Thin Limbs Research Articles

Transcript Deconvolution Reveals Heterogeneous Cell Populations in Microdissected Nephron Segments of the Rat Kidney

Introduction: The kidney tubule comprises distinct segments with specialized epithelium, including proximal tubules (PT) subsegments S1, S2, and S3, descending (DTL) and ascending (ATL) thin limbs, thick ascending limb (TAL) medullary (MTAL) and cortical (CTAL) subsegments, distal convoluted tubule (DCT), connecting tubule (CNT), and collecting ducts (CD) containing principal (PC) and intercalated (IC) cells. Single-cell RNA sequencing (scRNAseq) is a crucial tool in kidney research. However, it assigns cells to nephron segments based on transcriptome similarity, disregarding anatomical context and spatial information, critical for kidney function. Hypothesis: We hypothesized that cellular deconvolution of microdissected tubule segment transcriptomes would identify mixed cell populations coexisting on different parts of the nephron. Methods: We employed CibersortX to estimate cell proportions in microdissected rat nephron segment transcriptomes (GSE56743), using rat kidney epithelial cell transcriptomes from a scRNAseq dataset (GSE137869) as reference. Raw sequencing files from the Sequence Read Archive (SRA) were downloaded and reprocessed. The number of detected genes was 34,322 for bulk RNAseq and 15,282 for scRNAseq, with a shared gene space of 13,373 used for deconvolution. Results: We discovered that PT.S1 segments primarily consist of S1 cells (81%) and some S2 cells (12%). Microdissected PT.S2 segments contain a mix of S1 (18%), S2 (58%), and S3 (19%) cells. PT.S3 segments were primarily composed of S3 cells (75%). DTL and ATL segments displayed a high percentage of the corresponding cell type. Microdissected MTAL segments were a mix of MTAL (25%) and CTAL (69%) cells, while microdissected CTAL segments were almost exclusively composed of MTAL cells (95%). DCT segments exhibited 80% DCT cells with some principal (2%) and intercalated (1%) cells. In CNT, CCD, and OMCD, we observed a continuous increase in PC abundance (58%, 72%, and 78%, respectively) along with a decrease in IC (32%, 23%, and 11%, respectively). Microdissected IMCD contained PC (58%), IC (4%), and 18% of a distinct cell type with IC characteristics not found in other segments. Conclusions: Our analysis supports the well-established cell heterogeneity in connecting tubules and the collecting duct system, including the existence of a specialized IMCD cell. Additionally, we identified mixed cell populations in the S2 segment of the proximal tubule and the medullary thick ascending limb. In the case of S2, the high proportion of cells from adjacent S1 and S3 segments suggests a differentiation continuum from S1 to S3 with diffused anatomical boundaries. We identified two different TAL cell types, the MTAL phenotype restricted to the MTAL and the CTAL phenotype present in higher abundance in both subsegments. These cell types may correspond to the medullary "rough surface" and cortical "smooth surface" cells described in rat TAL. In summary, the terminology used in scRNAseq cluster assignment referring to specific nephron segments is misleading, as it encompasses anatomical regions with multiple coexisting cell types. This highlights the need for caution when interpreting such data. This work was supported in part by CWRU BGT670107 and NIH-NIDDK DK128304 grants to AGV. 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.

Transcriptomic, epigenomic, and spatial metabolomic cell profiling redefines regional human kidney anatomy

A large-scale multimodal atlas that includes major kidney regions is lacking. Here, we employed simultaneous high-throughput single-cell ATAC/RNA sequencing (SHARE-seq) and spatially resolved metabolomics to profile 54 human samples from distinct kidney anatomical regions. We generated transcriptomes of 446,267 cells and chromatin accessibility profiles of 401,875 cells and developed a package to analyze 408,218 spatially resolved metabolomes. We find that the same cell type, including thin limb, thick ascending limb loop of Henle and principal cells, display distinct transcriptomic, chromatin accessibility, and metabolomic signatures, depending on anatomic location. Surveying metabolism-associated gene profiles revealed non-overlapping metabolic signatures between nephron segments and dysregulated lipid metabolism in diseased proximal tubule (PT) cells. Integrating multimodal omics with clinical data identified PLEKHA1 as a disease marker, and its invitro knockdown increased gene expression in PT differentiation, suggesting possible pathogenic roles. This study highlights previously underrepresented cellular heterogeneity underlying the human kidney anatomy.

Identification of countercurrent tubule-vessel arrangements in the early development of mouse kidney based on immunohistochemistry and computer-assisted 3D visualization.

