Urinary small extracellular vesicles derived CCL21 mRNA as biomarker linked with pathogenesis for diabetic nephropathy

Background Diabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking. Methods Fourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ. Results The number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model. Conclusions Urinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.

MO1030THE FIRST CASE OF ATYPICAL HEMOLYTIC UREMIC SYNDROME (AHUS) FEATURES IN AN EXPERIMENTAL MODEL

Background and Aims Thrombotic Microangiopathies (TM) are three distinct clinical syndromes presenting the same histological renal pattern: typical and atypical Hemolytic Uremic Syndrome (SEU- aSEU) and Thrombotic Thrombocytopenic Purpura (TTP). So far, the first report of a TM has been generally attributed to Eli Moschowitz. In 1922 he described the case of a 16 years old girl who died after acute onset of fever with petechial lesions and autoptic findings of hyaline thrombosis of terminal arterioles and capillaries, thus profiling the first case of TTP. Only in the 1955, Conrad Gasser described the first medical record of HUS, describing the case of a patient with a manifestation of bilateral necrosis of the renal cortex. We describe the first reported case of a Thrombotic Microangiopathy, in particular an experimentally induced aHUS by Richard M. Pearce in 1909. In this case, the trigger leading to aHUS was represented by snake venom injection in an experimental rabbit model. Method Pearce described acute glomerular lesions produced in the rabbit using dried venom of rattlesnake Crotalus Adamanteus. It was dissolved in salt solution in the proportion of 0.25 of a milligram to one cubic centimeter, rising gradually to two milligrams, and then followed by doses of 0.5 of a milligram of fresh venom at various intervals. The intervals between injections depended on the general condition of the animal and the amount of albuminuria. Results Rabbit kidneys showed well marked hemorrhagic and exudative lesions in the glomeruli; hyaline, granular, blood, and hemoglobin casts in both convoluted and collecting tubules; and granular degeneration of the epithelium of the convoluted tubules and loops of Henle. Pearce also described a “penetration of the cells of the compressed glomerular tuft into the mass of hemorrhage lying either in the tuft itself or in the capsular space” (Figure 1). In animals surviving 20-30 days after the first injection of the venom, the acute lesions were demonstrated to subside at the microscopic examination and their earlier presence was marked by “occasional casts and compressed masses of red cells in the glomerular spaces and tufts”. At the same time other models also showed “extensive granular degeneration of the convoluted tubules and many casts”. The renal autoptic findings presented the features of a vascular nephritis with severe endothelial changes. Conclusion Glomerular and tubular lesions of rabbits’ kidneys induced by crotalus’s venom showed typical features of what is today defined as aHUS. In this first experiment, the author described a glomerular tuft as “more analogous to the organization of a red thrombus than it is to any form of glomerular lesion known in man”, so we can affirm that Pearce described, ante litteram TMA histological features many years before other scientists.

EFFECT OF NICOTINE SMOKE ON LIVER AND KIDNEYS OF ADULT MALE ALBINO RATS: AN EXPERIMENTAL STUDY

Apart from heart and lungs, being cited as the main affected organs by nicotine smoke and hence, contributing for the morbidity and mortality, other organs like kidneys, gastrointestinal tract, liver, reproductive organs, endocrine glands, skin etc. are also affected. In the current study we planned to evaluate the effects of nicotine smoke on liver and kidneys. The study was conducted on 12 inbred adult Wistar albino rats; 6 animals acting as control and remaining 6 acting as test group. The animals of control group were given only sterile water where as animals of test group were exposed to smoke produced from the nicotine wrapped in a cotton wool in the dose of 6mg/day three times a day for each session of 5 minutes each for 5 days. Each rat was exposed to the smoke produced due to nicotine separately in a closed inhalational chamber and not in groups. The rats were sacrificed after the period of experimentation and testis were dissected out and subjected further to tissue processing for histological examination. The histological examination of tissue sections of liver of test group animals revealed distorted or normal lobular architecture of hepatic lobules with dilated and congested central vein, portal venule and hepatic sinusoids. Also, there was presence of focal inflammatory aggregates especially around bile canaliculi at portal triads. On the other hand, the tissue sections of liver of control group showed normal hepatic architecture with normal hepatocytes. The renal specimens of the control group also demonstrated normal renal architectural pattern; with glomeruli surrounded by urinary space and renal tubules present in cortex and medulla composed of collecting tubules, loop of henle and convoluted tubules. On the contrary, the renal sections of test group showed dilation of urinary space, shrunken and distorted glomeruli with some renal tubules showing solid cord like pattern and some showing eosinophilic material in lumen, focal inflammatory infiltrates and renal congestion. Keywords: Nicotine smoke, liver, kidney

