Quantitative Enzyme Patterns in the Nephron of the Healthy Human Kidney

AbstractStudying isoform expression at the microscopic level has always been a challenging task. A classical example is kidney, where glomerular and tubulo-insterstitial compartments carry out drastically different physiological functions and thus presumably their isoform expression also differs. We aim at developing an experimental and computational pipeline for identifying isoforms at microscopic structure-level. We microdissed glomerular and tubulo-interstitial compartments from healthy human kidney tissues from two cohorts. The two compartments were separately sequenced with the PacBio RS II platform. These transcripts were then validated using transcripts of the same samples by the traditional Illumina RNA-Seq protocol, distinct Illumina RNA-Seq short reads from European Renal cDNA Bank (ERCB) samples, and annotated GENCODE transcript list, thus identifying novel transcripts. We identified 14,739 and 14,259 annotated transcripts, and 17,268 and 13,118 potentially novel transcripts in the glomerular and tubulo-interstitial compartments, respectively. Of note, relying solely on either short or long reads would have resulted in many erroneous identifications. We identified distinct pathways involved in glomerular and tubulointerstitial compartments at the isoform level.We demonstrated the possibility of micro-dissecting a tissue, incorporating both long- and short-read sequencing to identify isoforms for each compartment.

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Extensive preclinical evaluation of lutetium-177-labeled PSMA-specific tracers for prostate cancer radionuclide therapy

European Journal of Nuclear Medicine and Molecular Imaging ◽

10.1007/s00259-020-05057-6 ◽

2020 ◽

Author(s):

Eline A. M. Ruigrok ◽

Nicole van Vliet ◽

Simone U. Dalm ◽

Erik de Blois ◽

Dik C. van Gent ◽

...

Keyword(s):

Prostate Cancer ◽

Radionuclide Therapy ◽

Specific Binding ◽

Human Kidney ◽

Careful Evaluation ◽

Healthy Human ◽

Binding Characteristics ◽

Preclinical Evaluation

Abstract Purpose Various radiolabeled prostate-specific membrane antigen (PSMA)–targeting tracers are clinically applied for prostate cancer (PCa) imaging and targeted radionuclide therapy. The PSMA binding affinities, biodistribution, and DNA-damaging capacities of these radiotracers have not yet been compared in detail. A major concern of PSMA-targeting radiotracers is the toxicity in other PSMA-expressing organs, such as the salivary glands, thus demanding careful evaluation of the most optimal and safest radiotracer. In this extensive preclinical study, we evaluated the clinically applied PSMA-targeting small molecule inhibitors DOTA-PSMA-617 (PSMA-617) and DOTAGA-PSMA-I&T (PSMA-I&T) and the PSMA nanobody DOTA-JVZ-007 (JVZ-007) using PSMA-expressing cell lines, a unique set of PCa patient-derived xenografts (PDX) and healthy human tissues. Methods and results In vitro displacement studies on PSMA-expressing cells and cryosections of a PSMA-positive PDX revealed high and specific binding affinity for all three tracers labeled with lutetium-177 with IC50 values in the nanomolar range. Interestingly, [177Lu]Lu-JVZ-007 could not be displaced by PSMA-617 or PSMA-I&T, suggesting that this tracer targets an alternative binding site. Autoradiography assays on cryosections of human salivary and renal tissues revealed [177Lu]Lu-PSMA-617 to have the lowest binding to these healthy organs compared with [177Lu]Lu-PSMA-I&T. In vivo biodistribution assays confirmed the in vitro results with comparable tumor uptake of [177Lu]Lu-PSMA-617 and [177Lu]Lu-PSMA-I&T at all timepoints, resulting in induction of similar levels of DNA double-strand breaks in the tumors. However, [177Lu]Lu-PSMA-I&T demonstrated approximately 40× higher renal uptake at 4 and 8 h post injection resulting in an unfavorable tumor-to-kidney ratio. Conclusion [177Lu]Lu-PSMA-617 has the most favorable biodistribution in mice as well as more favorable binding characteristics in vitro in PSMA-positive cells and human kidney and salivary gland specimens compared with [177Lu]Lu-PSMA-I&T and [177Lu]Lu-JVZ-007. Based on our preclinical evaluation, [177Lu]Lu-PSMA-617 is the best performing tracer to be taken further into clinical evaluation for PSMA-targeted radiotherapeutic development although with careful evaluation of the tracer binding to PSMA-expressing organs.