Nephron loop-vessel countercurrent arrangement in the medulla provides the structural basis for the formation of concentrated urine. To date, the morphogenesis of it and relevant water and solutes transportation has not been fully elucidated. In this study, with immunohistochemistry for aquaporins (AQP) and Na-K-2Cl co-transporter (NKCC2), as well as 3D visualization, we noticed in embryonic day 14.5 kidneys that the countercurrent arrangement of two pairs of loop-vessel was established as soon as the loop and vessel both extended into the medulla. One pair happened between descending limb and ascending vasa recta, the other occurred between thick ascending limb and descending vasa recta. Meanwhile, the immunohistochemical results showed that the limb and vessel expressing AQP-1 such as descending thick and thin limb and descending vasa recta was always accompanied with AQP-1 negative ascending vasa recta or capillaries and thick ascending limb, respectively. Moreover, the thick ascending limb expressing NKCC2 closely contacted with descending vasa recta without expressing NKCC2. As kidney developed, an increasing number of loop-vessels in countercurrent arrangement extended into the interstitium of the medulla. In addition, we observed that the AQP-2 positive ureteric bud and their branches were separated from those pairs of tubule-vessels by a relatively large and thin-walled veins or capillaries. Thus, the present study reveals that the loop-vessel countercurrent arrangement is formed at the early stage of nephrogenesis, which facilitates the efficient transportation of water and electrolytes to maintain the medullary osmolality and to form a concentrated urine.

Mild dehydration effects on the murine kidney single-nucleus transcriptome and chromatin accessibility.

Daily, we may experience mild dehydration with a rise in plasma osmolality that triggers the release of vasopressin. Although the effect of dehydration is well characterized in collecting duct principal cells (CDPCs), we hypothesized that mild dehydration (<12 h) results in many kidney cell-specific changes in transcriptomes and chromatin accessibility. Single-nucleus (sn) multiome (RNA-assay for transposase-accessible chromatin) sequencing and bulk RNA sequencing of kidneys from male and female mice that were mildly water deprived or not were compared. Water-deprived mice had a significant increase in plasma osmolality. sn-multiome-seq resulted in 19,837 nuclei that were annotated into 33 clusters. In CDPCs, aquaporin 2 (Aqp2) and aquaporin 3 (Apq3) were greater in dehydrated mice, but there were novel genes like gremlin 2 (Grem2; a cytokine) that were increased compared with ad libitum mice. The transcription factor cAMP-responsive element modulator (Crem) was greater in CDPCs of dehydrated mice, and the Crem DNA motif was more accessible. There were hundreds of sex- and dehydration-specific differentially expressed genes (DEGs) throughout the kidney, especially in the proximal tubules and thin limbs. In male mice, DEGs were enriched in pathways related to lipid metabolism, whereas female DEGs were enriched in organic acid metabolism. Many highly expressed genes had a positive correlation with increased chromatin accessibility, and mild dehydration exerted many transcriptional changes that we detected at the chromatin level. Even with a rise in plasma osmolality, male and female kidneys have distinct transcriptomes suggesting that there may be diverse mechanisms used to remain in fluid balance.NEW & NOTEWORTHY The kidney consists of >30 cell types that work collectively to maintain fluid-electrolyte balance. Kidney single-nucleus transcriptomes and chromatin accessibility profiles from male and female control (ad libitum water and food) or mildly dehydrated mice (ad libitum food, water deprivation) were determined. Mild dehydration caused hundreds of cell- and sex-specific transcriptomic changes, even though the kidney function to conserve water was the same.

#5592 THE SODIUM PHOSPHATE COTRANSPORTER NAPI-IIB IS UPREGULATED AND EXPRESSED DE NOVO IN KIDNEYS OF PATIENTS WITH CHRONIC KIDNEYS DISEASE

