The Value of Myocardium and Kidney Histopathological and Immunohistochemical Findings in Accidental Hypothermia-Related Fatalities.

Background and Objectives: The post-mortem diagnosis of hypothermia is challenging in forensics. The aim of our study was to detect the kidney and heart histopathological changes that occurred in a group of hypothermia-related fatalities. Materials and Methods: The cohort included 107 cases identified in the database of our department between 2007 and 2021, which have been associated with extreme cold stress. Demographic and clinicopathological data were collected from the medico-legal reports. Archived tissue samples were evaluated to identify the histopathological features, in routine haematoxylin-eosin (H&E), Periodic acid-Schiff (PAS), and Masson's trichrome stainings, while cardiac sirtuin1 (SIRT1) and renal ubiquitin (Ub) immunostaining have been performed. Results: The majority of cases exposed to low temperatures were males (76%) from rural regions (68.2%) during the cold season. Paradoxical undressing was documented in 9.3% of cases. The common comorbidities included alcoholism (50.5%), neuropsychiatric diseases (10.3%), diabetes mellitus (3.7%), and lung tuberculosis (4.7%). The microscopic heart exam revealed areas of myocardial degeneration (100%), contraction bands (95.3%), fatty change (13.1%) and focal wavy contractile myocardial cells. Basal vacuolisation of renal tubular epithelial cells (Armanni-Ebstein lesions) (21.5%), focal tubular necrosis (7.5%), tubular renal cysts (7.5%), interstitial haemorrhages (5.6%), diabetic kidney disease (3.7%), background benign nephroangiosclerosis (42.1%), variable thickening of tubules and corpuscles basement membranes, capsular space amorphous material, and intratubular casts were identified in kidney tissue samples. Myocardial cells displayed SIRT1 weak expression, with a loss of immunopositivity correlated with areas with contraction bands, while a variable Ub expression was observed in renal corpuscles capsules, proximal, distal, and collecting renal tubules, Henle's loops, urothelium, and intratubular casts. Conclusions: In the context of the current concept that death associated with hypothermia is still a diagnosis of exclusion, our findings suggest that the microscopic exam provides relevant data that support the diagnosis of hypothermia-related fatalities in appropriate circumstances of death. A deeper insight into the histopathologic findings in hypothermic patients may lead to new therapeutic approaches in these cases.

A new view of macula densa cell microanatomy.

Although macula densa (MD) cells are chief regulatory cells in the nephron with unique microanatomical features, they have been difficult to study in full detail due to their inaccessibility and limitations in earlier microscopy techniques. The present study used a new mouse model with a comprehensive imaging approach to visualize so far unexplored microanatomical features of MD cells, their regulation, and functional relevance. MD-GFP mice with conditional and partial induction of green fluorescent protein (GFP) expression, which specifically and intensely illuminated only single MD cells, were used with fluorescence microscopy of fixed tissue and live MD cells in vitro and in vivo with complementary electron microscopy of the rat, rabbit, and human kidney. An elaborate network of major and minor cell processes, here named maculapodia, were found at the cell base, projecting toward other MD cells and the glomerular vascular pole. The extent of maculapodia showed upregulation by low dietary salt intake and the female sex. Time-lapse imaging of maculapodia revealed highly dynamic features including rapid outgrowth and an extensive vesicular transport system. Electron microscopy of rat, rabbit, and human kidneys and three-dimensional volume reconstruction in optically cleared whole-mount MD-GFP mouse kidneys further confirmed the presence and projections of maculapodia into the extraglomerular mesangium and afferent and efferent arterioles. The newly identified dynamic and secretory features of MD cells suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD cells and between MD and other target cells.NEW & NOTEWORTHY This study illuminated a physiologically regulated dense network of basal cell major and minor processes (maculapodia) in macula densa (MD) cells. The newly identified dynamic and secretory features of these microanatomical structures suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD and other target cells. Detailed characterization of the function and molecular details of MD cell intercellular communications and their role in physiology and disease warrant further studies.

Effect of forkhead box O1 in renal tubular epithelial cells on endotoxin-induced acute kidney injury.

