Lumen Of Proximal Tubules Research Articles

Analysis of thioredoxin 1 levels in a mouse model of unilateral renal ischemia-reperfusion injury

Kidney disease, acute and chronic, is associated with high morbidity and mortality. Currently, a major obstacle in the field of nephrology is the lack of novel and more sensitive biomarkers that would allow an early diagnose and a proper monitoring of disease progression. Oxidative stress and the increased production of reactive oxygen species (ROS) have been implicated in the pathophysiology of a variety of renal diseases. Proteins of the thioredoxin (Trx) family, among others thioredoxins (Trxs) and glutaredoxins (Grxs), represent one of the most important protective systems in the defense against ROS as well as in the repair of their intracellular effects. Thioredoxin 1 (Trx1) is a cytosolic redox protein that catalyzes the reduction of protein disulfides using the thiol groups of two cysteine residues on its CGPC active site. Trx1 is abundantly expressed in different segments of the nephron and has been shown to be released from cells under conditions of oxidative stress in vitro and in vivo. We hypothesized that renal IRI induces the release of Trx1 from cells into extracellular fluids such as serum and tubular fluid/urine and that Trx1 increase may correlate with the degree of damage in the kidney. We established an effective mouse model of unilateral renal ischemia-reperfusion injury (IRI) and studied Trx1 levels in the serum, urine, as well as in various nephron segments of mice following IRI or sham surgery. We compared different reperfusion times (4h, 8h, 16h, 24h, 48h, 96h, and 168h) to track Trx1 levels fluctuation over time. Serum levels of Trx1 increased 4h after renal IRI, while other markers such as blood urea nitrogen and creatinine remained low. Serum Trx1 showed a positive correlation with tubular injury score helping predict intrarenal damage over time. Additionally, immunohistochemistry studies revealed an increase in Trx1 signals in the proximal tubules and collecting ducts as well as a strong nuclear localization in the same nephron segments that became more intense by 96h, a time point characterized by an intense mitotic activity in the tubules. Between 16 and 24h, Trx1 appeared in the lumen of proximal tubules, however urinary levels of Trx1 did not significantly increase. Based on these findings we conclude that serum Trx1 may represent a novel biomarker for assessing renal damage. In addition to this, we hypothesize that intrarenal changes in Trx1 expression and localization might be related to the activation of regeneration mechanisms in the renal tubular epithelium. Further studies are needed to understand the role of Trx1 in the regeneration process after renal IRI. This work was funded by the Center of Integrative Mammalian Research of Ross University School of Veterinary Medicine and by a Startup funding from Long Island University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Impact of Uric Acid on Human Health: Beyond Gout and Kidney Stones

AbstractIn most primates, including humans, uric acid (UA) is the end product of purine metabolism due to the loss of hepatic uricase activity during evolution. This loss resulted in higher serum urate concentrations (3.5–7.5 mg/dL) than normally observed in other mammals (0.05–2 mg/dL). About 70% of the daily urate burden is eliminated via the kidneys and the remainder via the intestines, where gut bacteria break it down. Urate is freely filtered through the glomerular capillaries, and most of the filtered urate is reabsorbed so that only an amount equivalent to about 10% of the filtered load is excreted in the urine. Virtually all of the renal urate reabsorption takes place in proximal convoluted tubules. Many transport proteins connected with urate have been identified. However, the best studied are URAT1 and GLUT9, which function in concert to translocate urate from the proximal tubule lumen to the peritubular fluid, the first in the apical membrane and the second in the basolateral membrane. Genetic mutations, as well as drugs that alter the function of these transporters, can affect urate homeostasis resulting in abnormal serum levels, which may, in turn, be involved in the pathogenesis of chronic metabolic and inflammatory diseases, including most features of the metabolic syndrome, hypertension, cardiovascular disease, and chronic kidney disease. Several mechanisms are thought to provide the link between urate and these disorders, including reactive oxygen species (oxidative stress) and both acute and chronic inflammation. This mini-review summarizes the basic human biology of UA and its association with and potential involvement in developing chronic diseases beyond gout and nephrolithiasis.

Nephroprotection of p-coumaric acid against sublethal dose of carbon tetrachloride in Wistar rats: histological evidence

In Mexico, chronic kidney disease is a public health problem, when is diagnosed in advanced stages, the only treatment options are dialysis, hemodialysis or organ transplantation, however, the health system does not have the economic capacity or the infrastructure to fully cover these treatments. Therefore, the objective of this research work was to evaluate the activity of the p-coumaric acid (pCA) as a possible nephroprotective agent against toxic carbon tetrachloride (CCl4)-induced kidney damage in male Wistar rats. Renal parenchyma was evaluated using two stains, hematoxylin and eosin (H&E) and periodic acid with Schiff's reagent (PAS). The administration of CCl4 (4 g/kg, p.o., one dose) induced tubular necrosis and glomerular rupture within 24 h, with loss of microvilli and basement membranes, with widening of the lumen of the distal and proximal tubules. On the other hand, pCA (100 mg/kg, p.o., administered 24 h and 1 h before CCl4 and 1 h after this toxic agent) showed nephroprotective action by reducing the presence of these morphological changes. Our results suggest for the first time that pCA, when administered preventively, slows the deterioration of renal structure induced by acute exposure to a sublethal dose of CCl4.