Abstract Background and Aims Renal reabsorption of phosphate is mediated by 3 main transporters located in the luminal brush border membrane of the proximal tubule, namely NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit2 (SLC20A2). The expression of all three proteins has been shown to be reduced in animal models of kidney disease [1]. Recently, we demonstrated that NaPi-IIb, a paralogue of the SLC34 subfamily that is mostly expressed in small intestine and lungs, is also expressed in the loop of Henle. Its mRNA expression is enhanced in rodent models of kidney injury. However, the expression of NaPi-IIb has not been characterized in humans and it is unknown whether NaPi-IIb is also upregulated in chronic kidney disease (CKD). Method In this study, we examined the localization of NaPi-IIa, IIb, and IIc in kidneys of control healthy individuals (kidney donors) (eGFR: 111.6 ± 22.9 ml/min/1.73m2) and patients with diabetic nephropathy (eGFR: 31.9 ± 7.7 ml/min/1.73m2), anti-glomerular basement membrane nephritis (anti-GMB) (eGFR: 27.2 ± 22.6 ml/min/1.73m2), and hypertensive nephropathy (eGFR: 31.5 ± 5.3 ml/min/1.73m2) by immunofluorescence. Moreover, we analyzed data from various transcriptome data bases from healthy individuals and patients with CKD. Results In kidneys from healthy donors, NaPi-IIb staining was restricted to thin and thick limbs of the loop of Henle, while AQP1 was observed in proximal tubules and descending thin limbs. NaPi-IIc was abundantly found in proximal tubule cells. In biopsies from patients, NaPi-IIb signal was also found in addition to thin and thick limbs in cells of the collecting duct and in many proximal tubules. In contrast, the staining of NaPi-IIc was reduced in all groups of patients with CKD. RNA expression data from the European Renal cDNA Bank (ERCB) confirmed our immunofluorescence findings. SLC34A2 (NaPi-IIb) mRNA was progressively more expressed in kidneys of patients with kidney disease of tubulointerstitial origin from CKD stages 1 to 5, while it was strongly upregulated from stage 4 onwards in CKD of glomerular origin. Single cell RNA sequencing data of patients with acute kidney injury and CKD [2] showed elevation of SLC34A2 expression in injured thick ascending limb, degenerative proximal tubule cells (14x higher, adj. p≪0.0001) (de novo expression), and descending thin limb (3.7x higher, adj. p≪0.0001). SLC34A1 (NaPi-IIa) expression levels were reduced in whole kidneys (ERCB data) and SLC34A1 and SLC34A3 expression also decreased in proximal tubule cells of patients with CKD. Conclusion The phosphate transporter NaPi-IIb is highly upregulated in kidneys from patients with various etiologies of CKD and its expression spreads to proximal tubule and collecting duct. In contrast, the expression of the main physiological phosphate transporters NaPi-IIa and NaPi-IIc is reduced in CKD. These findings may have implications for the development and maintenance of hyperphosphatemia in CKD patients and limit the use of specific NaPi-IIa and/or NaPi-IIc inhibitors in patients with CKD.

PARAMETERS CONTROLLING THE GEOMETRY OF DETACHMENT AND FAULT‐BEND FOLDS: INSIGHTS FROM 3D FINITE‐ELEMENT MODELS APPLIED TO THE AHWAZ ANTICLINE IN THE DEZFUL EMBAYMENT, SW IRAN

Fault‐related folds are present in most tectonic settings and can serve as structural traps for hydrocarbons. These structures have therefore been widely studied by both structural and petroleum geologists using a range of techniques. Approaches include field‐ and seismic‐based methods, and numerical and analogue modelling. Geomechanical models attempt to examine the mechanical and geometric features of folds.This study investigates the effects of variations in a range of parameters, including detachment and ramp geometry, friction coefficient and internal friction angle, on the geometry and development of detachment folds and fault‐bend folds. For this purpose, we ran seven series of numerical, 3D elastic‐plastic finite element models using ABAQUS software (26 model runs in all). Each model set‐up consisted of five layers whose mechanical properties were based on those of stratigraphic units in the Zagros fold‐and‐thrust belt, SW Iran. The models were labelled series A to F and series H. Models in series A investigated the impact of concave, convex, wavy and oblique detachment surfaces on the development of detachment folds; those in series D examined the role of ramp dip and of listric, oblique and wavy ramps on the development of fault‐bend folds. Models in series B and E, and series C and F, examined the effects of variations in the friction coefficient and of the internal friction angle, respectively, on the development of these two classes of folds. Finally, hybrid models in series H were provided to evaluate the results.Major results were as follows. Firstly, the geometry of modelled detachment and fault‐bend folds was found to be influenced by the geometry of the associated ramps and detachment faults. Thus the crests of anticlines and the trough lines of synclines were located at points of maximum curvature and at inflexion points on a wavy detachment fault or wavy ramp, respectively. Second, two important additional factors controlling fold style were identified: the friction coefficient, and the presence of along‐strike geometric variations in the ramp or the detachment fault. Layers with low friction coefficients and high internal friction angles formed detachment folds with thick hinges and thin limbs; conversely, layers with high friction coefficients and low internal friction angles created detachment folds with thick limbs and thin hinges. Application of the results to modelling of the Ahwaz anticline in the Dezful Embayment, SW Iran, was successful, and in general the modelled structure was consistent with that observed in the field.

Lectin Histochemistry of the Normal Feline Kidney.