Mitochondrial damage in renal tubular epithelial cells (RTECs) is a hallmark of endotoxin-induced acute kidney injury (AKI). Forkhead box O1 (FOXO1) is responsible for regulating mitochondrial function and is involved in several kidney diseases. Here, we investigated the effect of FOXO1 on endotoxin-induced AKI and the related mechanism. In vivo, FOXO1 downregulation in mouse RTECs and mitochondrial damage were found in endotoxin-induced AKI. Overexpression of FOXO1 by kidney focal adeno-associated virus (AAV) delivery improved renal function and reduced mitochondrial damage. Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1-α), a master regulator of mitochondrial biogenesis and function, was reduced in endotoxin-induced AKI, but the reduction was reversed by FOXO1 overexpression. In vitro, exposure to LPS led to a decline in HK-2 cell viability, mitochondrial fragmentation, and mitochondrial superoxide accumulation, as well as downregulation of FOXO1, PGC1-α, and mitochondrial complex I/V. Moreover, overexpression of FOXO1 in HK-2 cells increased HK-2 cell viability and PGC1-α expression, and it alleviated the mitochondrial injury and superoxide accumulation induced by LPS. Meanwhile, inhibition of FOXO1 in HK-2 cells by siRNA treatment decreased PGC1-α expression and HK-2 cell viability. Chromatin immunoprecipitation assays and PCR analysis confirmed that FOXO1 bound to the PGC1-α promoter in HK-2 cells. In conclusion, downregulation of FOXO1 in RTECs mediated endotoxin-induced AKI and mitochondrial damage. Overexpression of FOXO1 could improve renal injury and mitochondrial dysfunction, and this effect occurred at least in part as a result of PGC1-α signaling. FOXO1 might be a potential target for the prevention and treatment of endotoxin-induced AKI.

Electron microscopy study of 496 cases of lupus nephritis: A single-center experience.

INTRODUCTION: Renal involvement is seen in about 40-82% of systemic lupus erythematosus (SLE) Asian patients. The exact diagnosis and classification of lupus nephritis are important for treatment and prognosis. This study aimed to investigate the value of electron microscopy (EM) in the diagnosis and classification of lupus nephritis compared with light microscopy. METHOD: In this cross-sectional referral-center 16-year study of lupus nephritis, the final diagnosis was based on the EM study. Primary light microscopy findings were compared with EM diagnosis. Moreover, Immunofluorescence patterns distribution was assessed. RESULTS: From 496 patients diagnosed with lupus nephritis based on EM, 225(45.4%) of patients were categorized in class IV, followed by 98(19.7%), 93(18.8%), 46(9.3%), and 14(2.8%) who were categorized into classes of II, III, V, and VI respectively. Only 1(0.2%) patient belonged to class I, and 19(3.8%) cases were diagnosed with mixed two classes. Using EM was essential for diagnosing 25.6% of cases taking the correct classification by light microscopy into account; however, disregarding correct classification, this could change to a 7.4% contribution rate of EM. The most common cause of misdiagnosis, disregarding incorrect classification, was inadequate or wrong tissue. Positive associations were detected between tubular atrophy and interstitial fibrosis of both electron and light microscopy with different classes (P < 0.001). CONCLUSION: While light microscopy is highly accurate for diagnosing lupus nephritis regardless of correct classification, EM contributes substantially to the correct classification of lupus nephritis types.

Ultrastructural Evidence for Direct Renal Infection with SARS-CoV-2.

BACKGROUND: A significant fraction of patients with coronavirus disease 2019 (COVID-19) display abnormalities in renal function. Retrospective studies of patients hospitalized with COVID-19 in Wuhan, China, report an incidence of 3%-7% progressing to ARF, a marker of poor prognosis. The cause of the renal failure in COVID-19 is unknown, but one hypothesized mechanism is direct renal infection by the causative virus, SARS-CoV-2. METHODS: We performed an autopsy on a single patient who died of COVID-19 after open repair of an aortic dissection, complicated by hypoxic respiratory failure and oliguric renal failure. We used light and electron microscopy to examine renal tissue for evidence of SARS-CoV-2 within renal cells. RESULTS: Light microscopy of proximal tubules showed geographic isometric vacuolization, corresponding to a focus of tubules with abundant intracellular viral arrays. Individual viruses averaged 76 µm in diameter and had an envelope studded with crown-like, electron-dense spikes. Vacuoles contained double-membrane vesicles suggestive of partially assembled virus. CONCLUSIONS: The presence of viral particles in the renal tubular epithelium that were morphologically identical to SARS-CoV-2, and with viral arrays and other features of virus assembly, provide evidence of a productive direct infection of the kidney by SARS-CoV-2. This finding offers confirmatory evidence that direct renal infection occurs in the setting of AKI in COVID-19. However, the frequency and clinical significance of direct infection in COVID-19 is unclear. Tubular isometric vacuolization observed with light microscopy, which correlates with double-membrane vesicles containing vacuoles observed with electronic microscopy, may be a useful histologic marker for active SARS-CoV-2 infection in kidney biopsy or autopsy specimens.