Efficacy of orally administered montmorillonite in myoglobinuric acute renal failure model in male rats.

Acute kidney injury can be associated with serious consequences and therefore early treatment is critical to decreasing mortality and morbidity rate. We evaluated the effect of montmorillonite, the clay with strong cation exchange capacity, on the AKI model in rats. Glycerol (50% solution, 10 ml/kg) was injected in the rat hind limbs to induce AKI. 24 hr after induction of acute kidney injury, the rats received oral doses of montmorillonite (0.5 g/kg or 1 g/kg), or sodium polystyrene sulfonate (1 g/kg) for three consecutive days. Glycine induced acute kidney injury in rats with high levels of urea (336.60± 28.19 mg/dl), creatinine (4.10± 0.21 mg/dl), potassium (6.15 ± 0.28 mEq/L), and calcium (11.52 ± 0.19 mg/dl). Both doses of montmorillonite (0.5 and 1 g/kg) improved the serum urea (222.66± 10.02 and 170.20±8.06, P<0.05), creatinine (1.86±0.1, 2.05± 0.11, P<0.05), potassium (4.68 ± 0.4, 4.73 ± 0.34, P<0.001) and calcium (11.15 ± 0.17, 10.75 ± 0.25, P<0.01) levels. Treatment with montmorillonite especially at a high dose reduced the kidney pathological findings including, tubular necrosis, amorphous protein aggregation, and cell shedding into the distal and proximal tubule lumen. However, administration of SPS could not significantly decrease the severity of damages. According to the results of this study, as well as the physicochemical properties of montmorillonite, such as high ion exchange capacity and low side effects, montmorillonite can be a low-cost and effective treatment option to reduce and improve the complications of acute kidney injury. However, the efficacy of this compound in human and clinical studies needs to be investigated.

The Number of Calcium Oxalate Deposits Significantly Correlate with Elevated Serum Creatinine Levels in Calcium Oxalate Nephropathy

Abstract Introduction/Objective Calcium oxalate nephropathy is defined as acute and/or chronic decrease in renal function due to deposition of calcium oxalate crystals in renal tubules leading to acute tubular injury, acute or chronic interstitial nephritis, and elevated serum creatinine levels. However, the correlation between serum creatinine (SCr) levels and the number of calcium oxalate deposits has not been well described in the literature. Methods/Case Report Eight controls with mild to moderate acute tubular injury and 10 cases with calcium oxalate nephropathy (calcium group) were selected for the study. The patient’s SCr values were obtained from electronic medical records and the number of polarizable calcium oxalate crystal deposits in the lumen of proximal tubules seen on Hematoxylin &amp; Eosin stained slides at 40x high power field were recorded for each biopsy. Umpired student T-test was used to compare the two groups. Linear regression analysis was used to correlate SCr and the number of calcium oxalate deposits. Results (if a Case Study enter NA) Mean ages were not significantly different between two groups (49 ± 8 years in controls and 64 ± 6 years in calcium group). SCr was significantly higher in the calcium group (5.97 ± 0.68 mg/dl*) than in the control group (2.59 ± 0.37 mg/dl). No calcium oxalate deposits were seen in any of the controls, but the number of calcium oxalate deposits ranged from 3 to 7 in the calcium group (Table 1). In our study, we found a significant linear correlation between SCr and the number of crystals/high power field, when all cases were taken together (r value = 0.783, p value = 0.0002*). Conclusion Our data suggest that there is a direct association between the number of calcium oxalate crystals in proximal tubules with the severity of renal dysfunction. Although the etiologies for calcium oxalate nephropathy can be multifactorial, counting the deposit number on renal biopsies is simple method to predict SCr levels.

Meprin β meidates Apoptosis/Survival Through The IL‐6‐Mediated JAK/STAT Signaling Pathway In Ischemia/Reperfusion‐Induced Kidney Injury