Lectins have a strict binding specificity to carbohydrate moieties of cellular components, and can thus indicate changes in the glycosylation of cells in diseases. However, lectin-binding patterns in nephron segments of feline kidneys have not been fully surveyed. The present study reported lectin-binding patterns in normal feline kidneys by histochemical investigations of eight commercially available lectin detection kits. Kidneys from four normal cats (intact males, 23-27 months old) were fixed in 4% paraformaldehyde, and embedded in paraffin; lectin histochemistry was performed for WGA, s-WGA, RCA-I, ConA, PNA, SBA, DBA, and UEA-I lectins. WGA, RCA, and ConA binding was observed from Bowman's capsule to the collecting ducts, while only WGA was detected in the glomerular capillary. s-WGA was observed from the proximal tubules to the collecting ducts, showing discriminative heterogeneous binding. PNA and SBA were detected in the distal nephrons, such as the thin limbs of the loops of Henle, distal tubules, and collecting ducts. UEA-I binding was observed in the thick ascending limbs of the loops of Henle, especially in the macula densa regions. DBA lectin showed no positive labeling in nephrons. The observed binding patterns may prove beneficial for the analysis of changes in glycosylation in feline kidney diseases.

LBODP030 A Rare Case Of Partial Lipodystrophy From Mpdl (mandibular Hypoplasia, Deafness, Progeroid, Lipodystrophy Syndrome) Due To Pold1 Gene Mutation

Abstract MDPL (Mandibular hypoplasia, Deafness, Progeroid, Lipodystrophy syndrome) caused by a mutation in the polymerase delta 1 (POLD1) gene is an extremely rare progeroid syndrome associated with partial lipodystrophy. Currently, there are about 24 cases reported worldwide(1), mostly in people of European descent. We are reporting one such rare case in a Hispanic patient. The patient is a 37 year old male from Honduras who was referred to us by genetics for diabetes management. Family members noted extremely thin limbs and he did not walk till 2 years of age. Family history was negative with 8 full siblings, all reportedly healthy. On examination, his BMI was 15.19 kg/m2. He was noted to have birdlike facies with mid-face hypoplasia, beaked nose, retrognathia, dental malocclusion, and protruding maxillary teeth. He also had bilateral proptosis. His extremities were thin, lacking subcutaneous fat and muscle bulk with distal joint contractures in elbow, hands and feet. Genitourinary showed low testicular volumes (&amp;lt;3 ml). Genetic testing revealed a recurring mutation in the active site of POLD1 gene, which is diagnostic for MDPL. His clinical presentation was classic with dysmorphism, deafness, lipodystrophy and metabolic abnormalities. Due to the rarity of the disorder, there is very limited data on management of these patients. A symptom-based management was recommended. In terms of his diabetes, it was diagnosed at age 33 when his HbA1c was 11.2 and he was started on insulin. Patients with this syndrome tend to have insulin resistance. C-peptide was 2.88 ng/ml (ref. range: 0.78 - 5.19 ng/mL) with glucose 160 mg/dL indicating adequate beta-cell reserve; GAD-65 and anti-pancreatic islet cell antibodies were negative. Insulin was weaned off and currently, his diabetes is being managed with diet and lifestyle changes. He was found to have elevated total cholesterol and triglycerides. Hepatic steatosis was seen on liver ultrasonography. He was started on fenofibrate and low-fat diet; lipid panel has normalized. His echocardiogram was normal. In addition, males are known to have hypogonadotropic hypogonadism however this patient had a low testosterone and high FSH and LH, indicative of primary hypogonadism. A scrotal ultrasound was ordered. DXA scan showed low bone mineral density with the lowest Z-score of -3.6 at the lumbar spine. He was started on testosterone replacement with Vitamin D. Testosterone therapy will help replace the testosterone deficiency in addition to slowing down bone loss. MDPL syndrome is an exceedingly rare clinical entity associated with multiple endocrinopathies. We plan to periodically monitor labs and imaging to continue managing this patient with a symptom-based approach. (1) Wang, Xiu-Wen; Lu, Ling-Yun; Xie, Ying1; Yu, Xi-Jie1 A Chinese girl with (MDPL) diagnosed via POLD1 mutation detection, Chinese Medical Journal: August 20, 2020 - Volume 133 - Issue 16 - p 2009-2011 Presentation: No date and time listed

Immunolocalization of intermediate filaments in the kidney of the dromedary camel (Camelus dromedarius)

Intermediate filaments belong to a large family of proteins which contribute to the formation of the cytoskeleton. The immunolocalization of cytoskeletal proteins has been used extensively in the diagnosis of various renal pathologies. The present study described the immunolocalization of the cytoskeletal proteins vimentin, desmin, smooth muscle actin, and cytokeratin 19 in the normal kidney of the dromedary camel. Kidney samples from eight adult camels were processed for histology and immunohistochemistry. The kidney was enclosed in a renal capsule composed of vimentin immunoreactive fibroblasts and smooth muscle actin immunoreactive smooth muscle cells. The smooth muscle cells in the renal capsule did not exhibit desmin immunoreactivity. Podocytes forming the visceral layer of the glomerular capsule were immunoreactive for vimentin. Immunoreactivity for vimentin and smooth muscle actin in the parietal layer of the glomerular capsule varied, with both reactive and non-reactive cells observed. Intraglomerular mesangial cells were immunoreactive for smooth muscle actin and desmin, but non-reactive to vimentin. The endothelial lining of blood vessels was vimentin immunoreactive, while smooth muscle actin and desmin were demonstrated in the smooth muscle cells of the vessels. The thin limbs of the loops of Henle in cortical nephrons displayed vimentin immunoreactivity. The proximal and distal convoluted tubules, as well as the collecting ducts were negative to vimentin, smooth muscle actin, desmin and cytokeratin 19 immunostaining. In conclusion, the present study has revealed that similarities and differences exist in the immunolocalization of cytoskeletal proteins in the camel when compared to other mammals. The presence of smooth muscle actin in the parietal cells of the glomerular capsule suggests a contractile function of these cells. The results of the study indicate that vimentin and smooth muscle actin can be used as markers for the identification of podocytes and intraglomerular mesangial cells, respectively, in the camel kidney.