A New Method of Isolation of Mouse Renal Primary Tubular Epithelial Cells.

Kidney diseases are becoming an emerging public health problem. In order to further explore the etiology of various kidney diseases, we improved the methods of isolation of primary cultures of mouse renal tubular epithelial cells. At the first stage, the kidneys were perfused with collagenase solution. To this end, the superior mesenteric artery, celiac artery and thoracic aorta were ligated and perfusion was performed through the abdominal aorta. Then, the cells were isolated ex vivo and their integrity, purity, viability, and concentration were evaluated. The proposed cost-effective and simple method provides high purity and high concentration of primary renal epithelial cells for molecular biology studies of the kidneys.

DsbA-L deficiency exacerbates mitochondrial dysfunction of tubular cells in diabetic kidney disease.

Excessive mitochondrial fission has been identified as the central pathogenesis of diabetic kidney disease (DKD), but the precise mechanisms remain unclear. Disulfide-bond A oxidoreductase-like protein (DsbA-L) is highly expressed in mitochondria in tubular cells of the kidney, but its pathophysiological role in DKD is unknown. Our bioinformatics analysis showed that tubular DsbA-L mRNA levels were positively associated with eGFR but negatively associated with Scr and 24h-proteinuria in CKD patients. Furthermore, the genes that were coexpressed with DsbA-L were mainly enriched in mitochondria and were involved in oxidative phosphorylation. In vivo, knockout of DsbA-L exacerbated diabetic mice tubular cell mitochondrial fragmentation, oxidative stress and renal damage. In vitro, we found that DsbA-L was localized in the mitochondria of HK-2 cells. High glucose (HG, 30 mM) treatment decreased DsbA-L expression followed by increased mitochondrial ROS (mtROS) generation and mitochondrial fragmentation. In addition, DsbA-L knockdown exacerbated these abnormalities, but this effect was reversed by overexpression of DsbA-L. Mechanistically, under HG conditions, knockdown DsbA-L expression accentuated JNK phosphorylation in HK-2 cells. Furthermore, administration of a JNK inhibitor (SP600125) or the mtROS scavenger MitoQ significantly attenuated JNK activation and subsequent mitochondrial fragmentation in DsbA-L-knockdown HK-2 cells. Additionally, the down-regulation of DsbA-L also amplified the gene and protein expression of mitochondrial fission factor (MFF) via the JNK pathway, enhancing its ability to recruit DRP1 to mitochondria. Taken together, these results link DsbA-L to alterations in mitochondrial dynamics during tubular injury in the pathogenesis of DKD and unveil a novel mechanism by which DsbA-L modifies mtROS/JNK/MFF-related mitochondrial fission.

Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.

How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin ß2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.

Electron microscopic findings can support multiple etiologies of nephrotoxicity in renal tubules.

Electron microscopy (EM) has been mainly used for identifying ultrastructural abnormalities such as fusion of foot processes and immune complex deposits in glomeruli. However, electron microscopic findings in renal tubules can provide either diagnostic evidence (unique finding) or supportive evidence (additional finding) for final diagnosis. Here we present multiple situations that EM can be used for drawing conclusions of various drug-associated nephrotoxicity. Multiple cases with drug-induced nephrotoxicity are reviewed, including clinical history, EM findings, and serum creatinine (sCr) levels, prior to renal biopsy and during follow-up. Two cases with nephrotoxicity by aminoglycoside antibiotics showed acute tubular injury with EM findings of myeloid bodies, characterized by laminated dense materials in lysosomes in both proximal and distal tubular epithelium (diagnostic evidence). Five cases of vancomycin associated nephrotoxicity presented with acute tubular injury and vancomycin casts in distal tubules, characterized by central laminated casts in the lumina of distal tubules (supportive evidence). Vedolizumab, a humanized monoclonal antibody used in treating Crohn's disease, can cause T-cell dominant acute interstitial nephritis, with EM revealing lymphocytic infiltration into tubules as tubulitis (supportive evidence). Four of Seven cases (5/8) cases had renal functional recovery upon follow-up check for sCr. EM findings of characteristic changes in renal tubules can be particularly useful as either diagnostic or supportive evidence, in correlation with clinical history and etiologies of nephrotoxicity. Therefore, EM should not only focus on glomerular changes, but renal tubular changes as well.