Meprin metalloproteinases have been implicated in the pathophysiology of IR‐induced kidney injury. Existing in vitro data show that meprins could modulate interleukin‐6 (IL‐6)‐mediated inflammation via proteolytic processing of IL‐6 and its receptor. However, it is not known whether meprin β cleaves IL‐6 in vivo, and how inactivation of IL‐6 modulates downstream mediators of the IL‐6 signaling pathway in kidney tissue. The goal of the current study was to determine how meprin β expression affects IL‐6 and its correlation to kidney injury and cellular apoptosis/survival in mice kidneys subjected to IR. We used the unilateral IR as a model of renal inflammation in wild‐type (WT) and meprin β knockout (βKO) male mice, with the contralateral kidneys serving as controls. The mice were sacrificed at 24 h post‐IR, and kidney tissue processed for evaluation by RT‐PCR, western blot, and immunohistochemistry (IHC). IHC staining for kidney injury molecule 1 (KIM‐1) was used to assess kidney injury. Statistical analysis utilized 2‐way ANOVA. Real‐time PCR data showed a significant increase (P ≤ 0.0001) in mRNA levels for IL‐6, Caspase3, and BCL‐2 in both WT and βKO mice subjected to IR when compared to counterpart control kidneys. Western blot data showed that protein levels for IL‐6, P‐STAT3β, Caspase3, and BCL‐2 significantly increased in βKO kidneys (P ≤ 0.0001) but not in WT counterparts at 24 h post‐IR, suggesting meprin β‐mediated decreases in IL‐6, Caspase3 and BCL‐2. Interestingly, immunohistochemical staining of kidney sections for IL‐6, P‐JAK2 and P‐STAT3, Caspase3 and BCL‐2 showed significant increases only in select kidney tubules for both genotypes. In the renal corpuscles, P‐JAK2 levels significantly increased only in the βKO (P ≤ 0.0001) and not in WT kidneys at 24h post‐IR. In contrast, BCL‐2 expression in the renal corpuscle was significantly higher in WT kidneys subjected to IR compared to control kidneys (P ≤ 0.0001) but not in βKO mice. To assess the correlations in localization of IL‐6 expression and the kidney injury and cellular apoptosis in kidney tubules, we used immunofluorescence counterstaining of IL‐6 with either kidney injury biomarker, KIM‐1, or cellular apoptosis biomarker, Caspase 3. Our data showed that IL‐6 expression was positively associated with both KIM‐1 and Caspase 3, and thus kidney injury and cellular apoptosis in several tubules in both genotypes subjected to IR. Data from immunofluorescence counterstaining of kidney tissues also showed that the levels of IL‐6, Caspase3 and BCL‐2 were higher in meprin β‐expressing proximal tubules (PTs), at 24 h post‐IR when compared to the distal kidney tubules (DTs), which lack meprins. High levels of IL‐6, Caspase3 and BCL‐2 were also detected at 24 h post‐IR in the lumen of PTs and DTs from WT and βKO kidneys, suggesting increased release into filtrate and subsequently into urine. However, P‐JAK2 and P‐STAT3 expression were high in PTs of both genotypes with accumulation in the lumen of PTs in βKO kidneys only at 24 h post‐IR. In conclusion, data showed that Meprin β expression modulates IR‐induced kidney injury and cellular apoptosis/survival in part through IL6‐ mediated JAK/STAT signaling.

Meprin β Modulates Cellular Proliferation Through The IL‐6 Signaling Pathway In IRInduced Kidney Injury

Inflammation plays a central role in the progression of kidney injury induced by ischemia/reperfusion (IR). Interleukin‐6 (IL‐6) trans‐signaling has been shown to play a protective role by promoting repair processes in IR‐induced renal injury. Meprin metalloproteases have been implicated in inflammation‐induced kidney injury. Meprins proteolytically process the proinflammatory cytokine, IL‐6, causing IL‐6 inactivation. It was recently reported that meprins also cleave the IL‐6 receptor (IL‐6R). IL‐6 binds to its receptor (IL‐6R) in two ways, membrane bound (mbIL‐6R), activating the classic IL‐6 signaling pathway, or the soluble form (sIL‐6R), activating the IL‐6 trans‐signaling pathway. IL‐6 trans‐signaling induces proliferation through either MAPK/ERK or PI3K/AKT pathway or in crosstalk with AKT/ERK. We previously showed that meprin β modulates cellular survival (BCL‐2) through IL‐6/JAK/STAT signaling pathway in IRinduced kidney injury. However, it’s not known how meprin β modulation of the IL‐6 signaling pathway impacts the cellular proliferation in IR‐induced acute kidney injury. Previous studies have shown that IL‐6 binds to its receptor forming IL‐6/IL6R complex, which binds to the membranebound gp130 dimer. This leads to activation phosphorylation of ERK and AKT pathways, which in turn leads to induction of cellular proliferation. PCNA is a cellular proliferation marker that is induced through activation of the IL‐6 signaling pathway. The goal of the current study was to determine how meprin β modulation of the IL‐6 signaling pathway impacts downstream cellular proliferation in IR‐induced kidney injury. We used the unilateral IR as a model of renal inflammation in wild‐type (WT) and meprin β knockout (βKO) male mice, with the contralateral kidneys serving as controls. The mice were sacrificed at 96 h post‐IR, and kidney tissue processed for evaluation by RT‐PCR and immunohistochemistry (IHC). To determine staining intensity, the tissue sections were evaluated for IL‐6 and PCNA levels using light microscopy and imaged using Image J analysis Software. Statistical analysis of the optical density (OD) data utilized two‐way ANOVA. Our PCR data showed a significant increase in mRNA levels for IL‐6 and PCNA in WT and βKO mice (P ≤ 0.01) at 96 h‐post IR when compared to WT control kidneys. However, the baseline mRNA levels for PCNA were significantly higher in βKO (P ≤ 0.01) when compared to WT kidneys. Immunohistochemical data showed significant increases (P&lt;0.05) in IL‐6 and PCNA in select tubules in both genotypes at 96 h post‐IR when compared to control kidneys for each genotype. Data from immunofluorescence counterstaining of kidney tissues showed that the levels of IL‐6, and PCNA were higher in meprin β‐expressing proximal tubules (PTs), at 96 h post‐IR when compared to the distal kidney tubules (DTs), which lack meprins. High levels of IL‐6 were also present in the lumen of PTs and DTs from WT and βKO kidneys at 96 h post‐IR, suggesting increased release into filtrate and subsequently into urine. However, high levels of PCNA were present in the lumen of PTs only from both genotypes at 96 h post‐IR. In conclusion, our data shows that meprin β expression modulates cellular proliferation through the IL‐6 signaling pathway in IR‐induced kidney injury.