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The Isoenzymes of Lactate Dehydrogenase in the Nephton of the Healthy Human Kidney

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.1968.6.3.132 ◽

1968 ◽

Vol 6 (3) ◽

Author(s):

K. G. THIELE ◽

H. MATTENHEIMER

Keyword(s):

Lactate Dehydrogenase ◽

Human Kidney ◽

Healthy Human

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The impact of preserved Klotho gene expression on antioxidative stress activity in healthy kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00486.2017 ◽

2018 ◽

Vol 315 (2) ◽

pp. F345-F352 ◽

Cited By ~ 11

Author(s):

Takaaki Kimura ◽

Kazuhiro Shiizaki ◽

Tetsu Akimoto ◽

Takahiro Shinzato ◽

Toshihiro Shimizu ◽

...

Keyword(s):

Gene Expression ◽

Renal Function ◽

Human Kidney ◽

Wild Type ◽

Healthy Human ◽

Expression Levels ◽

Klotho Gene ◽

Antioxidative Stress ◽

Deficient Mice ◽

Gene Expression Levels

Klotho, which was originally identified as an antiaging gene, forms a complex with fibroblast growth factor 23 receptor in the kidney, with subsequent signaling that regulates mineral metabolism. Other biological activities of Klotho, including antiaging effects such as protection from various types of cellular stress, have been shown; however, the precise mechanism of these effects of Klotho gene in the healthy human kidney is not well understood. In this study, we examined the relationships of Klotho and antioxidative stress gene expression levels in zero-hour biopsy specimens from 44 donors in kidney transplantation and verified them in animal models whose Klotho gene expression levels were varied. The nitrotyrosine expression level in the kidney was evaluated in these animal models. Expression levels of Klotho gene were positively correlated with the p53 gene and antioxidant enzyme genes such as catalase, superoxide dismutase 1 (SOD1), SOD2, peroxiredoxin 3 (PRDX3), and glutathione peroxidase 1 (GPX1) but not clinical parameters such as age and renal function or pathological features such as glomerulosclerosis and interstitial fibrosis tubular atrophy. The expression levels of all genes were significantly higher in mice with Klotho overexpression than in wild-type mice, and those except for catalase, PRDX3, and GPX1 were significantly lower in Klotho-deficient mice than in wild-type littermate mice. Nitrotyrosine-positive bands of various sizes were observed in kidney from Klotho-deficient mice only. The preservation of Klotho gene expression might induce the antioxidative stress mechanism for homeostasis of healthy human kidney independently of its general condition, including age, renal function, and histological findings.

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Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity

10.1101/2021.12.09.471943 ◽

2021 ◽

Author(s):

Caitriona M McEvoy ◽

Julia M Murphy ◽

Lin Zhang ◽

Sergi Clotet-Freixas ◽

Jessica A Mathews ◽

...

Keyword(s):

Single Cell ◽

Human Kidney ◽

Cell Types ◽

Significant Heterogeneity ◽

Proximal Tubular Cells ◽

Healthy Human ◽

Fresh Tissue ◽

Tubular Cells ◽

Distinct Cell ◽

Proximal Tubular

Maintaining organ homeostasis requires complex functional synergy between distinct cell types, a snapshot of which is glimpsed through the simultaneously broad and granular analysis provided by single-cell atlases. Knowledge of the transcriptional programs underpinning the complex and specialized functions of human kidney cell populations at homeostasis is limited by difficulty accessing healthy, fresh tissue. Here, we present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 high-quality cells to map healthy kidney at high resolution. Our sex-balanced dataset revealed sex-based differences in gene expression within proximal tubular cells, specifically, increased anti-oxidant metallothionein genes in females and the predominance of aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed between male and female proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. Within the immune niche, we identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions and validated findings by flow cytometry. We observed significant heterogeneity in resident myeloid populations and identified an MRC1+ LYVE1+ FOLR2+ C1QC+ population as the predominant myeloid population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, revealing novel insights into the complexity of renal parenchymal cells and kidney-resident immune populations.