Zebra-like bodies in COVID-19: is phospholipidosis evidence of hydroxychloroquine induced acute kidney injury?

COVID-19 (from SARS-CoV-2) is the cause of an ongoing pandemic, with an increasing number of cases and significant mortality worldwide. Clinical trials and extensive studies are being conducted on a large scale for a better understanding of the pathophysiology of this disease and its effect on different organs. Several experimental treatment protocols have been introduced, in which hydroxychloroquine (HCQ) was one of the first drugs used. While patients can develop many side effects of HCQ, studies have documented a rare association of long-term HCQ treatment with zebra-like bodies in the ultrastructural examination of kidney biopsies, a finding typically seen in Fabry's disease, as well as in association with chronic HCQ use, among other drugs. We present a similar finding in the postmortem examination of a male in his early seventies with COVID-19 infection, who received five days of HCQ treatment before stopping the medication due to cardiac and renal toxicity.

The combination of a neprilysin inhibitor (sacubitril) and angiotensin-II receptor blocker (valsartan) attenuates glomerular and tubular injury in the Zucker Obese rat.

OBJECTIVE: Diabetic nephropathy (DN) is characterized by glomerular and tubulointerstitial injury, proteinuria and remodeling. Here we examined whether the combination of an inhibitor of neprilysin (sacubitril), a natriuretic peptide-degrading enzyme, and an angiotensin II type 1 receptor blocker (valsartan), suppresses renal injury in a pre-clinical model of early DN more effectively than valsartan monotherapy. METHODS: Sixty-four male Zucker Obese rats (ZO) at 16 weeks of age were distributed into 4 different groups: Group 1: saline control (ZOC); Group 2: sacubitril/valsartan (sac/val) (68 mg kg-1 day-1; ZOSV); and Group 3: valsartan (val) (31 mg kg-1 day-1; ZOV). Group 4 received hydralazine, an anti-hypertensive drug (30 mg kg-1 day-1, ZOH). Six Zucker Lean (ZL) rats received saline (Group 5) and served as lean controls (ZLC). Drugs were administered daily for 10 weeks by oral gavage. RESULTS: Mean arterial pressure (MAP) increased in ZOC (+ 28%), but not in ZOSV (- 4.2%), ZOV (- 3.9%) or ZOH (- 3.7%), during the 10 week-study period. ZOC were mildly hyperglycemic, hyperinsulinemic and hypercholesterolemic. ZOC exhibited proteinuria, hyperfiltration, elevated renal resistivity index (RRI), glomerular mesangial expansion and podocyte foot process flattening and effacement, reduced nephrin and podocin expression, tubulointerstitial and periarterial fibrosis, increased NOX2, NOX4 and AT1R expression, glomerular and tubular nitroso-oxidative stress, with associated increases in urinary markers of tubular injury. None of the drugs reduced fasting glucose or HbA1c. Hypercholesterolemia was reduced in ZOSV (- 43%) and ZOV (- 34%) (p < 0.05), but not in ZOH (- 13%) (ZOSV > ZOV > ZOH). Proteinuria was ameliorated in ZOSV (- 47%; p < 0.05) and ZOV (- 30%; p > 0.05), but was exacerbated in ZOH (+ 28%; p > 0.05) (ZOSV > ZOV > ZOH). Compared to ZOC, hyperfiltration was improved in ZOSV (p < 0.05 vs ZOC), but not in ZOV or ZOH. None of the drugs improved RRI. Mesangial expansion was reduced by all 3 treatments (ZOV > ZOSV > ZOH). Importantly, sac/val was more effective in improving podocyte and tubular mitochondrial ultrastructure than val or hydralazine (ZOSV > ZOV > ZOH) and this was associated with increases in nephrin and podocin gene expression in ZOSV (p < 0.05), but not ZOV or ZOH. Periarterial and tubulointerstitial fibrosis and nitroso-oxidative stress were reduced in all 3 treatment groups to a similar extent. Of the eight urinary proximal tubule cell injury markers examined, five were elevated in ZOC (p < 0.05). Clusterin and KIM-1 were reduced in ZOSV (p < 0.05), clusterin alone was reduced in ZOV and no markers were reduced in ZOH (ZOSV > ZOV > ZOH). CONCLUSIONS: Compared to val monotherapy, sac/val was more effective in reducing proteinuria, renal ultrastructure and tubular injury in a clinically relevant animal model of early DN. More importantly, these renoprotective effects were independent of improvements in blood pressure, glycemia and nitroso-oxidative stress. These novel findings warrant future clinical investigations designed to test whether sac/val may offer renoprotection in the setting of DN.