Analysis of a Dog Skeleton Deposited in a Scythian Tomb-91/2016 from Sâncrai (Alba County)

The article presents the results of the analysis of a dog skeleton found over a human skeleton, in the Scythian tomb, 91/2016 in Sâncrai, Alba county. The dog was completely deposited on its right side, over the human corpse. Over time, the animal bones deteriorated, becoming crumbly, so that few complete pieces could be recovered. According to measurements, the animal is 43.5 cm tall with thin limbs. The proximal segment of the legs provided a slightly higher waist (45.4 cm) than the distal one, (39.8 c). The animal is slightly smaller compared to those from the Scythian world, with sizes over 50 cm. During the re-inhumation process, the human bones were mixed with those of the meat offering which was laid into the pit during the initial funerals. The meat offering consisted of a part from the left limb of a cattle, slaughtered towards 2 years.

A Novel Homozygous WRN Mutation Identified in a Middle Aged Man With Diabetes Mellitus Complicated By Multiple Features of Accelerated Aging

Background: Werner syndrome (WS; MIM# 277700) is a rare genetic disorder characterized by accelerated aging and predisposition to cancers. The causal gene, WRN, encodes a multifunctional nuclear protein that belongs to the RecQ family of DNA helicases. The earliest sign of WS is short stature due to the lack of pubertal growth spurt. In addition to the aged-appearance, WS patients typically develop functional declines of multiple organs including the endocrine system in their middle ages. Frequent Endocrine disorders include Type 2 Diabetes (T2D), dyslipidemia (DL), hypothyroidism, hypogonadism and osteoporosis. Most common causes of deaths are myocardial infarction and cancers in their 50s. Clinical Case: A 54-year old man was admitted for right wrist septic arthritis. His medical history was pertinent for premature cataract diagnosed in his 2nd decade. He had uncontrolled T2D (peak HbA1C level 10%) since the age of 32 years, treated with oral hypoglycemics including glimepiride, sitagliptin and metformin then shifted to insulin over the past 2 years. He also had DL since the age of 41 years, characterized by hypertriglyceridemia (766 mg/dL) with very low HDL (7 mg/dL). At age 50, he was diagnosed with hypothyroidism and negative anti-TPO antibodies. Moreover, he had refractory bilateral leg ulcers that manifested at the age of 35 years requiring multiple debridments, premature coronary artery disease at the age of 38 years and ischemic cardiomyopathy. On examination, his height was 154 cm and weight 42 kgs. He had prominent eyes, a pinched nose, grey and sparse scalp hair with absence of eyebrows and eyelashes. Hoarse voice was easily noticeable. He had very thin limbs, flat feet, central fat distribution and muscle wasting. Skin was shiny, tight, and atrophic with overlying hypermelanosis. Old bilateral hyperkeratotic deep feet ulcers were noticed with variable necrosis. Blood chemistry testing showed: HbA1C 8.2%, disturbed lipid profile, TG 341 mg/dL, HDL 17 mg/dL, LDL 155 mg/dL, and TSH 21 µU/mL (0.2 - 4.2 µU/mL). MRI of the foot revealed Achilles’ tendon calcification. His parents were 2nd degree cousins and had normal features. Three of his siblings had similar features and premature death. His diabetes was atypical due to his low BMI, high insulin requirements and the relatively early onset. WS was suspected based on the overall features of accelerated aging with characteristic deep leg ulcers and the family history. Genetic sequence analysis revealed a novel pathogenic homozygous nonsense variant mutation c.1111G>T, p.Glu371* of the WRN gene. Conclusion: Werner syndrome is a rare genetic disease, but it carries high morbidity and mortality burden, in addition to impaired quality of life. It should be highly suspected in patients with atypical T2D, DL, premature coronary disease, refractory leg ulcers with features of accelerated aging and short stature.