Meprin β mediates Osteopontin‐Signaling in IR‐Induced Acute Kidney Injury

Ischemia/reperfusion (IR) causes acute kidney injury (AKI) and is associated with high morbidity and mortality rates. Meprins metalloproteases are expressed abundantly in the brush border membranes of proximal kidney tubules and implicated in the progression of AKI. However, the underlying mechanisms are not fully understood. Meprins are capable to proteolytically processing osteopontin (OPN), a highly phosphorylated multifunctional and matricellular glycophosphoprotein. OPN mediates cell signaling pathways involved in apoptosis and extracellular matrix metabolism. The objective of the present study was to determine the effect of meprin β expression on OPN and downstream mediators of the OPN‐signaling pathway in IR‐induced renal injury. Surgical procedures were used to induce unilateral IR in wild‐type (WT) and meprin β knockout (βKO) mice. The mice were sacrificed at 24h post‐IR. Blood samples and kidney tissues were obtained for analysis. Neutrophil gelatinase associated lipocalin (NGAL) was used for biochemical assessment of kidney injury. Real‐time PCR, immunohistochemical staining, and western blot analysis were used to evaluate the levels of OPN, caspase‐3, Bcl‐2, and NFκB. Statistical analysis utilized 2‐way ANOVA. Our data show that NGAL levels were increased significantly in the kidney of WT (P=0.0001) and βKO (P=0.01) mice at 24h post‐IR when compared to their control counterparts. OPN mRNA increased in both genotypes at 24h post‐IR. Western blot analysis detected a full length OPN protein band at 66 kDa, with significant increases (p=0.001) in protein levels in WT kidney at 24h post‐IR when compared to levels in control kidneys. This increase in OPN was not observed in meprin βKO mice subjected to IR. Additionally, we detected a cleaved OPN fragment at 32 kDa, for which there were no significant changes in either genotype at 24h IR post‐IR. Immunohistochemical staining showed the increase in the levels of OPN occurred in select WT kidney tubules and in renal corpuscles. To determine whether meprin β expression correlates with the increased OPN, we used immunofluorescence counterstaining with proximal tubule (PT) markers (meprin β for WT and villin for βKO). Our data show that the increase in OPN occurred in both proximal and distal kidney tubules. Interestingly, higher levels of OPN were detected in the lumen of PTs from WT but not βKO kidneys, suggesting that meprin β is partly responsible for enhanced release of OPN into filtrate and ultimately into urine as previously reported. OPN functions as anti‐apoptotic protein in different diseases, therefore we evaluated its impact on caspase‐3, Bcl‐2, and NFκB. The mRNA levels for caspase‐3, Bcl‐2 and NFκB decreased in WT but increased in βKO mice at 24h post‐IR. Western blot detected higher levels of caspase‐3 and Bcl‐2 only in WT kidneys. Immunohistochemical staining for caspase‐3, Bcl‐2 and NFκB also showed higher levels in tubules and renal corpuscle of WT kidneys when compared to βKO kidneys at 24h post‐IR. Taken together, our data show that meprin β regulates OPN and down‐stream mediators of OPN‐signaling pathway in IR‐induced AKI.

Histopathological Impact of Carica Papaya Seed Extract in Rat Model of Gentamicin Nephrotoxicity

Background: Kidneys are susceptible to many commonly used drugs, owing to their property of filtering, reabsorbing, and excreting multiple drugs. Aim: To study the renoprotective effect of ethanolic seed extract of Carica papaya on renal histomorphology in nephrotoxicity caused by Gentamicin. Study design: Experimental study. Methodology: Rats (n=30) were subdivided into 3 equal groups. Group I (control group) and Group II &amp; III were administered Gentamicin (80 mg/kg) was given daily for 05 days continuously, intra-peritoneally to cause nephro-toxicity. Histomorphological examination of kidney tissue was done on day-6. In group-III, ethanolic extract of Carica papaya seeds (1000 mg/kg) was given daily for 5 days orally for its induction of nephro-protective effects. Data was entered and analyzed in computer using SPSS. Results: Acute Tubular Necrosis (ATN) and presence of Hyaline casts in the lumen of proximal tubules were the distinctive morphological characteristics. Histopathological specimens of the kidney in Ethanolic treated group showed moderate characteristics of renal damage as compared to that of the disease control group. Conclusion: Study concluded that that Carica papaya seed extract can significantly ameliorate the nephrotoxic effects of Gentamicin thus nephro-protective. Keywords: Gentamicin, Carica papaya, Nephrotoxicity and Histomorphology.