HIVAN associated tubular pathology with reference to ER stress, mitochondrial changes, and autophagy.

Human immunodeficiency virus associated nephropathy (HIVAN) is a unique form of a renal parenchymal disorder. This disease and its characteristics can be accredited to incorporation of DNA and mRNA of human immunodeficiency virus type 1 into the renal parenchymal cells. A proper understanding of the intricacies of HIVAN and the underlying mechanisms associated with renal function and disorders is vital for the potential development of a reliable treatment for HIVAN. Specifically, the renal tubule segment of the kidney is characterized by its transport capabilities and its ability to reabsorb water and salts into the blood. However, the segment is also known for certain disorders, such as renal tubular epithelial cell infection and microcyst formation, which are also closely linked to HIVAN. Furthermore, certain organelles, like the endoplasmic reticulum (ER), mitochondria, and lysosome, are vital for certain underlying mechanisms in kidney cells. A paradigm of the importance of said organelles can be seen in documented cases of HIVAN where the renal disorder results increased ER stress due to HIV viral propagation. This balance can be restored through the synthesis of secretory proteins, but, in return, the secretion requires more energy; therefore, there is a noticeable increase in mitochondrial stress. The increased ER changes and mitochondrial stress will greatly upregulate the process of autophagy, which involves the cell's lysosomes. In conjunction, we found that ER stress and mitochondrial changes are associated in the Tg26 animal model of HIVAN. The aim of our review is to consolidate current knowledge of important mechanisms in HIVAN, specifically related to the renal tubules' association with ER stress, mitochondrial changes and autophagy. Although the specific regulatory mechanism detailing the cross-talk between the various organelles is unknown in HIVAN, the continued research in this field may potentially shed light on a possible improved treatment for HIVAN.

Uric acid transporters BCRP and MRP4 involved in chickens uric acid excretion.

BACKGROUND: Breast cancer resistance protein (BCRP) and multidrug resistance protein 4 (MRP4) are involved in uric acid excretion in humans and mice. Despite evidence suggesting that renal proximal tubular epithelial cells participate in uric acid excretion in chickens, the roles of BCRP and MRP4 therein remain unclear. This study evaluated the relationship between BCRP and MRP4 expression and renal function in chickens. RESULTS: Sixty laying hens were randomly divided into four treatment groups: a control group (NC) fed a basal diet; a sulfonamide-treated group (SD) fed the basal diet and supplemented with sulfamonomethoxine sodium via drinking water (8 mg/L); a fish meal group (FM) fed the basal diet supplemented with 16% fishmeal; and a uric acid injection group (IU) fed the basal diet and intraperitoneally injected with uric acid (250 mg/kg body weight). The results showed that serum uric acid, creatinine, and blood urea nitrogen levels were significantly higher in the SD and IU, but not FM, than in the NC groups. Renal tubular epithelial cells in the SD and IU groups were damaged. Liver BCRP and MRP4 mRNA and protein levels were significantly decreased in the SD and IU groups, but slightly increased in the FM group. In the SD group, BCRP and MRP4 were significantly increased in the ileum and slightly increased in the kidney. In the FM group, BCRP and MRP4 were significantly increased in the kidney and slightly increased in the ileum. In the IU group, BCRP and MRP4 were significantly increased in the kidney and ileum. BCRP and MRP4 expression in the jejunum was not affected by the treatments. CONCLUSION: Together, these results demonstrate that BCRP and MRP4 are involved in renal and intestinal uric acid excretion in chickens and that BCRP is positively related to MRP4 expression. Further, impairment of renal function results in an increase in serum uric acid as well as a compensatory increase in BCRP and MRP4 in the ileum; however, under normal renal function, renal BCRP and MRP4 are the main regulators of uric acid excretion.