Extra-gastric expression of the proton pump H+/K+-ATPase in the gills and kidney of the teleost Oreochromis niloticus

ABSTRACTPotassium regulation is essential for the proper functioning of excitable tissues in vertebrates. The H+/K+-ATPase (HKA), which is composed of the HKα1 (gene: atp4a) and HKβ (gene: atp4b) subunits, has an established role in potassium and acid–base regulation in mammals and is well known for its role in gastric acidification. However, the role of HKA in extra-gastric organs such as the gill and kidney is less clear, especially in fishes. In the present study in Nile tilapia, Oreochromis niloticus, uptake of the K+ surrogate flux marker rubidium (Rb+) was demonstrated in vivo; however, this uptake was not inhibited with omeprazole, a potent inhibitor of the gastric HKA. This contrasts with gill and kidney ex vivo preparations, where tissue Rb+ uptake was significantly inhibited by omeprazole and SCH28080, another gastric HKA inhibitor. The cellular localization of this pump in both the gill and kidney was demonstrated using immunohistochemical techniques with custom-made antibodies specific for Atp4a and Atp4b. Antibodies against the two subunits showed the same apical ionocyte distribution pattern in the gill and collecting tubules/ducts in the kidney. Atp4a antibody specificity was confirmed by western blotting. RT-PCT was used to confirm the expression of both subunits in the gill and kidney. Taken together, these results indicate for the first time K+ (Rb+) uptake in O. niloticus and that HKA is implicated, as shown through the ex vivo uptake inhibition by omeprazole and SCH28080, verifying a role for HKA in K+ absorption in the gill's ionocytes and collecting tubule/duct segments of the kidney.

Pax2 and Pax8 Proteins Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult Kidney

BackgroundAs the glomerular filtrate passes through the nephron and into the renal medulla, electrolytes, water, and urea are reabsorbed through the concerted actions of solute carrier channels and aquaporins at various positions along the nephron and in the outer and inner medulla. Proliferating stem cells expressing the nuclear transcription factor Pax2 give rise to renal epithelial cells. Pax2 expression ends once the epithelial cells differentiate into mature proximal and distal tubules, whereas expression of the related Pax8 protein continues. The collecting tubules and renal medulla are derived from Pax2-positive ureteric bud epithelia that continue to express Pax2 and Pax8 in adult kidneys. Despite the crucial role of Pax2 in renal development, functions for Pax2 or Pax8 in adult renal epithelia have not been established.MethodsTo examine the roles of Pax2 and Pax8 in the adult mouse kidney, we deleted either Pax2, Pax8, or both genes in adult mice and examined the resulting phenotypes and changes in gene expression patterns. We also explored the mechanism of Pax8-mediated activation of potential target genes in inner medullary collecting duct cells.ResultsMice with induced deletions of both Pax2 and Pax8 exhibit severe polyuria that can be attributed to significant changes in the expression of solute carriers, such as the urea transporters encoded by Slc14a2, as well as aquaporins within the inner and outer medulla. Furthermore, Pax8 expression is induced by high-salt levels in collecting duct cells and activates the Slc14a2 gene by recruiting a histone methyltransferase complex to the promoter.ConclusionsThese data reveal novel functions for Pax proteins in adult renal epithelia that are essential for retaining water and concentrating urine.