Meprin β Activity Modulates IL‐6/JAK/STAT Signaling Pathway in Ischemia/Reperfusion Induced Acute Kidney Injury

Meprin metalloproteases have been implicated in inflammation-induce kidney injury. Meprins proteolytically processes the pro-inflammatory cytokine, IL-6, causing IL-6 inactivation. However, it's not known how IL-6 inactivation by meprin β impact the inflammatory response in kidneys. The goal of this study was to determine whether meprin β expression modulates IL-6 signaling in ischemia/reperfusion (IR)-induced kidney injury. IL-6 trans-signaling is initiated when IL-6 binds to its soluble receptor (sIL-6r) and gp130 receptor, resulting in activation of Janus Kinase-Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway and activation of its mediators (e.g. suppressor of cytokine signaling 3, SOCS3) and downstream target genes (e.g. pro-apoptosis, anti-apoptosis and proliferation). We therefore sought to determine if meprin β expression impacts IL-6 levels and downstream mediators of the IL-6 trans-signaling mediated JAK-STAT pathway. We used the unilateral IR as a model of renal inflammation in wild-type (WT) and meprin β knockout (βKO) male mice, with the contralateral kidneys serving as controls. The mice were sacrificed at 24h post-IR, and kidney tissue processed for evaluation by RT-PCR, western blot, and immunohistochemistry (IHC). IHC staining for kidney injury molecule 1 (KIM-1) was used to assess kidney injury. Statistical analysis utilized two-way ANOVA. Our PCR data showed that meprin β deficiency associated with a significant upregulation of IL-6 mRNA (40%) in βKO mice at 24 h post-IR compared to WT. Similarly, Western blot data showed increased IL-6 protein levels. We then evaluated downstream mediators of the IL-6 signaling pathway and showed that WT kidneys which express meprin β had a 50% and 90% upregulation in the expression of the JAK/STAT negative regulator gene (SOCS3) and proliferating cell nuclear antigen (PCNA) gene, respectively when compared to βKO. However, meprin β expression did not affect the expression of the pro-apoptotic gene (BCL-2) or the anti-apoptotic gene (caspase3) at 24 h post-IR. Western Blot data showed increased levels of P-JAK2, P-STAT3, Caspase3, BCL2, SOCS3 and PCNA in βKO at 24 h post-IR compared to controls, but not in WT counterparts. Immunohistochemical analysis showed significant increases (P<0.001) in percentage of cells expressing IL-6, P-JAK2, P-STAT3, Caspase3 and BCL2 in some tubules in both genotypes subjected to IR when compared to their controls (OD levels >1.0). However, βKO kidneys had significantly higher expression level of IL-6, P-JAK2, P-STAT3, Caspase3 and BCL2 total proteins compared to WT 24h post-IR (P<0.001). Data from immunofluorescence counterstaining of kidney tissues showed that the levels of IL-6, P-STAT3, P-JAK-2, Caspase3 and BCL-2 were higher in meprin β-expressing proximal tubules (PT), at 24 h post-IR when compared to the distal kidney tubules (DT), which lack meprins. High level of IL-6, P-STAT3, P-JAK-2, Caspase3 and BCL-2 was also detected 24 post IR in the lumen of PT and DT from WT and βKO kidneys, suggesting increased release into filtrate and subsequently into urine. Taken together, our data demonstrates that meprin β expression modulates the IL-6/JAK/STAT signaling pathway in renal IR-induced inflammation.

Meprin β Regulates Osteopontin‐Signaling in IR‐Induced Acute Kidney Injury

Ischemia/reperfusion (IR) is a leading cause of acute kidney injury (AKI) and associated with high morbidity and mortality rate. The underlying mechanisms involved in IR-induced AKI are not fully understood. Meprins, zinc metalloproteases that are abundantly expressed in the brush border membranes of proximal kidney tubules, have been implicated in the pathology of IR. Meprins are capable to proteolytically process several mediators of cell signaling pathways mediators involved in apoptosis and extracellular matrix (ECM) metabolism such as osteopontin (OPN). The objective of the current study was to determine how meprin β expression affects OPN and downstream mediators of the OPN-signaling pathway. Surgical procedures were used to induce unilateral IR with contralateral nephrectomy in wild-type (WT) and meprin β knockout (βKO) mice which are deficient in the meprin B (β-β) and the heteromeric isoform of meprin A (α-β). The mice were sacrificed at 24 h post-IR. Blood samples and kidney tissues were obtained for analysis. Neutrophil gelatinase associated lipocalin (NGAL) was used for biochemical assessment of kidney injury. Real-time PCR, immunohistochemical staining, and western blot analysis were used to evaluate the levels of OPN, caspase-3, Bcl-2, and NFκB. Statistical analysis utilized 2-way ANOVA. Our data show NGAL level were elevated significantly in WT (P=0.0001) and βKO (P=0.01) at 24 h post-IR compare to their control counterparts. However, the fold change was higher in WT when compared to βKO mice indicating that meprin β enhances kidney injury. OPN mRNA increased in both genotypes at 24 h post-IR. In contrast, OPN protein levels only increased in WT kidneys. Immunohistochemical staining showed the increase in the levels of OPN occurred in select WT kidney tubules. To determine whether meprins expression correlates with the increased OPN, we used immunofluorescence counterstaining with proximal tubule (PT) markers (meprin β for WT and villin for βKO). Our data show that the increase in OPN occurred in distal tubules (DTs), which do not express meprins. Interestingly, higher levels of OPN were detected in the lumen of PTs from WT but not βKO kidneys, suggesting that meprin β is responsible for enhanced release of OPN into filtrate and ultimately into urine as previously reported. OPN functions as anti-apoptotic molecules in different diseases, therefore we evaluated its impact on caspase-3, Bcl-2, and NFκB. The mRNA levels for caspase-3, Bcl-2 and NFκB decreased in WT but increased in βKO mice at 24 h post-IR. Immunohistochemical staining for caspase-3, Bcl-2 and NFκB also showed high staining levels in tubules of WT kidney compared to βKO at 24 h post-IR. These findings suggest that meprin β expression has a direct impact on the levels of OPN in the kidney and affects apoptotic genes regulated by the OPN signaling pathway.