The inhibition of Nrf2 accelerates renal lipid deposition through suppressing the ACSL1 expression in obesity-related nephropathy.

Background: Obesity has become a worldwide epidemic, and the incidence of obesity is increasing year by year. Obesity-related nephropathy (ORN) is a common kidney complication of obesity. Long-chain acyl-CoA synthetases-1, (ACSL1), is a key enzyme in the oxidative metabolism of fatty acids in mitochondria and ACSL1 may play a direct role in renal lipid deposition and promote the progress of ORN. In this study, we focus on the renoprotective role of ACSL1 in ORN. Methods: Electron microscopy, immunohistochemical (IHC) staining, Western blot, and real-time PCR were used to detect the expression of ACSL1and Nrf2 in ORN patients, ob/ob mice and palmitic acid (PA)-treated HK-2 cells. Oil red staining and Elisa Kit were used to detect the intracellular FFA and TG contents in ob/ob mice and PA-treated HK-2 cells. Dihydroethidium (DHE) staining and the MDA/SOD measurement were used to detect the ROS production. In order to demonstrate the role of ACSL1 and the interaction between ACSL1 and Nrf2 in ORN, related siRNA and plasmid were transfected into HK-2 cells. Results: More ROS production and renal lipid deposition have been found in ORN patients, ob/ob mice and PA-treated HK-2 cells. Compared with control, all the expression of ACSL1and Nrf2 were down-regulated in ORN patients, ob/ob mice and PA-treated HK-2 cells. The Nrf2 could regulate the expression of ACSL1 and the ACSL1 played the direct role in renal lipid deposition. Conclusions: The Nrf2 is inhibited in ORN, resulting more ROS production and oxidative stress. Increased oxidative stress will suppress the expression of ACSL1, which could increase the intracellular FFA and TG contents, ultimately leading to renal lipid deposition in renal tubulars and accelerating the development of ORN.

Nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant results in albuminuria and mesangial expansion.

Intracellular trafficking processes play a key role for the establishment and maintenance of membrane surfaces in renal epithelia. Therefore, dysfunctions of these trafficking processes could be key events and important determinants in the onset and progression of diseases. The presence of cellular vacuoles-observed in many histologic analyses of renal diseases-is a macroscopic hint for disturbed intracellular trafficking processes. However, how vacuoles develop and which intracellular pathways are directly affected remain largely unknown. Previous studies showed that in some cases, vacuolization is linked to malfunction of the Vac14 complex. This complex, including the scaffold protein Vac14, the lipid kinase PIKfyve, and its counteracting lipid phosphatase Fig4, regulates intracellular phosphatidylinositol phosphate levels, which in turn, control the maturation of early-into-late endosomes, as well as the processing of autophagosomes into autophagolysosomes. Here, we analyzed the role of Vac14 in mice and observed that the nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant led to albuminuria, accompanied by mesangial expansion, increased glomerular size, and an elevated expression of several kidney injury markers. Overexpression of this Vac14 variant in podocytes did not reveal a strong in vivo phenotype, indicating that Vac14-dependent trafficking processes are more important for tubular than for glomerular processes in the kidney. In vitro overexpression of Vac14L156R in Madin-Darby canine kidney cells had no impact on apico-basal polarity defects but resulted in a faster reassembly of junctional structures after Ca2+ depletion and delayed endo- and transcytosis rates. Taken together, our data suggest that increased albuminuria of Vac14L156R-overexpressing mice is a consequence of a lowered endo- and transcytosis of albumin in renal tubules.

Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

Acute kidney injury (AKI), characterized by aggressive inflammatory responses and destruction of renal resident cells, can cause abrupt kidney dysfunction. To date, effective therapy for AKI is lacking. In this study, we evaluated the renoprotective effect of wogonin, an herbal active compound, using a cisplatin-induced AKI mouse model. In vivo results show that wogonin substantially suppressed the increased levels of serum creatinine and blood urea nitrogen (BUN) almost to the normal level. Wogonin also attenuated tubular damage, shown by PAS staining, electron microscopy and molecular analysis of KIM-1. In addition, wogonin suppressed kidney inflammation as indicated by a >60% decrease in macrophage infiltration, a >50% reduction in inflammatory cytokine production and inhibited NF-κB activation in the injured kidney. Mechanistically, molecular docking results show that wogonin effectively inhibited RIPK1 by occupying the ATP-binding pocket of the enzyme, which is a key regulator of necroptosis. Moreover, inhibition of RIPK1, or RIPK3, reversed the protective effects of wogonin in cisplatin-treated HK2 cells, indicating wogonin works in a RIPK1/RIPK3-dependent manner. Surprisingly, wogonin enhanced the anti-proliferative effect of cisplatin on human hepatoma HepG2 cells. Thus, our findings suggest wogonin may be a renoprotective adjuvant for cisplatin-based anticancer therapy.

Perturbations in mitochondrial dynamics by p66Shc lead to renal tubular oxidative injury in human diabetic nephropathy.

Renal tubular injury is increasingly being recognized as an early characteristic of diabetic nephropathy (DN). Mitochondrial dynamic alterations and redox protein p66Shc-mediated oxidative stress are both critical for ensuing diabetic tubular cell injury and apoptosis; whether these two processes are interlinked remains unclear. In the present study, we observed changes in mitochondrial morphology and expression of associated proteins in tubules of patients with DN. We demonstrated mitochondrial fragmentation as an important pathogenic feature of tubular cell injury that is linked to oxidative stress and p66Shc up-regulation. In renal proximal tubular cells, alterations in mitochondrial dynamics and expression of fission-fusion proteins were observed under high glucose (HG) ambience, along with p66Shc Ser36 phosphorylation. Gene ablation of p66Shc alleviated HG-induced mitochondrial fragmentation, down-regulated Fis1 and reduced p66Shc-Fis1 binding, increased Mfn1 expression, and disrupted interactions between Mfn1 and proapoptotic Bak. Overexpression of p66Shc exacerbated these changes, whereas overexpression of dominant-negative p66Shc Ser36 mutant had a marginal effect under HG, indicating that p66Shc phosphorylation as a prerequisite in the modulation of mitochondrial dynamics. Disrupted mitochondrial dynamics and enhanced Mfn1-Bak interactions modulated by p66Shc led to loss of mitochondrial voltage potential, cytochrome C release, excessive ROS generation, and apoptosis. Taken together, these results link p66Shc to mitochondrial dynamic alterations in the pathogenesis of DN and unveil a novel mechanism by which p66Shc mediates HG-induced mitochondrial fragmentation and proapoptotic signaling that results in oxidative injury and apoptosis in the tubular compartment in human diabetic nephropathy.

Tubular basement membrane immune complex deposition is associated with activity and progression of lupus nephritis: a large multicenter Chinese study.

Tubulointerstitial injury is found frequently in lupus nephritis. Immune complex deposits can occur in the tubular basement membranes (TBMs), although its significance in lupus nephritis patients remains unclear. This study assessed the clinical and prognostic features of lupus nephritis patients with TBM deposits in a large Chinese multicenter cohort. Complete data were collected from 195 patients with renal biopsy-proven lupus nephritis diagnosed in the Peking University First Hospital as the discovery cohort. A total of 102 lupus nephritis patients were enrolled from another four centers as the validation cohort. The status of TBM deposits was retrospectively assessed using electron microscopy, and the associations of the deposits with clinical data, pathological characteristics and renal outcomes were further analyzed. The percentage of positive TBM deposits was nearly 30% in the lupus nephritis patients. Using immuno-gold labeling, we found that 10/10 patients were positive for IgG, 7/10 were C3d positive, 6/10 were C1q positive, and 1/10 were C4d positive. Patients with TBM deposits presented with more active features, including a higher SLEDAI score (SLE Disease Activity Index) ( p < 0.001), higher serum creatinine level ( p = 0.001) and lower serum C3 level ( p < 0.001). These patients also presented with higher scores for most renal pathological indices, including the total activity indices score ( p < 0.001) and total chronicity indices score ( p = 0.001). TBM deposits affected renal outcomes in the univariate Cox hazards regression analysis (HR = 4.2, 95% CI = 1.3-14.3, p = 0.02). In conclusion, TBM deposits were common in lupus nephritis patients and correlated closely with the clinical disease activity and renal outcome.