MON-LB65 Lithium: The Culprit of Multiple Endocrinopathies

Background: Lithium, commonly used to treat various psychiatric disorders such as bipolar disorder, can cause acute toxicity that presents with nausea, vomiting and diarrhea. Lithium can also cause life-threatening endocrine abnormalities, including hypercalcemia, hypernatremia, and both hypo- and hyperthyroidism. Clinical Case: A 61-year old female with hypothyroidism, bipolar disorder, hyperparathyroidism with two-gland parathyroidectomy on lithium for over 30 years presented with altered mental status. Initial labs revealed elevated creatinine 1.92 mg/dL (0.8-2.00mg/dL) compared to baseline 0.82 mg/dL, sodium 154 mg/dL (135-147 mg/dL), Corrected calcium 11.7 mg/dL (8.5-10.5 mg/dL), PTH 96 pg/mL (15-65 pg/mL), and high lithium levels 1.45 mmol/L (0.60-1.20 mmol/L). Further studies showed hypotonic polyuria with no increase in urine osmolality after desmopressin, consistent with nephrogenic diabetes insipidus. Lithium was held and she was treated with aggressive intravenous hydration with dextrose 5% water. Hypercalcemia is thought to result from increased secretion of PTH due to an increased set point at which calcium suppresses PTH release; this often resolves once lithium is stopped. Lithium can also unmask previously unrecognized mild hyperparathyroidism, and/or raise serum PTH concentrations independent of calcium levels.1 The drug interferes with the kidneys’ ability to concentrate urine in the collecting tubules by desensitizing response to antidiuretic hormone, causing diabetes insipidus. The resulting volume depletion from excessive urinary water loss in turn lead to acute kidney injury and hypernatremia.2 Hypothyroidism results from lithium-inhibited synthesis and release of thyroid hormones and decreases iodine trapping. Conclusion: Although these are infrequent complications of lithium use, they remain pertinent clinical findings to consider due to their morbidity. In this case, our patient may have avoided multiple chronic electrolyte abnormalities leading to altered mental status if lithium toxicity had been recognized earlier. References:1. García-Maldonado, Gerardo, and Rubén de Jesús Castro-García. “Endocrinological Disorders Related To The Medical Use Of Lithium. A Narrative Review”. Revista Colombiana De Psiquiatría (English Ed.), vol 48, no. 1, 2019, pp. 35-43. Elsevier BV, doi:10.1016/j.rcpeng.2018.12.005. 2. Tasci, E. “Lithium-Induced Nephrogenic Diabetes Insipidus Responsive To Desmopressin”. Acta Endocrinologica (Bucharest), vol 15, no. 2, 2019, pp. 270-271. ACTA Endocrinologica Foundation, doi:10.4183/aeb.2019.270.

ESTIMATION OF TIME SINCE DEATH BY HISTOLOGICAL EXAMINATION OF COLLECTING TUBULES IN HUMAN KIDNEY.

Background: The current research histological changes in the Collecting tubules were examined at various postmortem intervals in the deceased human. In these research histological changes of tissue after death is influenced by P.M.I. atmospheric temperature and humidity, external and internal factors. Aim: To study the time since death by post mortem histological changes of collecting tubules in human Kidney. Materials and Methods: Present research was carried out, 40 cases of deceased kidney sample in between the temperature 17.3/22.3-31.3/450C,humidity 11/36 to 75/95 and duration range 4hrs to 52.30hr. The collecting tubules were examined to establish their correlation ship with hours PMI., temperatures, humidity or time since death by studied histological (H& E, PAS staining) in Pt.J.N.M.Medical College and Dr. B.R.Ambedkar Memorial Hospital Raipur (C.G.). Result: In collecting tubules (CT) after 4hrs PMI (27.5/42.20C T) disruption of epithelium was observed in most of the place. while after 46hrs PMI( 24.3/25.90C,T) disruption of epithelium, debris in the lumen with enucleated epithelial cells and pyknotic nuclei were seen, after 52.30hrs PMI(24.5/320C,T) disruption of epithelium, individualization of cells, debris in the lumen with pyknotic changes and enucleated cells were seen, Conclusion: Retraction of epithelium, disruption with individualization of cells, nuclear pyknosis, karyolysis and loss of tubular architecture with debris in the lumen were observed in collecting tubules. These criteria’s presented in this study could be used to determine the time after death. Keywords: Collecting tubules, P.M.I. (Post mortem interval), Medullary rays, Interstitium, Enucleated and Debris.