Regulation of Megalin membrane traffic by AMP‐activated protein kinase in kidney proximal tubule epithelial cells

Megalin is a transmembrane receptor protein that belongs to a family of proteins known as low-density lipoprotein receptors (LDLRs) that functions in reabsorption of various molecules from the urine in the proximal tubule epithelial cells (PTECs) of the kidney. Disruption of the regulation of Megalin is associated with various stages of renal disease. Megalin mediates reabsorption by functioning as a scavenger receptor: binding of molecules (cargo) within the kidney proximal tubule lumen to the ectodomain of Megalin followed by internalization of cargo-bound Megalin via clathrin-mediated endocytosis, the first stage in transcytosis of cargo molecules across the kidney epithelium. Kidney proximal tubule cells experience metabolic stress due to low oxygen availability. We have recently shown that metabolic stress resulting in activation of AMP-activated protein kinase (AMPK) regulates the endocytosis of a large number of cell surface proteins, including that of beta1-integrin, that has a similar requirement for dab2 for internalization as does Megalin. How AMPK may regulate Megalin internalization in kidney PTECs is not well understood. Megalin internalization occurs via recruitment of the receptor to clathrin-coated pits (CCPs), which are ~100 nm structures comprised of clathrin and other proteins at the cell surface. Upon assembly and receptor recruitment, CCPs mediate membrane budding and eventually scission, leading to an internalized clathrin-coated vesicle (CCV). Megalin recruitment to CCPs is mediated by the clathrin adaptor protein dab2 that binds to NPXY motif that is present on Megalin and other LDLR family proteins. Here, we examine how metabolic stress and AMPK regulate the formation of CCPs and the dab2-dependent recruitment of Megalin and related receptors for internalization. Using cells engineered to express fluorescently tagged clathrin and other proteins, together with total internal reflection fluorescence microscopy and automated image analysis, we uncovered that AMPK activation triggers a reduction in the size and rate of initiation of CCPs, but an increased efficiency of formation of clathrin-coated vesicles. In addition, AMPK activation resulted in a robust increase in the recruitment of dab2 to CCPs. We have complemented this approach with assays that monitor the cell surface membrane traffic of Megalin and related receptors such as integrins. Collectively, these studies reveal a novel signaling pathway that mediates the regulation of the clathrin internalization of several receptors by AMPK and metabolic stress. A better understanding of signals responsive to cell metabolism and metabolic stress in kidney PTECs and how these lead to remodeling of endocytic membrane traffic may reveal novel ways to treat certain forms of kidney diseases.

PKB is a central molecule in the modulation of Na+-ATPase activity by albumin in renal proximal tubule cells

Evidence points to a possible role of tubular sodium reabsorption in worsening renal injury. Proximal tubule (PT) albumin overload is a critical process in the development of tubule-interstitial injury (TII), and consequently in progression of renal disease. We studied the possible correlation between changes in albumin concentration in the lumen of PT with modification of Na+-ATPase activity. An albumin overload animal model and LLC-PK1 cells as a model of PT cells were used. Albumin overload was induced by intraperitoneal injection of BSA in 14-week-old male Wistar rats. An increase in sodium clearance, fractional excretion of sodium, proteinuria, ratio between urinary protein and creatinine, and albuminuria were observed. These observations indicate that there could be a correlation between an increase in albumin in the lumen of PTs and renal sodium excretion. We observed that the activity of both Na+-ATPase and (Na++K+)ATPase decreased in the renal cortex of an albumin overload animal model. Using LLC-PK1 cells as a model of PT cells, inhibition of Na+-ATPase activity was observed with higher albumin concentrations, similar to that observed in the animal model. The inhibition of protein kinase B by higher albumin concentration was found to be a critical step in the inhibition of Na+-ATPase activity. Interestingly, activation of the ERK1/2/mTORC1/S6K pathway was required for protein kinase B inhibition. This mechanism leads to a decrease in protein kinase C activity and, consequently to inhibition of Na+-ATPase activity. Taken together, our results indicate that the molecular mechanism underlying the modulation of PT Na+-ATPase activity by albumin overload involves activation of the ERK1/2/mTORC1/S6K pathway, which leads to inhibition of the mTORC2/PKB/PKC pathway. Our findings contribute to better understanding regarding handing of renal Na+ induced by albumin overload in the lumen of PTs and, consequently, in the progression of renal disease.