Pyruvate dehydrogenase kinase 4 deficiency attenuates cisplatin-induced acute kidney injury.

Clinical prescription of cisplatin, one of the most widely used chemotherapeutic agents, is limited by its side effects, particularly tubular injury-associated nephrotoxicity. Since details of the underlying mechanisms are not fully understood, we investigated the role of pyruvate dehydrogenase kinase (PDK) in cisplatin-induced acute kidney injury. Among the PDK isoforms, PDK4 mRNA and protein levels were markedly increased in the kidneys of mice treated with cisplatin, and c-Jun N-terminal kinase activation was involved in cisplatin-induced renal PDK4 expression. Treatment with the PDK inhibitor sodium dichloroacetate (DCA) or genetic knockout of PDK4 attenuated the signs of cisplatin-induced acute kidney injury, including apoptotic morphology of the kidney tubules along with numbers of TUNEL-positive cells, cleaved caspase-3, and renal tubular injury markers. Cisplatin-induced suppression of the mitochondrial membrane potential, oxygen consumption rate, expression of electron transport chain components, cytochrome c oxidase activity, and disruption of mitochondrial morphology were noticeably improved in the kidneys of DCA-treated or PDK4 knockout mice. Additionally, levels of the oxidative stress marker 4-hydroxynonenal and mitochondrial reactive oxygen species were attenuated, whereas superoxide dismutase 2 and catalase expression and glutathione synthetase and glutathione levels were recovered in DCA-treated or PDK4 knockout mice. Interestingly, lipid accumulation was considerably attenuated in DCA-treated or PDK4 knockout mice via recovered expression of peroxisome proliferator-activated receptor-α and coactivator PGC-1α, which was accompanied by recovery of mitochondrial biogenesis. Thus, PDK4 mediates cisplatin-induced acute kidney injury, suggesting that PDK4 might be a therapeutic target for attenuating cisplatin-induced acute kidney injury.

Injury induced expression of caveolar proteins in human kidney tubules - role of megakaryoblastic leukemia 1.

BACKGROUND: Caveolae are membrane invaginations measuring 50-100 nm. These organelles, composed of caveolin and cavin proteins, are important for cellular signaling and survival. Caveolae play incompletely defined roles in human kidneys. Induction of caveolin-1/CAV1 in diseased tubules has been described previously, but the responsible mechanism remains to be defined. METHODS: Healthy and atrophying human kidneys were stained for caveolar proteins, (caveolin 1-3 and cavin 1-4) and examined by electron microscopy. Induction of caveolar proteins was studied in isolated proximal tubules and primary renal epithelial cells. These cells were challenged with hypoxia or H2O2. Primary tubular cells were also subjected to viral overexpression of megakaryoblastic leukemia 1 (MKL1) and MKL1 inhibition by the MKL1 inhibitor CCG-1423. Putative coregulators of MKL1 activity were investigated by Western blotting for suppressor of cancer cell invasion (SCAI) and filamin A (FLNA). Finally, correlative bioinformatic studies of mRNA expression of caveolar proteins and MKL1 were performed. RESULTS: In healthy kidneys, caveolar proteins were expressed by the parietal epithelial cells (PECs) of Bowman's capsule, endothelial cells and vascular smooth muscle. Electron microscopy confirmed caveolae in the PECs. No expression was seen in proximal tubules. In contrast, caveolar proteins were expressed in proximal tubules undergoing atrophy. Caveolar proteins were also induced in cultures of primary epithelial tubular cells. Expression was not enhanced by hypoxia or free radical stress (H2O2), but proved sensitive to inhibition of MKL1. Viral overexpression of MKL1 induced caveolin-1/CAV1, caveolin-2/CAV2 and SDPR/CAVIN2. In kidney tissue, the mRNA level of MKL1 correlated with the mRNA levels for caveolin-1/CAV1, caveolin-2/CAV2 and the archetypal MKL1 target tenascin C (TNC), as did the MKL1 coactivator FLNA. Costaining for TNC as readout for MKL1 activity demonstrated overlap with caveolin-1/CAV1 expression in PECs as well as in atrophic segments of proximal tubules. CONCLUSIONS: Our findings support the view that MKL1 contributes to the expression of caveolar proteins in healthy kidneys and orchestrates the induction of tubular caveolar proteins in renal injury.