Histogenesis of human fetal kidney from 14 weeks to 36 weeks: a study

Background: The knowledge of fetal human Kidney morphology and developmental anatomy is very important for prenatal diagnosis of disorders such as Wilm’s tumor, hydronephroses and congenital malformation etc.Methods: The study was carried out on 40 kidneys procured from 20 spontaneously aborted fetuses (11males and 9 females) ranging from 14wks-36wks of gestation, after confirming their age through CRL they were grouped and then processed to form slides and stained with haemtoxylin and eosin and seen under light microscope.Results: All kidneys were lobulated at early gestational age and became fused by 36 wks. Corticomedullary junction and preformed collecting tubules were seen clearly by 18wks. Well differentiated PCT and DCT were formed by 19-23 wks. Well-formed pyramids by 28 wks and medullary rays by 29 weeks were clearly distinguished. Loop of Henle developed and distinguished by 28 wks. Increased vascularity was seen by 32-36 wks. Nephrogenic zone and undifferentiated mesenchyme decreased and matured glomeruli increased by 36 wks.Conclusions: The present study gave emphasis to the development of each component in medulla and cortex of kidney.

Contiguous gene deletion in a Chinese family with X-linked nephrogenic diabetes insipidus: challenges in early diagnosis and implications for affected families

Nephrogenic diabetes insipidus (NDI) is a rare disorder of the renal collecting tubules, characterized by an inability to concentrate urine due to an impaired response to arginine vasopressin (AVP), resulting in dilute urine and polyuria. Causes of NDI are heterogeneous and diagnosing congenital NDI (cNDI) in young infants is clinically challenging, as typical symptoms are often unappreciated or inconspicuous. Instead, young infants may present with non-specific signs such as vomiting, poor feeding, failure to thrive, unexplained fevers, irritability, constipation or diarrhea. We report a 37-day-old infant who presented with polyuria and severe hypernatremic dehydration that was unresponsive to vasopressin. The patient was treated with amiloride, indomethacin and hydrochlorothiazide. Genetic analysis revealed a novel contiguous deletion involving the entire AVPR2 gene and the last exon of the adjacent ARHGAP4 gene. A study of the family confirmed the carrier status in the mother. This case illustrates the importance of molecular testing in confirming the diagnosis in the index patient, as well as in identifying asymptomatic at-risk female carriers so that appropriate genetic counselling can be given for reproductive planning. All pediatric patients with suspected cNDI should undergo genetic analysis for a definitive diagnosis.

With no lysine kinase 4 modulates sodium potassium 2 chloride cotransporter activity in vivo

With no lysine kinase 4 (WNK4) is essential to activate the thiazide-sensitive NaCl cotransporter (NCC) along the distal convoluted tubule, an effect central to the phenotype of familial hyperkalemic hypertension. Although effects on potassium and sodium channels along the connecting and collecting tubules have also been documented, WNK4 is typically believed to have little role in modulating sodium chloride reabsorption along the thick ascending limb of the loop of Henle. Yet wnk4−/− mice (knockout mice lacking WNK4) do not demonstrate the hypocalciuria typical of pure distal convoluted tubule dysfunction. Here, we tested the hypothesis that WNK4 also modulates bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) function along the thick ascending limb. We confirmed that w nk4−/− mice are hypokalemic and waste sodium chloride, but are also normocalciuric. Results from Western blots suggested that the phosphorylated forms of both NCC and NKCC2 were in lower abundance in wnk4−/− mice than in controls. This finding was confirmed by immunofluorescence microscopy. Although the initial response to furosemide was similar in wnk4−/− mice and controls, the response was lower in the knockout mice when reabsorption along the distal convoluted tubule was inhibited. Using HEK293 cells, we showed that WNK4 increases the abundance of phosphorylated NKCC2. More supporting evidence that WNK4 may modulate NKCC2 emerges from a mouse model of WNK4-mediated familial hyperkalemic hypertension in which more phosphorylated NKCC2 is present than in controls. These data indicate that WNK4, in addition to modulating NCC, also modulates NKCC2, contributing to its physiological function in vivo.