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Quantitative histochemistry of the nephron. VII. Various phosphatases in the healthy human kidney

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(60)90465-3 ◽

1960 ◽

Vol 91 (1) ◽

pp. 130-137 ◽

Cited By ~ 39

Author(s):

S.L. Bonting ◽

Alta D. Tsoodle ◽

Hendrina de Bruin ◽

B.R. Mayron

Keyword(s):

Human Kidney ◽

Healthy Human ◽

Quantitative Histochemistry

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Iron sucrose (“rbt-3”) activates the hepatic and renal hamp1 gene, evoking renal hepcidin loading and resistance to cisplatin nephrotoxicity

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa348 ◽

2020 ◽

Author(s):

Richard A Zager ◽

Ali C M Johnson ◽

Renibus Therapeutics

Keyword(s):

Human Kidney ◽

Time Lapse ◽

Healthy Human ◽

Protein Levels ◽

Cisplatin Nephrotoxicity ◽

Human Volunteers ◽

Nrf2 Activation ◽

Plasma And Urine Samples

Abstract Background Fe sucrose (FeS) administration induces a state of renal preconditioning, protecting against selected forms of AKI. Recent evidence suggests that recombinant hepcidin also mitigates acute renal damage. Hence, the goals of this study were as follows: i) Determine whether a new proprietary FeS formulation (“RBT-3”), can acutely activate the hepcidin (HAMP1) gene in humans, raising plasma and renal hepcidin concentrations; ii) assess whether the kidney participates in this posited RBT-3-hepcidin generation response; iii) test whether RBT-3 can mitigate a clinically relevant AKI model (experimental cisplatin toxicity); and iv) explore whether mechanisms in addition to hepcidin generation are operative in RBT-3’s cytoprotective effects. Methods Healthy human volunteers (n, 9) and subjects with stage 3-4 CKD (n, 9) received 120, 240, or 360 mg of RBT-3 (IV over 2 hrs). Plasma and urine samples were collected and assayed for hepcidin levels (0-72 hrs post RBT-3 injection). In complementary mouse experiments, RBT-3 effects on hepatic vs. renal hepcidin (HAMP1) mRNA and protein levels were compared. RBT-3’s impact on the mouse Nrf2 pathway, and on experimental cisplatin nephrotoxicity, were assessed. Direct effects of exogenous hepcidin on in vivo and in vitro (HK-2 cells) cisplatin toxicity were also tested. Results RBT-3 induced rapid, dose dependent, and comparable plasma hepcidin increases in both HVs and CKD subjects (∼15x baseline within 24 hrs). Human kidney hepcidin exposure was confirmed by 4 fold urinary hepcidin increases. RBT-3 up-regulated mouse hepcidin mRNA, but much more so in kidney (>25x) vs. liver (∼2x). RBT-3 also activated kidney Nrf2 (increased Nrf2 nuclear binding; increased Nrf2-responsive gene mRNAs: HO-1, SrXN1, GCLC, NQO1). RBT-3 preconditioning (18 hr time lapse) markedly attenuated experimental cisplatin nephrotoxicity (∼50% BUN/creatinine decrements), in part, by reducing renal cisplatin uptake by 40%. Exogenous hepcidin (without RBT-3) treatment conferred protection against mild in vivo (but not in vitro) cisplatin toxicity. Conclusions RBT-3 acutely and dramatically up-regulates cytoprotective hepcidin production, increasing renal hepcidin levels. However, additional cytoprotective mechanisms are activated by RBT-3 (e.g., Nrf2 activation; reduced cisplatin uptake). Thus, RBT-3-induced preconditioning likely confers renal resistance to cisplatin via an interplay of multiple cytoprotective activities.

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Contribution of the Human Kidney to the Metabolic Clearance of Drugs

Annals of Pharmacotherapy ◽

10.1177/106002809202601116 ◽

1992 ◽

Vol 26 (11) ◽

pp. 1421-1428 ◽

Cited By ~ 33

Author(s):

Tom B. Vree ◽

Yechiel A. Hekster ◽

Patricia G. Anderson

Keyword(s):