Genetic Deletion of the Prostaglandin EP3 Receptor in the Kidney Tubule of Adult Mice Has No Impact on Kidney Water Handling

Prostaglandin E2 (PGE2) is an important lipid mediator modulating various aspects of kidney function. PGE2 exerts its effects via four PGE2 receptors, EP1-EP4. The EP3 receptor is expressed in the thick ascending limb (TAL) and the collecting duct, where it is proposed to inhibit cAMP generation and NaCl and water reabsorption. However, EP3 is also expressed in endothelial cells of arteries and arterioles, which also play a role in kidney function. Therefore, to assess the tubular role of EP3 in adult mice we generated a mouse model based on the Pax8Cre system with doxycycline-dependent deletion of EP3 along the renal tubule and assessed their renal phenotype in respect to water handling. RNAscope confirmed that EP3 was highly expressed in cortical and medullary TAL and collecting ducts, but it was non-detected in proximal tubule and thin limbs. Two weeks after treatment with doxycycline, EP3 mRNA expression was reduced by >80% in whole kidney (RT-q-PCR) and non-detectable (RNAscope) in tubules of knockout mice compared to control mice. There were no compensatory changes in other EP receptors. Under basal conditions, there were no significant differences in food and water intake, bodyweight, urinary output or plasma and urine biochemistries in both male or female control and knockout mice. There were no differences between genotypes in their renal handling of water during an acute water load, or in their response to the vasopressin V2 receptor agonist dDAVP. Rats drinking 1% NaCl for several days in combination with an EP3 antagonist have increased urine output (Hao et al., 2016). However, relative to controls, we could not detect significant differences in urine volume or osmolality in tubular EP3 knockout mice during 1% NaCl intake. In conclusion, EP3 in the renal tubule is not important for renal water handling or compensatory mechanisms exist. This new model provides a novel tool for examination of the role of EP3 in other aspects of renal function or kidney disease independently of potential developmental abnormalities or systemic effects.

Forehead Swelling and Fever in a 12-year-old Ugandan Boy.

1. Ashley R.B. Bjorklund, MD*,† 2. Tina M. Slusher, MD*,† 3. Abigail Faulman, MD‡ 4. Cynthia R. Howard, MD† 5. Beatrice Odongkara, MBChB§ 1. *Hennepin Healthcare, Minneapolis, MN 2. †University of Minnesota, Minneapolis, MN 3. ‡Children’s Minnesota, Minneapolis, MN 4. §Gulu University/Gulu Regional Referral Hospital, Gulu, Uganda A 12-year-old boy with a history of sickle cell disease (SCD) presents to a government regional referral hospital in Northern Uganda with complaints of feeling ill with fever and a painful lump on his forehead. He also notes pain and swelling of his left upper arm. The fever, forehead lump, and painful swelling of the left arm had been present for at least 2 months. He denies any trauma. On presentation to the hospital he is febrile, tachycardic, and ill appearing. Initial physical examination is remarkable for frontal bossing with multiple tender, fluctuant, round (2- to 3-cm-diameter) lesions over his forehead and scalp; respiratory distress; a prominent systolic murmur; thin limbs; a tender, indurated lesion (approximately 5 × 3 cm) draining purulent fluid on his left upper arm; and no noted focal neurologic deficits. In the emergency department, incision and drainage of the forehead lesion yields scant drainage despite what appears to be a visibly purulent and fluctuant swelling. The fluid obtained is serous and bloody, without overt purulence. There is concern that the lesions may involve more than the subcutaneous tissues, with possible extension to the bone. Laboratory tests show leukocytosis with a neutrophil predominance (white blood cell count, 23,360/μL [23.36×109/L]; neutrophils, 77.5%), thrombocytosis, and anemia (hemoglobin level, 7.7 g/dL [4.78 mmol/L]). Rapid diagnostic testing for malaria is negative. Urine is remarkable for dark coloration without red blood cells on microscopic examination. Cultures from the wound are sent to the laboratory. Radiographs of the cranium (Fig 1) and humerus (Fig 2) are obtained. Figures 1. Radiograph demonstrating lytic lesions of the frontal and parietal bones. Figure 2. Radiograph demonstrating …

Management guideline for Werner syndrome 2020. 2. Sarcopenia associated with Werner syndrome.

Sarcopenia is defined as a condition that combines decreased skeletal muscle mass with weakness or decreased physical function. It is well known that in older adults, the presence of sarcopenia is a risk of frailty, falls and physical dysfunction. Patients with Werner syndrome are characterized by visceral fat accumulation and thin limbs, but the prevalence of sarcopenia in patients with Werner syndrome has not been investigated. A literature search was conducted using Werner syndrome and skeletal muscle as keywords. We also analyzed data from our 7 Werner syndrome patients. A literature search on the relationship between Werner syndrome and skeletal muscle yielded only one article reported from Japan. According to this paper, a decrease in skeletal muscle mass (appendicular skeletal muscle index) was observed in all 9 Werner syndromes investigated. On the other hand, in our 7 Werner syndrome patients, their appendicular skeletal muscle indexes were below the standard value except for one male patient who had continued resistance exercise. The decrease in skeletal muscle mass frequently occurs in patients with Werner syndrome. However, resistance exercise may prevent the appearance of sarcopenia and requires early intervention in patients with Werner syndrome. Geriatr Gerontol Int 2021; 21: 139-141.

Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka

The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.

SO040ROLE OF INTRACELLULAR CA2+ IN THE REGULATION OF THE RENAL CHLORIDE CHANNEL CLC-KA

Abstract Background and Aims It has been recognized that the physiological role of ClC-Ka and its mouse homolog ClC-K1 in the nephron is to confer high Cl- permeability in the thin Ascending Limb of loop of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla in the countercurrent system. Accordingly ClC-K1 KO mice display a severe polyuria resembling a Nephrogenic Diabetes Insipidus (NDI) phenotype. Thus, ClC-Ka in human nephron could be an interesting target for diuretic and anti-hypertensive drugs. Unfortunately the regulation of ClC-Ka in and its electrophysiological properties are still not completely known. Method We analyzed, by whole-cell voltage-clamp, Cl- currents in HEK293 cells co-expressing GFP-ClC-Ka and the accessory m-Cherry-tagged barttin subunit, upon intracellular Ca2+ mobilization. The patch clamp protocol consists of a holding potential from 0 mV, which is stepped to various test potentials from -195 and +125 mV for 150 ms. Pulses ended with a tail pulse to -125 mV for 20 ms. Concomitantly we monitored by live confocal microscopy, the membrane expression of either GFP-ClC-Ka or barttin-mCherry. Results In control conditions we found a linear current/voltage relationship over a broad range of membrane voltages except of hyperpolarization below -100 mV, where the channel open probability was reduced, providing the well-known hook-shaped appearance of the I-V curve for ClC-Ka channel. Interestingly, both 100 µM ATP and 400 µg/ml of dandelion root extract (DRE), both increasing intracellular Ca2+ in HEK293, significantly inhibited Cl- currents in a time dependent way. Pre-treatment of cells with either the Ca2+ chelator BAPTA-AM or the PCK inhibitor Calphostin C, reverted the inhibitory effects of both ATP and DRE. Interestingly, 1µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of ATP and DRE on Cl- currents. We finally demonstrated that 30 µM Heclin, an inhibitor of E3 ubiquitin ligase, did not revert the DRE- and ATP- induced Cl- currents inhibition, thus suggesting that the ClC-Ka inhibition is independent of channel ubiquitination. Accordingly, live confocal analysis showed that all the Ca2+ mobilizing compounds tested did not induced neither ClC-Ka nor Barttin internalization. We hypothesized that phosphorylation in the putative PKC consensus sites present in ClC-Ka amino acidic sequence may be involved in the inhibition of channel activity. Conclusion In conclusion, we demonstrated for the first time that the activity of ClC-Ka in renal cells could by inhibited by either receptor agonists or natural molecules able to activate an intracellular Ca2+/PKC pathway, thus opening venues for the development of new diuretic drugs.

Acute Kidney Injury in COVID-19: Emerging Evidence of a Distinct Pathophysiology.

Acute Kidney Injury in COVID-19: Emerging Evidence of a Distinct Pathophysiology.

New Insights into the Role of Intracellular Ca2+ in the Regulation of the Human Kidney Chloride Channel ClC‐Ka