Hemoglobin alters vitamin carrier uptake and vitamin D metabolism in proximal tubule cells: implications for sickle cell disease.

Kidney disease, including proximal tubule (PT) dysfunction, and vitamin D deficiency are among the most prevalent complications in sickle cell disease (SCD) patients. Although these two comorbidities have never been linked in SCD, the PT is the primary site for activation of vitamin D. Precursor 25-hydroxyvitamin D [25(OH)D] bound to vitamin D-binding protein (DBP) is taken up by PT cells via megalin/cubilin receptors, hydroxylated to the active 1,25-dihydroxyvitamin D [1,25(OH)2D] form, and released into the bloodstream. We tested the hypothesis that cell-free hemoglobin (Hb) filtered into the PT lumen impairs vitamin D uptake and metabolism. Hb at concentrations expected to be chronically present in the ultrafiltrate of SCD patients competed directly with DBP for apical uptake by PT cells. By contrast, uptake of retinol binding protein was impaired only at considerably higher Hb concentrations. Prolonged exposure to Hb led to increased oxidative stress in PT cells and to a selective increase in mRNA levels of the CYP27B1 hydroxylase, although protein levels were unchanged. Hb exposure also impaired vitamin D metabolism in PT cells, resulting in reduced ratio of 1,25(OH)2D:25(OH)D. Moreover, plasma levels of 1,25(OH)2D were reduced in a mouse model of SCD. Together, our data suggest that Hb released by chronic hemolysis has multiple effects on PT function that contribute to vitamin D deficiency in SCD patients.

Pathological Changes of Urinary System in a Dog with Urolithiasis and Renal Papillary Necrosis

Background: Urolithiasis is one of the important lower urinary tract diseases in dogs. Uroliths develop when urine becomes "supersaturated”. Struvite urolithiasis in dogs is formed when urine saturated with magnesium, ammonium and phosphate. Renal papillary necrosis is a form of nephropathy involving the necrosis of the renal papilla. The aim of this case is the evaluation of histopathological changes in kidney and urinary bladder caused by urolithiasis with sitruvite, a pathology commonly seen in dogs. Renal papillary necrosis that encountered in the case was evaluated histopathologically. We intend to discuss the relationship between urolithiasis and renal papillary necrosis.Case: The material of the case was a 7.5 year-old Rottweiler female dog that had been operated for the urolithiasis treatment and died after surgery. The animal sent to pathology department was necropsied and evaluated histopathologically. Also the uroliths removed during the surgery were sent for analysis postoperatively. Qualitative composition of uroliths was determined by a series of chemical analyses in Department of Biochemistry. X-ray diffraction was performed as well in General Directorate of Mineral Research and Exploration in the objective of result verification. Macroscopically, 30 pieces uroliths with varying size from 1 mm to 3 cm were seen in urinary bladder. They were 110 g in weight with smooth surface and round and/or ovoid shapes. After necropsy, specimens from kidney and urinary bladder were fixed in 10% neutral buffered formalin, embedded in paraffin wax, cut at 5 μm, and stained with hematoxylin and eosin (HE). Bladder wall thickening and hemorrhage on mucosa were noticed. Kidneys had roughened surface with the capsule being difficult to peel off. Also, renal papillary necrosis, reddish in color was observed in papilla of the right kidney. Microscopically, crystal formations were observed proximal tubule lumens of kidneys. Interstitial nephritis in cortex and medulla had been noticed in both kidneys. Interstitial nephritis has been noticed in both kidneys. Hemorrhagic necrotic area demarcated by connective tissue was encountered in medulla of the right kidney. In the lamina propria of urinary bladder hemorrhage were noted. Desquamation and degeneration of lamina epithelialis and wide hemorrhage area in lamina propria were noted in urinary bladder. Results obtained from biochemical analysis were verified with x-ray diffraction method, uroliths were determined as magnesium ammonium phosphate (struvite).Discussion: Renal papillary necrosis is a form of nephropathy involving the necrosis of the renal papilla, caused by ischemia of the inner medulla of the kidney. According to the first report of renal papillary necrosis, symmetrical soft calyceal concretions and their appearances are typical lesions for diagnosis of renal papillary necrosis. The presence of crystals in the tubule lumen and the formation of struvite stone (which has the same chemical composition with calyceal concretions) established a distinct relationship between urolithiasis and renal papillary necrosis. This case showed that struvite stones can reach large volumes in urinary bladder, and caused bladder wall thickness and hemorrhages. Also, the lower urinary tract obstruction or urolithiasis should be remembered in the renal papillary necrosis together with the risk of hydronephrosis.

Calcium receptor signaling and citrate transport.