Drug Metabolism ◽

Nalidixic Acid ◽

Renal Excretion ◽

Dominant Role ◽

Plasma Concentrations ◽

Human Kidney ◽

Pharmacokinetic Studies ◽

Metabolic Clearance ◽

Healthy Human ◽

Renal Metabolism

OBJECTIVE: To demonstrate that the human kidney is capable not only of filtering and secreting drugs and their metabolites, but also of carrying out conjugation reactions such as acyl glucuronidation, N-glucuronidation, and glycination. DATA SOURCES: Plasma concentrations and renal excretion rates of drugs are measured and renal clearance is calculated in a series of selected pharmacokinetic studies in healthy human volunteers (some studies were conducted in the authors' laboratory and others were reported in the literature). BACKGROUND THEORY: It is generally agreed that the liver plays the dominant role in drug metabolism, and that the function of the kidneys is limited to excretion of parent drug and metabolites. This can be easily understood when a metabolite is present in both plasma and urine. When the metabolite is present in urine but is not measurable in plasma, then the possibility exists that the metabolite is formed by the kidneys. RESULTS: “Simple” excretion by the kidneys is demonstrated for sulfatroxazole/sulfamethoxazole. Ether glucuronides of codeine are formed in the liver, and the resulting glucuronide is excreted by the kidneys. Possible formation of N1- and N2-glucuronides by the kidneys is demonstrated for sulfadimethoxine, sulfametomidine, and sulfaphenazole. Acyl glucuronidation of probenecid and nalidixic acid is carried out by the kidneys. The acyl glucuronidation of probenecid shows a capacity-limited formation/excretion rate of 46 mg/h, which is subject dependent. During this process, the acyl glucuronidation of co-administered nalidixic acid is reduced from 53 to 16 percent compared with that of nalidixic acid alone. Probenecid and its acyl glucuronidation do not inhibit the ether glucuronidation of codeine in the liver, but only interfere with the active tubular secretion process. The acyl glucuronidation of the nonsteroidal antiinflammatory drug naproxen and its metabolite, O-desmethylnaproxen, may be carried out by the liver and kidneys. Glycination of benzoic acid and salicylic acid is carried out in both the liver and kidneys. CONCLUSIONS: It is difficult to recognize renal drug metabolism in the intact human body (in vivo); the glucuronides or conjugates must be measured via direct HPLC analysis. In cases where the metabolite is present in high concentrations in urine but not in blood, there may be an indication that the kidneys are responsible for the formation of the metabolite. Impaired kidney function not only affects renal excretion but may also affect renal metabolism.

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Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1720140115 ◽

2018 ◽

Vol 115 (12) ◽

pp. E2819-E2828 ◽

Cited By ~ 29

Author(s):

Vasilios Kalas ◽

Michael E. Hibbing ◽

Amarendar Reddy Maddirala ◽

Ryan Chugani ◽

Jerome S. Pinkner ◽

...

Keyword(s):

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Bacterial Infections ◽

Kidney Tissue ◽

Human Kidney ◽

Salt Bridge ◽

Healthy Human ◽

Acetyl Group

Treatment of bacterial infections is becoming a serious clinical challenge due to the global dissemination of multidrug antibiotic resistance, necessitating the search for alternative treatments to disarm the virulence mechanisms underlying these infections. Uropathogenic Escherichia coli (UPEC) employs multiple chaperone–usher pathway pili tipped with adhesins with diverse receptor specificities to colonize various host tissues and habitats. For example, UPEC F9 pili specifically bind galactose or N-acetylgalactosamine epitopes on the kidney and inflamed bladder. Using X-ray structure-guided methods, virtual screening, and multiplex ELISA arrays, we rationally designed aryl galactosides and N-acetylgalactosaminosides that inhibit the F9 pilus adhesin FmlH. The lead compound, 29β-NAc, is a biphenyl N-acetyl-β-galactosaminoside with a Ki of ∼90 nM, representing a major advancement in potency relative to the characteristically weak nature of most carbohydrate–lectin interactions. 29β-NAc binds tightly to FmlH by engaging the residues Y46 through edge-to-face π-stacking with its A-phenyl ring, R142 in a salt-bridge interaction with its carboxylate group, and K132 through water-mediated hydrogen bonding with its N-acetyl group. Administration of 29β-NAc in a mouse urinary tract infection (UTI) model significantly reduced bladder and kidney bacterial burdens, and coadministration of 29β-NAc and mannoside 4Z269, which targets the type 1 pilus adhesin FimH, resulted in greater elimination of bacteria from the urinary tract than either compound alone. Moreover, FmlH specifically binds healthy human kidney tissue in a 29β-NAc–inhibitable manner, suggesting a key role for F9 pili in human kidney colonization. Thus, these glycoside antagonists of FmlH represent a rational antivirulence strategy for UPEC-mediated UTI treatment.

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