + AB). Intra-renal T (CD3+) and B (CD20+) cells, their proliferation (Ki67+), and IgG+ plasma cells were analyzed by immunofluorescence microscopy. Formation of T and B cell zones and TLOs was assessed. Intra-renal expression of TLO-promoting factors, molecules of T:B crosstalk, and B cell differentiation was analyzed by qPCR. Intra-renal B and T cell zones and TLOs were detected in CR and were associated with elevated intra-renal mRNA expression of TLO-promoting factors, including CXCL13, CCL19, lymphotoxin-β, and BAFF. Intra-renal plasma cells were also elevated in CR. Anti-BAFF treatment significantly decreased intra-renal B cell zones and TLO, as well as intra-renal B cell-derived TLO-promoting factors and B cell differentiation markers. We conclude that BAFF-dependent intra-renal B cells promote TLO formation and advance local adaptive alloimmune responses in chronic rejection.

Angiotensin inhibition and cellular senescence in the developing rat kidney

Cellular senescence is important for the maintenance of tissue homeostasis during normal development. In this study, we aimed to investigate the effect of renin angiotensin system (RAS) blockade on renal cell senescence in the developing rat kidney. Newborn rat pups were treated with enalapril (30 mg/kg/day) or vehicle for seven days after birth. We investigated the intrarenal expressions of cell cycle regulators p21 and p16 with immunoblots and immunohistochemistry at postnatal day 8. For the determination of renal cellular senescence, immunostaining for senescence-associated β-galactosidase (SA-β-gal) and telomerase reverse transcriptase (TERT) was also performed. Enalapril treatment showed significant alterations in cellular senescence in neonatal rat kidneys. In the enalapril-treated group, intrarenal p16 and p21 protein expressions decreased compared to controls. The expressions of both p21 and p16 were reduced throughout the renal cortex and medulla of enalapril-treated rats. The immunoreactivity of TERT in enalapril-treated kidneys was also weaker than that in control kidneys. Control kidneys revealed a clear positive SA-β-gal signal in the cortical tubules; however, SA-β-gal activity was noticeably lower in the enalapril-treated kidneys than in control kidneys. Interruption of the RAS during postnatal nephrogenesis may disrupt physiologic renal cellular senescence in the developing rat kidney.