The calcium sensing receptor (CaSR) in the distal nephron decreases the propensity for calcium stones. Here we investigate if the apical CaSR in the proximal tubule also prevents stone formation acting via regulation of apical dicarboxylate and citrate transport. Urinary citrate, partially reabsorbed as a dicarboxylate in the proximal tubule lumen, inhibits stone formation by complexing calcium. We previously demonstrated a novel apical calcium-sensitive dicarboxylate transport system in OK proximal tubule cells. This calcium-sensitive process has the potential to modulate the amount of citrate available to complex increased urinary calcium. Using isotope labeled succinate uptake in OK cells along with various pharmacologic tools we examined whether the CaSR alters apical dicarboxylate transport and through which signal transduction pathways this occurs. Our results indicate that in the proximal tubule CaSR adjusts apical dicarboxylate transport, and does so via a CaSR→Gq→PKC signaling pathway. Thus, the CaSR may decrease the propensity for stone formation via actions in both proximal and distal nephron segments.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

α-Ketoglutarate drives electroneutral NaCl reabsorption in intercalated cells by activating a G-protein coupled receptor, Oxgr1

This review describes the recent discoveries about a powerful electroneutral NaCl reabsorption mechanism in intercalated cells, and its regulation by an intrarenal metabolite paracrine, α-ketoglutartate, and the G-protein coupled receptor, Oxgr1. The distal nephron fine-tunes sodium, chloride, potassium, hydrogen, bicarbonate and water transport to maintain electrolyte homeostasis and blood pressure. Intercalated cells have been traditionally viewed as the professional regulators of acid-base balance, but recent studies reveal that a specific population of intercalated cells, identified by the pendrin-transporter, have a surprising role in the regulation of salt balance. The pendrin-positive intercalated cells (PP-ICs) facilitate electroneutral NaCl reabsorption through the cooperative activation of multitransport protein network. α-Ketoglutartate is synthesized and secreted into the proximal tubule lumen in the combined state of metabolic alkalosis and intravascular volume contraction to activate Oxgr1 in PP-IC, which in turn activates the multitransport protein network to drive salt reabsorption and bicarbonate secretion by these cells. Recent studies identify a novel salt transport pathway in intercalated cells that is activated by an intrarenal paracrine system, α-ketoglutartate/Oxgr1. Activation of the paracrine system and transport pathway, particularly during alkalosis and volume contraction, mitigates deleterious salt wasting while restoring acid-base balance.

Crystalline cast nephropathy in a patient with IgD lambda myeloma

A 72-year-old woman was admitted at our department for renal dysfunction with general fatigue and loss of appetite for 2 months. She had no remarkable past medical history except hypertension, which was treated with angiotensin-converting enzyme inhibitor and a calcium channel blocker. Physical examination was unremarkable, except for pale conjunctivae. Urinary protein to creatinine ratio was 9.5 g/gCr, revealing a large discrepancy from the result of mild proteinuria by urinary dipstick (2?; indicating 100–300 mg/L). No hematuria was found. Serum creatinine level was increased to 1.90 mg/ dL associated with anemia (hemoglobin 7.3 g/dL). Other findings were total protein 6.8 g/dL, serum albumin 4.67 g/ dL, serum IgG 516 mg/dL (normal 870–1700 mg/dL), serum IgA 17 mg/dL (normal 110–410 mg/dL), serum IgM 5 mg/ dL (normal 34–220 mg/dL), and serum IgD 306 mg/dL (normal 0–9 mg/dL). Serum and urine electrophoresis revealed a monoclonal spike and an IgD-type monoclonal k band, respectively. The free k light chain level was 3720 mg/ L; serum j/k ratio was 0.0017. On the 5th day after admission, kidney biopsy was performed (Fig. 1). On light microscopy, minor glomerular abnormalities were observed; furthermore, diffuse mononuclear cell infiltration in the interstitium and crystalline casts in the proximal tubules were observed. These crystalline casts were hematoxylin and eosin positive but periodic acid-Schiff (PAS) negative. Moreover, only k light chain staining was positive. On electron microscopy, crystalline casts were found to form gigantic plate-shaped crystalline structures. Bone marrow aspiration cytology confirmed the diagnosis of multiple myeloma. She received bortezomib–dexamethasone therapy and achieved complete remission with normal renal function for 2 years. Cast nephropathy is the most common renal complication [1]. In rare cases, the casts exclusively comprise crystals. Myeloma-free light chains (MFLCs) are usually absorbed by proximal tubular cells, leading to acute proximal tubular damage associated with needle-shaped crystals in epithelial cells. This condition is well known as acquired light chain-related Fanconi syndrome [2]. Conversely, MFLCs that are not absorbed by the proximal tubules are distributed to the distal tubules and the ascending loop of Henle, where Tamm–Horsfall protein (THP) casts are exclusively synthesized [3]. These casts usually comprise needle-shaped or rhomboid crystals. THP is one of the glycoproteins, which is invariably PAS positive. The crystalline casts that were observed in this case were PAS negative and were distributed in the proximal tubule lumen. This is understandable, considering the site of THP synthesis. Some reports described crystal formation in organs except the kidneys. In these organs, crystal formation occurred in microvasculature due to low tissue temperature and/or blood circulation stasis; however, the precise mechanism is uncertain [4]. The characteristics of this case emphasized that MFLCs may form crystals independently or at least, without THP participation. Some have reported factors that may contribute to explain MFLC precipitation for crystal formation: a large MFLC amount & Satoshi Kumakura satoshi.kuma.714@gmail.com

Editorial: The emerging discipline of quantitative systems pharmacology.

Editorial: The emerging discipline of quantitative systems pharmacology.