It has been recognized that the physiological role of ClC‐Ka and its mouse homolog ClC‐K1 in the nephron is to confer high Cl− permeability in the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla in the countercurrent system. Accordingly ClC‐K1 KO mice display a severe polyuria resembling a Nephrogenic Diabetes Insipidus (NDI) phenotype. Thus, ClC‐Ka in human nephron could be an interesting target for diuretic and anti‐hypertensive drugs. Unfortunately the regulation of ClC‐Ka in and its electrophysiological properties are still not completely known. We analyzed, by whole‐cell voltage‐clamp, Cl− currents in HEK293 cells expressing GFP‐ClC‐Ka and the accessory m‐Cherry Barttin subunit upon intracellular Ca2+mobilization. The patch clamp protocol consists of a holding potential from 0 mV, which is stepped to various test potentials from −195 and +125 mV for 150 ms. Pulses ended with a tail pulse to −125 mV for 20 ms. In control conditions we found a linear current/voltage relationship over a broad range of membrane voltages except of hyperpolarization below −100 mV, where the channel open probability was reduced, providing the well‐known hook‐shaped appearance of the I‐V curve for ClC‐Ka channel. Interestingly, both 100 μM ATP and 400 μg/ml of dandelion root extract (DRE), both increasing intracellular Ca2+ in HEK293, significantly inhibited Cl− currents in a time dependent way. Pretreatment of cells with either the Ca2+ chelator BAPTA‐AM or the PCK inhibitor Calphostin C, reverted the inhibitory effects of both ATP and DRE. Interestingly, 1μM of Phorbol Meristate Acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of ATP and DRE on Cl− currents. We finally demonstrated that 30 μM Heclin, an inhibitor of E3 ubiquitin ligase, did not revert the DRE‐ and ATP‐ induced Cl− currents inhibition, thus suggesting that the ClC‐Ka inhibition is independent of channel ubiquitination. Accordingly, live confocal analysis showed that all the Ca2+mobilizing compounds tested did not induced neither ClC‐Ka nor Barttin internalization. We hypothesized that phosphorylation in the putative PKC consensus sites present in ClC‐Ka amino acidic sequence may be involved in the inhibition of channel activity. In conclusion, we demonstrated for the first time that the activity of ClC‐Ka in renal cells could by inhibited by either receptor agonists or natural molecules able to activate an intracellular Ca2+/PKC pathway, thus opening venues for the development of new diuretic drugs.Support or Funding InformationGrant n# ARS01_01081 to MC from The National Operational Program (PON) of the Ministry of Education, ITALYFigure 1

Combinatorial expression of claudins in the proximal renal tubule and its functional consequences

The proximal renal tubule (PT) is characterized by a highly conductive paracellular pathway, which contributes to a significant amount of solute and water reabsorption by the kidney. Claudins are tight junction proteins that, in part, determine the paracellular permeability of epithelia. In the present study, we determined the expression pattern of the major PT claudins. We found that claudin-2 and claudin-10 are coexpressed throughout the PT, whereas claudin-3 is coexpressed with claudin-2 predominantly in the proximal straight tubule. Additionally, claudin-2 and claudin-3 are expressed separately within mutually exclusive populations of descending thin limbs. We developed a novel double-inducible Madin-Darby canine kidney I cell model to characterize in vitro the functional effect of coexpression of PT claudins. In keeping with previous studies, we found that claudin-2 alone primarily increased cation (Na+ and Ca2+) permeability, whereas claudin-10a alone increased anion (Cl-) permeability. Coexpression of claudin-2 and claudin-10a together led to a weak physical interaction between the isoforms and the formation of a monolayer with high conductance but neutral charge selectivity. Claudin-3 expression had a negligible effect on all measures of cell permeability, whether expressed alone or together with claudin-2. In cells coexpressing a claudin-2 mutant, S68C, together with claudin-10a, inhibition of cation permeability through the claudin-2 pore with a thiol-reactive pore blocker did not block anion permeation through claudin-10a. We conclude that claudin-2 and claudin-10a form independent paracellular cation- and anion-selective channels that function in parallel.

Fitness Programs Features According to the Body Types of High School Girls

The Actuality of the Research. Fitness is one of the most innovative approaches in the establishment of the physical training programme, which is good for shaping the body and body mass. Fitness contributes to the harmonization of physical development and improves the functional statement of body’s system. In the contemporary scientific researches it is expected that theoretical-methodological basis of fitness integration in the school system of physical education will contribute to increasing of physical preparedness level and motivation to practice sports among the teenagers, their physical development, health promotion, socialization and self-determination. The Methods of the Research. The analysis of academic papers, questionnaire, testing, medical examination of students, pedagogical experiment; determination of the level of physical development and physical preparedness; mathematical statistics method. The study was conducted on the basis of the Lutsk Middle School of General Education I–III levels № 18, Lutsk Middle School of General Education I– III levels № 25, Lutsk Jurisprudential Lyceum of Heavy Physical Training. 120 high school girls were involved in the study. The Results of the Research. Different physical types were taken into account by developing fitness programs (asthenic, hypersthenic, normosthenic). The results of the study show that 22 % of high school girls are asthenic body type, 19 % are hypersthenic, 59 % of high school girls are normosthenic body type. The strength exercises to increase the body mass and improve the muscle tone are recommended for girls of asthenic body type with slight stature, above average height, narrow shoulders, thin limbs and underweight. The high school girls of hypersthenic body type are with a massive body, average height, broad shoulders, short limbs and they are overweight. Physical exercises for them were focused on decrease in weight of a body, the reduction of body types (shoulders, thorax, stomach, pelvis, hips), the reduction of adipose tissue. The high school girls of normosthenic body type have relatively proportional body. That is why the physical activities are aimed at improvement of muscle tone, slowdown of body mass and reduction of the pelvis. Висновки . Результати дослідження будуть застосовані до створення програми з метою підвищення фізичної активності та поліпшення функціональної та фізичної підготовленості для дівчат середньої школи.